Site-specific N-glycosylation of alpha-2-macroglobulin reflects the progression of Schistosoma japonicum-induced liver fibrosis

Site-specific N-glycosylation of alpha-2-macroglobulin reflects the progression of Schistosoma japonicum-induced liver fibrosis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Site-specific N-glycosylation of alpha-2-macroglobulin reflects the progression of Schistosoma japonicum-induced liver fibrosis Xiao-Dong Gao, Song Zhao, Ganglong Yang, Hanjie Li, Guofang Li, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8453779/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Schistosomiasis leads to liver fibrosis through an egg-induced granulomatous inflammatory response in the host. Although parasite glycosylation is known to contribute to immune evasion and pathogenesis, alterations in host protein glycosylation during infection progression and liver fibrosis development remain poorly understood. Using a rabbit model specifically established to study Schistosoma japonicum -induced liver fibrosis (SjLF), we performed comprehensive glycoprofiling of serum glycomes and glycoproteomes via lectin microarrays and high-resolution mass spectrometry. Our analysis revealed dynamic changes in N-glycosylation of proteins involved in the humoral immune response, with particular emphasis on site-specific glycosylation of alpha-2-macroglobulin (A2M). Ten glycosylation sites on A2M were identified, exhibiting significant variations across different stages of infection and liver fibrosis in SjLF, suggesting their potential as indicators for monitoring SjLF progression. Further validation demonstrated that the N 1424 QT glycosylation site of human A2M in clinical serum samples serves as a promising diagnostic biomarker for SjLF, underscoring the value of targeted glycoproteomics in precise disease monitoring. This study provides insights into host glycosylation remodeling during SjLF and highlights its critical role in schistosome–host interactions. Biological sciences/Immunology/Infectious diseases/Parasitic infection Biological sciences/Cell biology/Glycobiology Schistosomiasis liver fibrosis alpha-2-macroglobulin intact glycopeptide site-specific glycosylation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Schistosoma infection continues to impose a significant global health burden as a persistent neglected tropical disease, with an estimated 230 million individuals at risk worldwide, particularly in the endemic regions of Asia and the Western Pacific 1 , 2 . The central pathological mechanism of schistosomiasis is characterized by the host's immune response to the parasite's eggs, which are highly immunogenic and capable of inducing vigorous circulating and local immune reactions 3 . Schistosomes are dioecious trematodes that reside in the host's blood vessels as paired male and female adults. When schistosome eggs become trapped in the portal venules of the liver, they elicit a T-cell-mediated granulomatous inflammatory response, initiating a cascade of immunopathological events. In the early stages, this inflammatory process is reversible; however, with disease progression, excessive collagen deposition ensues, culminating in portal fibrosis, portal hypertension, and potentially irreversible organ damage 4 , 5 . Advanced liver fibrosis occurs in 5–10% untreated individuals, manifesting as life-threatening complications including ascites and esophageal varices 6 , 7 . Notably, elimination of adult worm burdens from the host fails to mitigate established granuloma-mediated liver damage 8 . Although praziquantel remains the cornerstone of schistosomiasis management, the persistence of reinfection cycles and the presence of irreversible fibrotic sequelae in endemic populations highlight critical gaps in our understanding of fibrogenesis and its regulatory checkpoints 9 . Therefore, effective prevention strategies, coupled with precise diagnosis and timely intervention, represent the most efficacious approaches for the control and management of this disease. As the final common pathway of all chronic inflammatory injuries, liver fibrosis precise diagnosis was established with the fibrosis scores and non-invasive serum biomarkers. The Fibrosis-4 (FIB-4) index, AST-to-platelet ratio index (APRI), NAFLD fibrosis score, and BARD score were the most commonly employed with alanine aminotransferase (ALT) and aspartate transferase (AST) level serving as central components 10 . However, the diagnosis of schistosome-induced liver fibrosis remains challenging because the level of ALT and AST do not change significantly during the progression 11 . It was found that soluble egg antigen (SEA) and S. japonicum eggs release exosomal miR-33 play critical role in the schistosomiasis japonica but not enough for the diagnosis 12 , 13 . Concurrently, glycosylation, one of the key post-translational modifications, play an important role in schistosome infection and liver fibrosis, which include the mediating host-parasite interactions and host immunity 14 – 17 . As a powerful tool, glycoproteomics can systematically unveil the protein glycosylation modifications during the liver fibrosis progression. Clinical glycoproteomics studies of virus-induced and MASLD-related liver fibrosis have revealed stage-specific glycosylation changes, including the increasing core fucosylation of haptoglobin, sialic acid truncation on α-1-antitrypsin, and bisected N-glycan accumulation on IgA 18 – 20 . Moreover, N-glycosylation heterogeneity of Mac-2 binding protein serves as a superior prognostic marker for portal hypertension risk stratification in liver fibrosis, outperforming conventional biomarkers 21 , 22 . Yet, the glycosylation alteration of schistosome-induced liver fibrosis is largely unclear. In this study, a well-characterized rabbit model was established for studying the entire progression of S. japonicum -induced liver fibrosis (SjLF). We utilized a multi-omics approach, combining lectin microarrays and high-resolution mass spectrometry (MS), to comprehensively profile glycopatterns and identify intact glycopeptides (IGPs) of the host serum across distinct stages of the SjLF. Our findings reveal dynamic changes in branched sialylated and fucosylated glycopatterns as well as the site-specific N-glycosylation heterogeneity of serum proteins. Particularly, the alpha-2-macroglobulin (A2M) was found to exhibit significant alterations in site-specific glycosylation, with specific glycan structures emerging as candidate biomarkers for the progression of SjLF. Targeted parallel reaction monitoring (PRM)-MS in clinical cohorts confirmed the diagnostic utility of these A2M glycosylation. Our study highlights the role of glycosylation in schistosome-host interplay and offers a tool for clinically diagnosing the progression of SjLF. Results Establishment of a rabbit model for the study of Schistosoma japonicum -induced liver fibrosis (SjLF) To systematically investigate the glycosylation landscape during Schistosoma japonicum -induced liver fibrosis (SjLF), a rabbit model was established to faithfully recapitulate the entire process, including the infection and liver fibrosis phases (Fig. 1 a). New Zealand rabbits were percutaneously infected with S. japonicum cercariae via abdominal application, following a standardized protocol 23 . Infection was confirmed by detection of ciliated miracidia in fecal samples at four weeks post-infection. At seven weeks post-infection, rabbits were treated with praziquantel (PZQ) for two consecutive days and then maintained for an additional seven weeks to allow progression of liver fibrosis. Serum samples were collected at multiple time points throughout the SjLF timeline: three days before infection served as the healthy control (Control); The one week and six weeks post-infection represented the early (I-1w) and late (I-6w) stages of infection, respectively; The twelve weeks and fourteen weeks post-infection corresponded to the early (I-12w) and late (I-14w) stages of liver fibrosis (Fig. 1 a). Noteworthy, twelfth week post-infection (I-12w) is also the fifth week after the PZQ treatment, reflecting the cure effect. These longitudinal serum samples were utilized to analyze dynamic changes in glycosylation profiles associated with the progression of SjLF, which includes the infection of S. japonicum (Sj infection) and liver fibrosis. The model rabbits were euthanized in the fourteenth week, and liver samples were collected for histopathological analysis (Fig. 1 a). The H&E and Masson's trichrome staining revealed that liver fibrosis was characterized by egg-induced granulomas, inflammatory tissue damage, and subsequent reparative fibrosis resulting from the deposition of S. japonicum eggs in the hepatic vasculature (Fig. 1 b). Additionally, elevated expression levels of α-smooth muscle actin (α-SMA) in the SjLF samples indicated activation of hepatic stellate cells and progressive fibrogenesis (Fig. 1 b). These findings demonstrated marked structural alterations, including increased collagen deposition and tissue stiffening in the livers of SjLF rabbits compared to healthy controls, thereby validating the reliability of the rabbit model for recapitulating human-like SjLF. Lectin microarray analysis reveals alterations in glycosylation patterns during the progression of SjLF. To explore the alterations in glycosylation patterns in the serum of the SjLF rabbit model, lectin microarray analysis was utilized to profile and quantify glycopatterns based on normalized relative fluorescence intensities (NFIs, Supplementary Table 1). As a reference, serum glycopatterns from the “Control” were initially examined, which exhibited high levels of T-antigen (Gal β1,3 GalNAc α-Ser/Thr, detected by Jacalin), oligomannosylation (α-D-Man, recognized by ConA), core fucosylation (Fuc α1,6 GlcNAc, bound by LCA), and terminal Gal β1,4 GlcNAc structures (identified by RCA120) (Supplementary Fig. 1). From a comprehensive perspective, our lectin microarray analysis unveiled substantial alterations in glycopatterns during the progression of SjLF. Hierarchical clustering indicated that glycosylation profiles from the early (I-1w) and late (I-6w) stages of infection, as well as the late liver fibrosis stage (I-14w), were significantly distinct from those of the control group (Fig. 2 a), suggesting dynamic changes associated with Sj infection and liver fibrosis phases. Noteworthy, serum samples collected at I-12w (the fifth week after PZQ treatment, see Fig. 1 a) exhibited a glycosylation profile highly similar to that of the “Control”, implying functional recovery likely due to the therapeutic effects of drug treatment (Fig. 2 a). In detail, a total of 17 lectin-recognized glycopatterns exhibited statistically significant alterations during SjLF (p < 0.05; Fig. 2 b). Glycopatterns with terminal fucosylation, branched (LacNAc)n structures, and sialylation (Neu5Ac), recognized by EEL, LTL, UEA-1 and MAL-I respectively, showed marked upregulation at the late stage of liver fibrosis (I-14w). Meanwhile, higher levels of branched (LacNAc)n and H(O) antigen (Fuc α1,2 Gal β1,4 GlcNAc) were detected by PWM and UEA-I at the early infection stage (I-1w). Additionally, the WFA-recognized epitope GalNAc α/β 1,3/6 Gal was elevated at the late stage of infection (I-6w) but rapidly normalized following PZQ treatment, indicating its potential as a prognostic biomarker for infection status and therapeutic response (Supplementary Fig. 2). Collectively, these findings underscore the crucial role of branched, sialylated, and fucosylated glycopatterns in the process of SjLF, highlighting their potential as candidates for early diagnosis of SjLF. Comprehensive glycoproteomics delineates N-glycosylation features throughout the entire process of SjLF. To achieve a deeper comprehension of the alterations in N-glycan structures throughout the SjLF process, we employed a high-resolution glycoproteomics technique, utilizing liquid chromatography-tandem mass spectrometry (LC-MS/MS) to directly investigate intact glycopeptides (IGPs) within the serum samples of model rabbits. A total of 2,987 unique intact glycopeptides (IGPs) were identified. The Venn diagram analysis revealed that approximately 458 N-linked IGPs were consistently present across all stages (Fig. 3 a). Conversely, a substantial number of SjLF-specific IGPs, which only emerge during the infection and liver fibrosis phases, were also observed (Fig. 3 a). Hierarchical clustering of the normalized IGP abundances demonstrated distinct group separations driven by site-specific N-linked glycosylation features (Fig. 3 b). Based on the dynamic changes occurring during the infection and liver fibrosis phases, IGPs can be categorized into nine clusters (Supplementary Fig. 3). Among these clusters, glycopeptides in clusters 1 and 2 exhibited a rapid response to Sj infection, whereas clusters 3 and 4 showed a delayed response. Notably, IGPs in cluster 2, which increased in both the early and late stages of infection (I-1w and I-6w, Supplementary Fig. 3), garnered our attention. It was discovered that, among the 231 IGPs in cluster 2, 33 glycopeptides originated from alpha-2-macroglobulin (A2M) (Fig. 3 c), indicating significant alterations in site-specific glycosylation on A2M. Similar unique IGPs were also identified as cluster 3, which increased during the late infection stage and subsequently returned to a low level during the early liver fibrosis stage (I-12w) following treatment with praziquantel (PZQ) (Supplementary Fig. 3). Among the 556 IGPs in cluster 3, 37 glycopeptides were derived from ceruloplasmin (CP), and 19 were from A2M (Fig. 3 d). In comparison to the IGPs that responded to the Sj infection, we were particularly intrigued by the IGPs that exhibited increased intensity during the liver fibrosis phase (Fig. 3 , Supplementary Fig. 3 and Supplementary Table 2). Specifically, IGPs in cluster 5 remained unchanged during the stages of Sj infection (I-1w and I-6w) and early liver fibrosis (I-12w) but showed an increase in the late stage of liver fibrosis (I-14w). According to our Venn diagram and hierarchical clustering analyses, the 250 IGPs in cluster 5, which were exclusive to the late stage of liver fibrosis (I-14w), were predominantly fucosylated and sialylated (Fig. 3 b), highlighting the role of site-specific glycosylation in liver fibrosis phase. Among these, 11 IGPs were derived from A2M, and 8 were from immunoglobulin heavy constant mu (IGHM) (Fig. 3 e). Additionally, IGPs in cluster 4 also showed a significant increase in the late stage of liver fibrosis, despite their distinct response to Sj infection and subsequent restoration in the early stage of liver fibrosis following PZQ treatment (Fig. 3 e, and Supplementary Fig. 3). The main contributors to this cluster of IGPs included IGHM (15 IGPs), haptoglobin (HP, 11 IGPs), and A2M (8 IGPs) (Fig. 3 f). In summary, IGPs with distinct enhancements were identified throughout the SjLF process, many of which originated from proteins related to humoral immune responses (e.g., CP, IGHM, HP, and A2M, as shown in Fig. 3 ). Notably, among these proteins, the glycosylation of A2M changed markedly across all stages, representing the interactions between its glycosylation remodeling and the progression of SjLF. Site-specific N-glycosylation on alpha-2-macroglobulin (A2M) reflects the progression of SjLF The preceding analysis unveiled dynamic alterations in alpha-2-macroglobulin (A2M)-derived IGPs across the phases of Sj infection and liver fibrosis. To elucidate the causes of these changes in IGP intensities, we initially examined the global glycosylation profile of A2M. As depicted in Fig. 4 a, the glycopeptide spectrum matches (GPSM) of IGPs from A2M remained relatively stable during the progression of SjLF, including the early and late stages of Sj infection and liver fibrosis. Additionally, a previous study has demonstrated consistent protein expression levels of A2M across these stages 24 . Based on these findings, we confirmed that the global N-glycosylation and protein expression of A2M are not the primary factors driving the dynamic changes observed in A2M-derived IGPs. To gain insights into the site-specific glycosylation of A2M and assess its potential clinical applications, we further analyzed the mass spectrometry data of A2M-derived IGPs. We identified 10 glycosylation sites with 110 unique IGPs and 49 N-linked glycan structures in host serum, representing the site-specific glycosylation heterogeneity of this protein across all stages (Fig. 4 b and 4 c). Overall, 84% of the glycans on A2M were fucosylated and sialylated. Regarding site-specific glycosylation subtypes, nearly all glycosylation sites exhibited high sialylation, except for the N 1399 RT site, which was predominantly occupied by high mannose-type glycans (Fig. 4 b and 4 c). Moreover, the N 381 KT motif was identified as the most N-glycosylated site, harboring approximately 25 glycan structures (Fig. 4 c). From a diagnostic perspective, IGPs such as N 55 ET-N4H5S2, N 247 VS-N4H5F2, N 381 KT-N5H5F1S1, N 410 TT-N4H5S1, N 410 TT-N4H5F2, and N 940 ES-N4H5S1 (highlighted in red, Fig. 4 c) exhibited increased levels in both Sj infection stages (I-1w and I-6w) and the late stage of liver fibrosis phase (I-14w). In contrast, IGPs like N 247 VS-N3H3F1, N 381 KT-N5H5S2, and N 1417 QT-N2H5 (highlighted in orange, Fig. 4 c) were specifically elevated in the late stage of liver fibrosis (I-14w, Fig. 4 c). These novel IGPs reflect the development of SjLF. Additionally, IGPs such as N 1359 LS-N5H5F1 and N 1399 RT-N2H7 (highlighted in blue, Fig. 4 c) were exclusively detected in the infection stages, indicating the presence of S. japonicum infection. Collectively, our results suggest that the site-specific glycosylation of A2M may serve as potential biomarkers for the progression of SjLF. Clinical validation of the site-specific glycosylation on human A2M identified diagnostic biomarkers for the SjLF To determine whether the site-specific glycosylation of A2M can serve as a biomarker for diagnosing SjLF, a cohort study was conducted using serum samples from SjLF patients. The clinical serum cohort included 12 S. japonicum -infected patients who underwent three follow-up sessions, as well as 12 healthy volunteers as controls. The baseline characteristics of the patients are detailed in Table 1 , where the severity of SjLF patients were categorized into three stages (normal, mild, and severe) based on liver parenchyma grading. The ALT and AST levels in serum samples from patients were not significantly elevated during the three follow-up detections (Table 1 and Supplementary Fig. 4). It is important to note that, as classic biomarkers of liver fibrosis, the standard ALT and AST levels for healthy individuals in women/men are typically ≤ 35/≤50 U/L and ≤ 40/≤60 U/L, respectively 25 . Most of the ALT and AST values of our patients fall within the normal range (Table 1 and Supplementary Fig. 4). However, liver fibrosis progression, as determined by liver parenchyma grading, was detected in 58% of patients during the second follow-up and in 100% of patients during the third follow-up (Supplementary Fig. 4). This discrepancy suggests that the classic ALT and AST biomarkers may not be suitable for diagnosing liver fibrosis induced by S. japonicum. Table 1 The clinical information and diagnostic performance of SjLF patients Case Name Gender Follow-up Liver parenchyma grading* Fibrosis stage ALT(U/L)** AST(U/L)*** P1 M 1 0 normal 34.6 28.1 2 0 normal 22.7 29.8 3 II severe 77.2 82.6 P2 M 1 0 normal 40.9 27.3 2 0 normal 39.9 33.4 3 I Mild 34.2 44.2 P3 M 1 0 normal 29.7 19.4 2 I mild 20.7 23.9 3 I mild 15.2 31.4 P4 F 1 0 normal 23.2 29.7 2 0 normal 25 32.9 3 I mild 38.3 55.7 P5 M 1 I mild 25.7 31.3 2 II severe 21.2 23.1 3 III severe 28 30 P6 M 1 II severe 21.4 27.1 2 I mild 12 23 3 III severe 16 25 P7 F 1 I mild 26.6 24.8 2 II severe 14.4 17.4 3 II severe 22 16 P8 F 1 I mild 14.7 21.3 2 II severe 10.6 12.4 3 II severe 7 17 P9 F 1 0 normal 22.2 19.6 2 0 normal 24.3 21.4 3 I mild 8.9 30.1 P10 F 1 0 normal 42.1 25 2 0 normal 30.8 27.8 3 I mild 46.8 35.2 P11 M 1 I mild 16.8 32.5 2 II severe 22.3 13.7 3 II severe 16 16 P12 F 1 I mild 15.5 22.6 2 II severe 37.1 25.7 3 II severe 15 14 * The liver parenchyma grading of SjLF patients. Grade 0 : Normal. Uniform echo pattern with moderate brightness and homogeneous distribution. Grade I : Coarse echo texture. Characterized by thickened and hyperechoic foci with heterogeneous distribution. Grade II : Prominent hyperechoic bands forming a "fish-scale" pattern, with mesh diameters mostly 2 cm. * * The levels of alanine aminotransferase (ALT) in patients with SjLF are presented. For healthy individuals, the standard ALT concentrations are usually ≤ 35 U/L for females and ≤ 40 U/L for males. Values exceeding 35 U/L in female or 40 U/L in male are indicated in red. * * * The levels of aspartate aminotransferase (AST) in patients with SjLF are presented. For healthy individuals, the standard AST concentrations are usually ≤ 50 U/L for females and ≤ 60 U/L for males. Elevated values above 50 U/L in females or 60 U/L in males are highlighted in blue. To validate the site-specific glycosylation of A2M in the patient cohort, an integrated MS strategy was developed for targeted detection of A2M-derived IGPs, instead of the conventional assay using bifunctional antibodies. Initially, non-targeted site-specific glycosylation analysis of A2M was performed using parallel accumulation-serial fragmentation (PASEF) MS/MS to create a spectral library for selecting A2M-derived IGPs. Subsequently, the selected IGPs were quantified using parallel reaction monitoring (PRM) (Fig. 5 a). In the non-targeted analysis, 39 IGPs and 5 glycosylation sites from human A2M were identified (Supplementary Tables 3). Glycopeptides were selected for PRM quantification based on four criteria: (i) detection in all samples from rabbit infection and liver fibrosis phases, (ii) significant alteration during the progression of SjLF, (iii) identification in clinical cohort samples via DDA analysis, and (iv) detection in over 97% of clinical cohort samples in PRM analysis (Supplementary Table 4). Consequently, two glycosylation sites (N 55 ET and N 1424 QT) of human A2M, which matched with the N 55 ET and N 1417 QT sties of rabbit serum (Fig. 4 c), were identified through this selection process. The human peptide containing the N 1424 QT site, VSN 1424 QTLSLFFTVLQDVPVR, was modified with 19 glycan structures, while the N55ET site only carried 6 glycans (Fig. 5 b). Ultimately, three N 1424 QT-containing IGPs, featuring sialylated bi-antennary glycans N4H5S1 and N4H5S2, and a sialo-fucosylated bi-antennary glycan N4H5F1S1, were selected for further PRM quantification (Fig. 5 b and 5 c). In the PRM analysis, oxonium ions (204.0866, 138.0550, 292.1019, and 274.0920), b/y and B/Y ions were used for glycopeptide identification, while Y1 ions were used for target quantification (Fig. 5 d). The three selected IGPs (N 1424 QT-N4H5S1, N 1424 QT-N4H5F1S1, and N 1424 QT-N4H5S2) exhibited differential abundance across normal, mild, and severe SjLF (Fig. 5 e and Supplementary Fig. 5). Importantly, PRM was able to distinguish glycan structures, such as core- and terminal-fucosylation, and also differentiate glycan isomers, like α2,3 and α2,6 linked sialic acid, using MS/MS spectra and retention time drifts 26 , 27 . In our study, the sialylated glycosylation of SGP was confirmed with three isomers (Supplementary Fig. 6). The isomers of A2M-N 1424 QT-N4H5F1S1 were identified as core-fucosylated with α2,6 (isomer 1) and α2,3 (isomer 2) sialic acid, showing a 4s gradient shift and a core-fucosylated peak (Y3-Fuc). These isomers also differed between SjLF patients and healthy volunteers (Fig. 5 f). The three individual A2M IGPs demonstrated strong diagnostic discrimination (AUC > 0.6), with combined analysis showing higher discrimination (AUC > 0.7) (Fig. 5 g). It should be noted that, prior to analyzing the glycopeptides of A2M, the protein expression and global glycosylation of A2M in clinical serums were examined and compared with those of healthy volunteers. Western blotting results indicated no significant changes in A2M protein expression in mild and severe SjLF patients (Supplementary Fig. 7a). For global glycosylation, lectin blotting with Con A and AAL demonstrated that the N-linked glycosylation of A2M was also not significantly different between healthy volunteers and patients (Supplementary Fig. 7b). These findings ruled out the possibility that global N-glycosylation and protein expression of A2M caused the alteration of glycosylation at the A2M-N 1424 QT site. Overall, our studies demonstrated that the site-specific glycosylation panel of A2M-N 1424 QT enhances the diagnostic accuracy of SjLF, indicating that PRM can serve as a targeted detection tool for the clinical diagnosis of site-specific glycosylation. Discussion Schistosomiasis affects over 300 million people globally, with a significant proportion at risk of developing liver fibrosis and cirrhosis. Currently, there are no molecular diagnostic markers available for the early detection of schistosome-induced liver fibrosis. Given the well-established association between protein glycosylation and liver fibrosis, we hypothesized that a systematic analysis of glycosylation changes during infection and disease progression could identify novel biomarkers for early diagnosis. Here, we developed a rabbit model of S. japonicum –induced liver fibrosis (SjLF), which recapitulates key features of human SjLF. Through the comprehensive analysis of N-glycosylation in this model, a set of potential glycoprotein biomarkers was discovered for the early detection of SjLF. Our lectin microarrays analysis unveiled a significant increase in branched, sialylated, and fucosylated glycan structures during the progression of SjLF (Fig. 2 ). This finding was further validated and explored through site-specific glycoproteomics, which directly analyzes IGPs while preserving glycan structural integrity and enables precise mapping of glycosylation events to specific proteins and sites 28 . We identified distinct enhancements in site-specific sialylated and fucosylated glycosylation during the progression of SjLF, including the Sj infection and liver fibrosis phases, particularly on humoral immune response-related proteins such as A2M, HP, and IGHM (Fig. 3 – 4 ). Previous studies on parasitic infections and the immunopathology of schistosomiasis have highlighted the physiological significance of humoral immunity in host defense against parasites 29 . Consistent with these observations, our analysis revealed alterations in N-glycan modifications on these proteins. Among them, the site-specific glycosylation of A2M exhibited the most unique features, showing dynamic alterations throughout the entire SjLF process (Fig. 3 ). A2M, an extracellular macromolecule primarily synthesized in the liver, is well-known as a broad-spectrum protease inhibitor and plays a crucial role in immune responses and the removal of damaged extracellular proteins 30 . Alterations in site-specific N-glycosylation on A2M during the progression of SjLF were confirmed in both rabbit models and human clinical cohorts (Fig. 4 – 5 ). The high-abundance glycans N4H5S1, N4H5S2, and N4H5F1S1 on the N 1424 QT site of human A2M could clearly distinguish normal, mild, and severe SjLF stages, with a combined AUC exceeding 0.7 as a panel (Fig. 5 ), highlighting the clinical relevance of A2M's site-specific glycosylation and glycan structures. Notably, A2M has been proposed as a serum fibromarker for chronic hepatitis C infection and liver fibrosis due to its upregulated protein levels in liver biopsies and serum of chronic hepatitis C patients 31 , 32 . However, A2M protein expression did not change across the progression of Sj infection and liver fibrosis 24 , indicating that its expression level is not suitable as a serologic biomarker for S. japonicum -induced liver fibrosis. In contrast, our findings on the alterations of site-specific N-glycosylation on A2M, especially IGPs containing the N 1424 QT site, could fill the gap for early SjLF diagnosis, an area not previously emphasized in clinical determinations. Several other potentially valuable discoveries were made from our comprehensive analysis of serum glycosylation. In the rabbit model, only the N 1399 RT glycosylation site of A2M was specifically modified with high mannose-type glycans, with IGPs containing N2H7 significantly increasing during infection stages (Fig. 4 ). It has been reported that mannose-binding protein (MBP) directly binds to Man5-7 high mannose of A2M to opsonize and activate the lectin pathway of the complement cascade, eliminating infection. This binding triggers the lectin pathway via MASP-2, cleaving complement components C4 and C2 to activate the C3 convertase C4b2a 33 . Thus, the N 1399 RT site may contribute to activating the lectin pathway of the complement cascade through its binding to MBP. It is conceivable that similar N-glycosylation sites with specific high-mannose modifications also exist in human A2M. Moreover, we detected several glycans typically considered schistosome-specific (data not shown), such as xylose-containing pentose glycans 34 – 37 . This raises the possibility of glycan transfer from the parasite to host proteins, a phenomenon that may contribute to immune modulation or pathogenesis. In conclusion, our study has comprehensively delineated and confirmed the site-specific glycosylation alterations induced by S. japonicum infection and the development of liver fibrosis. Notably, the glycosylation of human A2M at the N 1424 QT site emerges as a promising biomarker for the diagnosis of schistosomiasis infection and the early detection of SjLF. Further investigation into the molecular mechanisms underlying how site-specific glycosylation on A2M responds to SjLF may pave the way for the development of a vaccine against the S. japonicum infection. Material and Methods Ethics statement The animal experiments were performed in strict accordance with the Laboratory Animal Guideline for Ethical Review of Animal Welfare (GB 14925 − 2010). The experimental protocol was reviewed and approved by the Ethics Review Committee of the Jiangsu Institute of Parasitic Diseases, under the approval number JIPD − 2020–013. The human serum samples utilized in this study were sourced from individuals participating in a prospective cohort study involving exposure to S. japonicum . This cohort study was granted approval by the Ethics Committee of the Jiangsu Institute of Parasitic Diseases (approval number JIPD − 2019–008) 38 . Prior to their involvement, written informed consent was obtained from all participants. Pretreatment and protein preparation of sera from the rabbits New Zealand rabbits (weighing 2.0–2.5 kg, n = 4) were experimentally infected with 1000 cercariae of S. japonicum sourced from the Jiangsu Institute of Parasitic Diseases in China using the abdominal patch method. To verify the success of infection, fecal samples from these rabbits were subjected to the miracidium hatching test 23 . Sera were collected from S. japonicum -infected rabbits at 1 week post-infection (I-1w, early infection) and 6 weeks post-infection (I-6w, late infection) to represent the infection group. Following 7 weeks of S. japonicum infection, rabbits were orally administered praziquantel (PZQ, 150 mg/kg) for two consecutive days. Sera were further collected from rabbits at 12 weeks post-infection (I-12w, early liver fibrosis) and 14 weeks post-infection (I-14w, late liver fibrosis) to investigate liver fibrosis progression (Fig. 1 a). Additionally, sera were also obtained from uninfected New Zealand rabbits to serve as a healthy control group (Control). Finally, all 20 rabbits were euthanized for morphological examination. After incubation at 37℃ for 4 h, sera were isolated by centrifugation at 200 × g for 5 min and subsequently stored at − 80℃. Equal volumes of serum protein from each sample were mixed and combined 39 . Albumin and IgG were depleted from the sera to simplify the protein composition, following the instructions provided with the ProteoExtract albumin/IgG removal kit (Merck Millipore, Billerica, MA, USA) 40 . The serum proteins were then concentrated using 10 kDa centrifuge ultrafiltration tubes (Amicon ultra − 0.5, Millipore, MA, USA) by centrifugation at 13,000 × g for 5 min. Patients and sera sample collection The study cohort consisted of 12 healthy controls and 12 patients with confirmed S. japonicum infection. All serum samples were derived from participants enrolled in the prospective cohort study of S. japonicum exposure 38 . The cohort-related follow-up procedures, including questionnaires, physical examinations, and biospecimen collection, were conducted in strict adherence to the established work manual. The patients were recruited from Yangzhou, Jiangsu, China, an area endemic for S. japonicum , between August 2017 and July 2019. Each participant provided three serum samples collected during follow-ups in 2019, 2021, and 2025. Additionally, 12 control serum samples were collected in 2025 from age- and gender-matched healthy individuals with no history of S. japonicum infection from the same region. All patients had been treated with praziquantel (PZQ) upon diagnosis of S. japonicum infection during the 1960s to 1970s. Patients with the following conditions were excluded: mixed liver disease; coinfection with other viruses, such as hepatitis A virus (HAV), HBV, HCV, or HDV; decompensated liver disease; primary liver cancer or other malignant tumors; autoimmune or immunologically mediated diseases; organ transplantation; or severe cardiac, hepatic, or renal dysfunction. Basic physical examination information was documented. Liver parenchyma and size, the width of the main portal vein, spleen size, and the presence of ascites were assessed via ultrasound examinations. Hepatic parenchymal status (Grade 0 - III) and stages (normal, mild, and severe) were determined by community physicians. Relevant clinical information of the patients is summarized in Table 1 and supplementary Table 5. Lectin microarray determination and data analysis Briefly, the lectin microarray procedure was conducted as follows: 37 lectins were spotted in triplicate using the CapitalBio SmartArrayer 48 Microarray Spotter (CapitalBio, China). The microarrays were incubated and dried at 37°C, followed by storage at 4°C in the dark. Approximately 100 µg of serum proteins were labeled with Cy3 fluorescent dye at room temperature and purified using a G-25 column. The microarray was blocked in an LF-IIIA hybridization oven (SCIENTZ, Ningbo, China), and then 6 µg of the fluorescence-labeled proteins were applied. The lectin microarray was subsequently read using a Genepix 4000B confocal microarray scanner (Axon Instruments, USA). The median values of all lectins were normalized. One-way ANOVA was performed on the five groups of lectin-normalized fluorescence intensities (NFIs), with post-hoc analysis conducted using Tukey’s method to adjust p-values. A lectin NFI was considered significantly elevated if the NFI ratio between groups was > 1.5 and p < 0.05, and significantly decreased if the NFI ratio was < 0.67 and p < 0.05. SDS-PAGE and lectin blotting A 10% separating gel and a 5% stacking gel were prepared, and samples containing 20 µg of protein were loaded onto the gel. The gel was subsequently stained with Coomassie brilliant blue. For lectin staining, the proteins were transferred to a PVDF membrane (0.45 µm) using transfer buffer. To minimize non-specific binding, the membrane was blocked with Carbo-free blocking solution for 1 hour at room temperature. Subsequently, 2 µg/mL solutions of biotinylated-PWM (pokeweed mitogen), biotinylated-UEA-Ⅰ (Ulex europaeus agglutinin I), biotinylated-WFA (Wisteria floribunda agglutinin), biotinylated-ConA (concanavalin A), and biotinylated-AAL (Aleuria aurantia lectin) were prepared by diluting with blocking solution and incubated with the membrane overnight at 4°C. After washing the membrane, streptavidin-HRP (Horseradish Peroxidase, diluted 1:10,000) was added and incubated for 1 hour at room temperature. Following electrochemiluminescence (ECL) development for 1–2 minutes, the bands were visualized using the Molecular Imager ChemiDoc XRS + System and Image Lab Version 3.0. The results were analyzed using ImageJ software, with the relative intensity of the bands, which was calculated as the ratio of the grayscale value sum of the bands to the sum of the corresponding Coomassie brilliant blue (CB) staining 41 . Serum protein digestion and glycopeptide enrichment The serum proteins from rabbits and patients were pretreated and denatured in an 8 mM urea/40 mM NH4HCO3 buffer. They were then treated with 10 mM dithiothreitol (DTT) at room temperature, followed by 20 mM iodoacetamide (IAM) for 45 minutes in the dark. Sequencing-grade modified trypsin (Promega, Wisconsin, USA) was added, and the proteins were digested using filter-aided protein digestion in a 10 kDa centrifugation ultrafiltration tube. The reaction was terminated with 50% formic acid (FA) (v/v), and the peptide concentration was measured using NanoDrop. The peptides were purified using a combination of C18 and mixed anion exchange (MAX) solid - phase extraction columns 42 . Specifically, 600 µg of the peptide solution was transferred to the prepared column. The mixed column was washed sequentially with acetonitrile (ACN), 100 mM triethylammonium acetate buffer (TEAB), 1% trifluoroacetic acid (TFA) (v/v), and 95% ACN (v/v). After injection, the sample was washed three times with 1% TFA (v/v) in 95% ACN (v/v) and finally eluted with 50% ACN (v/v) in 1% FA to collect the enriched glycopeptide samples. The samples were then lyophilized using a freeze dryer (Labconco, USA). The intact glycopeptide (IGP) analysis by high-resolution liquid chromatography-tandem mass spectrometry (LC-MS/MS) Intact glycopeptides (IGPs) were resuspended in a 0.1% FA with 2% ACN solution at a concentration of 0.5 µg/µL. The rabbit serum samples were analyzed using an EASY - nLC 1200 system (Thermo Fisher Scientific, USA) coupled to a high - resolution Orbitrap Fusion Lumos Mass Spectrometer (Thermo Fisher Scientific, USA), with an injection volume of 3 µL per sample. The samples were initially separated on the EASY-nLC 1200 system using mobile phase buffer A (water) and buffer B (90% ACN) at a flow rate of 550 nL/min. The liquid phase gradient was programmed as follows: 0–1 min, buffer B linear gradient from 2% to 6%; 1–91 min, buffer B linear gradient from 6% to 30%; 91–113 min, buffer B linear gradient from 30% to 38%; 113–118 min, buffer B linear gradient from 38% to 80%; 118–123 min, buffer B maintained at 80%. Data-dependent high energy collision dissociation (HCD) was performed on the 20 most abundant ions. The AGC target was set at 4.0×10 5 , the maximum injection time was 250 ms, and the acquisition was conducted within the resolution range of m/z 350–2000. MS data analysis The protein databases of S. japonicum species in Uniprot database (downloaded on 09/23/2020) were downloaded and searched using Byonic software 43 , and the combined N-glycan databases of New Zealand rabbits 44 and S. japonicum 37 , 45 were constructed, containing a total of 121 N-glycans. Byonic software parameters are set as follows: restriction enzyme digestion site is R/K, maximum missed cleavage is 2, precursor mass tolerance is 10 ppm, fragment type is HCD, fragment mass tolerance is 20 ppm, cysteine alkylate is set to fixed modification, the false discovery rate (FDR) < 1%, and cut-off score was set to 300. Student's t-test (two-tailed, unpaired, and equal variance) was performed and the significant increased (adjusted p 2) or decreased (adjusted p < 0.05, FC < 0.5) protein glycosylation in each experimental group compared to the control group was set. Clinical serum IGPs analysis using LC-MS/MS The IGPs from clinical sera DDA based on parallel accumulation-serial fragmentation (PASEF) model was carried out using a timsTOF Pro2 (Bruker Daltics, Bremen, Germany) coupled to a nanoElute UPLC system (Bruker Daltics, Bremen, Germany). Water with 0.1% formic acid (mobile phase A) and ACN with 0.1% formic acid (mobile phase B) were used for LC separation. One microliter was injected onto a C18 trap column (Bruker Daltic; particle size 1.9 µm, 70 µm i.d. × 150 mm) and subjected to a gradient elution over 0–45 min, which ACN with 0.1% formic acid (2–22%), wash step (45–50 min) with ACN with 0.1% formic acid (22–37%), and equilibration with 80% acetonitrile with 0.1% formic acid (55–60 min). The column temperature was set at 50°C. LC-PRM only performs MS/MS on precursors shown on the preloaded target list. PRM glycopeptide quantification The IGPs analysis with DDA strategy were analyzed using MSFragger-Glyco 46 . The PRM analysis were performed using Skyline software (MacCoss Lab Software) 47 , which requires peptide and transition settings to generate a spectral library for patient sample quantification. A library was generated by uploading raw data and a list of glycopeptides identified by MSFragger-Glyco from the same data set. The spectral library was generated by oxonium ions, Y1, B, and b/y ions. The *.ssl format (.txt-based) list included raw data file names, scan numbers, charge states, peptide sequences with [+ glycan mass] at modified Asn, and retention times. Digestion parameters and peptide length filters were configured accordingly, with all possible glycan side chains added as structural modifications. For glycopeptide quantification, Skyline utilized Y1 ions (peptide + GlcNAc), oxonium ions (138.055, 204.087), and b/y ions for identification, with Y1 ions specifically used for quantification. Transitions were set for b, y, and special ions (oxonium 138.055, 204.087, and Y1 ions), with a 0.05 Da tolerance for parent ions and transitions. Area under receiver operating characteristic curve (AUC) values were obtained using pROC package (version 1.18.5). Declarations Acknowledgements This work was supported by One Hundred Person Project of the Chinese Academy of Sciences (Project No. 292024000225). This work was carried out using the facilities at Huairou Interdisciplinary Research Center for Engineering Mesoscience, CAS-IPE. Author contributions G. X.-D., Y. G., H. N., and Z. S. conceptualized and designed the study. With assistance from G. X.-D. and Y.G., Z. S., L. H., L.G., N.B. and X. L. conducted the experiments. Z. S., G.Y. and G. X.-D. wrote the manuscript with input from all authors. All authors have read and agreed to the published version of the manuscript. Competing interests There are no conflicts of interest to declare. Data availability The raw data and identification results have been deposited at the GlycoPOST (announced ID: GPST000660; https://glycopost.glycosmos.org/preview/6695917376946047b66aa9) References McManus, D.P. et al. Schistosomiasis. Nat Rev Dis Primers 4, 13 (2018). LoVerde, P.T. Schistosomiasis. Adv Exp Med Biol 1454, 75–105 (2024). Li, Q.-F. et al. The egg ribonuclease SjCP1412 accelerates liver fibrosis caused by Schistosoma japonicum infection involving damage-associated molecular patterns (DAMPs). Parasitology 151, 260–270 (2023). Xu, F. et al. Prior toxoplasma gondii infection ameliorates liver fibrosis induced by schistosoma japonicum through inhibiting Th2 response and improving balance of intestinal flora in mice. Int J Mol Sci 21 (2020). Olds, G.R., Griffin, A. & Kresina, T.F. 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Schneider, C.A., Rasband, W.S. & Eliceiri, K.W. NIH image to imageJ: 25 years of image analysis. Nat Methods 9, 671–675 (2012). Yang, G. et al. Comprehensive glycoproteomic analysis of chinese hamster ovary cells. Anal Chem 90, 14294–14302 (2018). Bern, M., Kil, Y.J. & Becker, C. Byonic: advanced peptide and protein identification software. Current Protocols in Bioinformatics 13, 13.20.11–13.20.14 (2012). Zhang, H. et al. Targeted glycomics by selected reaction monitoring for highly sensitive glycan compositional analysis. Proteomics 12, 2510–2522 (2012). Khoo, K.H., Chatterjee, D., Caulfield, J.P., Morris, H.R. & Dell, A. Structural mapping of the glycans from the egg glycoproteins of Schistosoma mansoni and Schistosoma japonicum: identification of novel core structures and terminal sequences. Glycobiology 7, 663–677 (1997). Polasky, D.A., Yu, F., Teo, G.C. & Nesvizhskii, A.I. Fast and comprehensive N- and O-glycoproteomics analysis with MSFragger-Glyco. Nat Methods 17, 1125–1132 (2020). MacLean, B. et al. Skyline: an open source document editor for creating and analyzing targeted proteomics experiments. Bioinformatics 26, 966–968 (2010). Additional Declarations There is NO Competing Interest. Supplementary Files supplementaryTables1223.xlsx Supplementary Table 1-5 SjLFSupplementaryFigures.docx Supplementary figures1-7 Cite Share Download PDF Status: Under Review Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8453779","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":572078224,"identity":"b91c288b-b52d-4008-af8b-a2fbf2339e81","order_by":0,"name":"Xiao-Dong Gao","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAuElEQVRIiWNgGAWjYFCCBCCukCBZyxmStTC2kaLB4Hj6xQ8f51nk8TMwP/zAUHOHsBbJnjfFkjO3SRRLNrAZSzAce0ZYC79EToI07zaJxA0HGMwYGBsOE9bCJpGT/Jt3jkTi/gPs34jTwi+RfkyatwFoCwMPkbYA/cJmOeOYRLHEYZ5iiYRjRGgBhtjjGx9q6vL429s3fvhQQ4QWBgYeAxCZwMDMAIlWIgD7AwbiFY+CUTAKRsGIBAD8djaZVuNQLAAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0002-8575-5095","institution":"Institute of Process Engineering, Chinese Academy of Sciences","correspondingAuthor":true,"prefix":"","firstName":"Xiao-Dong","middleName":"","lastName":"Gao","suffix":""},{"id":572078225,"identity":"d94df45d-0562-4aff-b410-fed566128696","order_by":1,"name":"Song Zhao","email":"","orcid":"","institution":"Jiangnanuniversity","correspondingAuthor":false,"prefix":"","firstName":"Song","middleName":"","lastName":"Zhao","suffix":""},{"id":572078226,"identity":"648ede75-aa2c-4e11-b1e5-6408afe473b1","order_by":2,"name":"Ganglong Yang","email":"","orcid":"https://orcid.org/0000-0001-5361-7344","institution":"State Key Laboratory of Biochemical Engineering, Institution of Process engineering,Chinese Academy of Science","correspondingAuthor":false,"prefix":"","firstName":"Ganglong","middleName":"","lastName":"Yang","suffix":""},{"id":572078227,"identity":"8a96329c-28d0-4613-9ae4-87a2745bc26e","order_by":3,"name":"Hanjie Li","email":"","orcid":"https://orcid.org/0009-0007-6721-1397","institution":"Jiangnanuniversity","correspondingAuthor":false,"prefix":"","firstName":"Hanjie","middleName":"","lastName":"Li","suffix":""},{"id":572078228,"identity":"3377169b-4b2e-4a66-a858-e02899919a52","order_by":4,"name":"Guofang Li","email":"","orcid":"","institution":"Institute of Process Engineering, Chinese Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Guofang","middleName":"","lastName":"Li","suffix":""},{"id":572078229,"identity":"c5912146-fdf3-489f-8d35-35f4edb88e54","order_by":5,"name":"Lu Xu","email":"","orcid":"","institution":"Institute of Process Engineering, Chinese Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Lu","middleName":"","lastName":"Xu","suffix":""},{"id":572078230,"identity":"322652f2-d948-41f2-8da1-5b8d52b1e225","order_by":6,"name":"Niannian Bi","email":"","orcid":"","institution":"Jiangsu Institute of Parasitic Diseases","correspondingAuthor":false,"prefix":"","firstName":"Niannian","middleName":"","lastName":"Bi","suffix":""},{"id":572078231,"identity":"943977a5-f7a5-444a-a4fe-b863a6496ef8","order_by":7,"name":"Nakanishi Hideki","email":"","orcid":"","institution":"Jiangnan University","correspondingAuthor":false,"prefix":"","firstName":"Nakanishi","middleName":"","lastName":"Hideki","suffix":""}],"badges":[],"createdAt":"2025-12-26 08:55:17","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8453779/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8453779/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":103067364,"identity":"0953bf23-4404-482a-90d4-818260871857","added_by":"auto","created_at":"2026-02-20 11:28:30","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1963405,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA rabbit model for the study of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eSchistosoma japonicum\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e-induced liver fibrosis (SjLF).\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(a).\u003c/strong\u003eSchematic illustration of the rabbit experiments procedure for \u003cem\u003eS. japonicum\u003c/em\u003einfection and liver fibrosis phases. New Zealand rabbits were infected with cercariae of \u003cem\u003eS. japonicum\u003c/em\u003e by abdominal patch method (dashed arrow). Fecal samples were examined by miracidium hatching to confirm the infection when the schistosome matures and lay eggs 4 weeks after inoculation (dashed line). Praziquantel (PZQ) was administered orally to rabbits for two days after 7 weeks of infection (dashed arrow). The rabbits were euthanized after 14 weeks. Downward solid arrows: the collection of peripheral serum samples at pre-infection 3 days as health control (Control), 1 week as early infection stage (I-1w) and 6 weeks as late infection stage (I-6w), as well as 12 weeks as early SjLF stage (I-12w) and 14 weeks as late SjLF stage (I-14w), respectively. Upward-pointing solid arrows: the dissection of liver tissue for histopathological examination to observe the status of fibrosis. \u003cstrong\u003e(b). \u003c/strong\u003eImages and representative H\u0026amp;E, Masson, ɑ-SMA IHC staining of liver tissues from the rabbits in control and SjLF.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8453779/v1/1cdb4da09b199705798fe4fc.png"},{"id":103504192,"identity":"e7baf329-dff0-4207-ab5d-be06f84a4e97","added_by":"auto","created_at":"2026-02-26 13:18:17","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":278198,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLectin microarray analysis reveals alterations in glycosylation patterns during the progression of SjLF. (a).\u003c/strong\u003e Hierarchical clustering of the significant different lectins in the serum glycoproteins from healthy control, early infection (I-1w), late infection (I-6w), early SjLF (I-12w) and late SjLF stages (I-14w). \u003cstrong\u003e(b).\u003c/strong\u003e The normalized relative binding intensities of significant different lectins with post-hoc test using Tukey's method, in which * means P\u0026lt;0.05, ** means P\u0026lt;0.01, *** means P\u0026lt;0.005 and **** means P\u0026lt;0.001. The glycan structures are depicted using the Consortium for Functional Glycomics (CFG) system\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8453779/v1/57a0b9cc60455f3aa05f9848.png"},{"id":103067366,"identity":"dd177f59-a50d-4d67-bce9-3de03b9f34f4","added_by":"auto","created_at":"2026-02-20 11:28:30","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1273127,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMS-based glycoproteomics delineates N-glycosylation features across the entire process of SjLF. (a).\u003c/strong\u003eVenn diagram illustration of the identified IGPs overlap from the five groups. \u003cstrong\u003e(b).\u003c/strong\u003eThe heatmap illustration of the relative abundance distribution of 2987 IGPs associated with glycosylation types. \u003cstrong\u003e(c-f).\u003c/strong\u003e Fuzzy c-means clustering of the identified IGPs associated with the \u003cem\u003eS. japonicum\u003c/em\u003e infection and liver fibrosis, in which all the IGPs were clustered into nine different clustering according to their relative abundance features as Supplementary Fig. 3 and Supplementary Table 2. Based on the response to all stages of \u003cem\u003eS. japonicum\u003c/em\u003e infection and liver fibrosis phases, the IGPs were categorized into four groups: rapid response to infection glycopeptides (\u003cstrong\u003ec\u003c/strong\u003e), late response to infection glycopeptides (\u003cstrong\u003ed\u003c/strong\u003e), liver fibrosis-associated glycopeptides (\u003cstrong\u003ee\u003c/strong\u003e), and SjLF-associated glycopeptides (\u003cstrong\u003ef\u003c/strong\u003e). The x-axis indicates the five stages (Control, I-1w, I-6w, I-12w and I-14w), while the y-axis shows the log2-transformed, normalized intensity ratios for each stage. GO enrichment analysis was conducted to identify the corresponding proteins of the clustered IGPs.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8453779/v1/ddd721153abc4693dd1e3816.png"},{"id":103067368,"identity":"32e8e355-ac6d-40b4-b20b-6fdf86dc6b35","added_by":"auto","created_at":"2026-02-20 11:28:30","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":694593,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSite-specific N-glycosylation on rabbit alpha-2-macroglobulin (A2M) reflects the progression of\u003c/strong\u003e \u003cstrong\u003eSjLF\u003c/strong\u003e. (\u003cstrong\u003ea).\u003c/strong\u003e The violin plots for relative abundances of rabbit A2M global glycosylation. GPSM: glycopeptide spectrum matches. (\u003cstrong\u003eb).\u003c/strong\u003e Site-specific N-linked glycan subtypes across 10 glycosylation sites of rabbit A2M. (\u003cstrong\u003ec).\u003c/strong\u003eDot plots of site-specific glycosylation illustrate the dynamic changes across 10 glycosylation sites of rabbit A2M. Glycan compositions highlighted in red signify the progression of SjLF. Those in orange indicate liver fibrosis but not \u003cem\u003eS. japonicum\u003c/em\u003e infection, while those in blue reflect \u003cem\u003eS. japonicum\u003c/em\u003einfection.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8453779/v1/db78f4db8a52aa3960b5b522.png"},{"id":103504045,"identity":"5d9d91e3-cb4f-4f9c-ab2c-15a673da51a4","added_by":"auto","created_at":"2026-02-26 13:11:03","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":633748,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cbr\u003e\n\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical validation of the site-specific glycosylation on human A2M identified diagnostic biomarkers for the SjLF. (a).\u003c/strong\u003e The clinical validation workflow for site-specific glycosylation on human A2M using a parallel reaction monitoring (PRM)-MS approach. The enriched IGPs from the clinical cohort underwent untargeted analysis employing a conventional glycopeptide analytical strategy, consistent with our previously established method in rabbit models. Subsequently, IGPs identified from human A2M were selected as targets for PRM-based quantitative analysis to evaluate their diagnostic performance in the diagnosis of the SjLF. \u003cstrong\u003e(b).\u003c/strong\u003e The site-specific glycans identified from the clinical cohorts using DDA analysis. The glycosylation sites N\u003csub\u003e55\u003c/sub\u003eET and N\u003csub\u003e1424\u003c/sub\u003eQT in the human A2M correspond to the homologous sites N\u003csub\u003e55\u003c/sub\u003eET and N\u003csub\u003e1417\u003c/sub\u003eQT identified in the rabbit model. Glycans observed at the N\u003csub\u003e55\u003c/sub\u003eET site are highlighted with a cyan background, while those at N\u003csub\u003e1424\u003c/sub\u003eQT are indicated in purple. Glycans shared between both sites are represented in pink. \u003cstrong\u003e(c).\u003c/strong\u003e The selected IGPs for target verification and their precursor ions feature for PRM, which contains the m/z, charges, and retention time, were uploaded to the Skyline as spectra library. \u003cstrong\u003e(d).\u003c/strong\u003e The ions fragment distribution for the targeted IGPs identification, including oxinium ions, b/y ions and B/Y ions from the A2M-N\u003csub\u003e1424\u003c/sub\u003eQT-N4H5S1, A2M-N\u003csub\u003e1424\u003c/sub\u003eQT-N4H5F1S1 and A2M-N\u003csub\u003e1424\u003c/sub\u003eQT-N4H5S2. \u003cstrong\u003e(e).\u003c/strong\u003e Scatter plot based on relative abundance of the three IGPs in normal (n = 22), mild SjLF (n =12) and severe SjLF (n =13) samples, respectively. \u003cstrong\u003e(f).\u003c/strong\u003e The discrimination of the isomers with α2,3 (right) and α2,6 (left) linked sialic acid and fucosylated IGP A2M-N\u003csub\u003e1424\u003c/sub\u003eQT-N4H5F1S1 based on the MS/MS fragments and retention time drifts, and the scatter plot based on relative abundance of the isomers in the normal, mild and severe SjLF. \u003cstrong\u003e(g).\u003c/strong\u003e The receiver operating characteristic (ROC) curves and area under the curves (AUC) for the performance of SjLF diagnosis (left), early SjLF diagnosis (middle), and the mild and severe SjLF discrimination (right) using the three glycans on A2M-N\u003csub\u003e1424\u003c/sub\u003eQT in verification clinical cohort.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-8453779/v1/7d4874483bd4131cf27a9209.png"},{"id":103509168,"identity":"6f1c9280-a018-449a-b911-22d155a2c8cc","added_by":"auto","created_at":"2026-02-26 13:56:59","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5968331,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8453779/v1/3ca0b0c7-3778-4a37-ac80-12437641bb2a.pdf"},{"id":103067363,"identity":"e75cbf6a-8b2a-4448-a7be-cda67450489c","added_by":"auto","created_at":"2026-02-20 11:28:30","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":315833,"visible":true,"origin":"","legend":"Supplementary Table 1-5","description":"","filename":"supplementaryTables1223.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8453779/v1/ad480c18a83ba5f33dfb5553.xlsx"},{"id":103067369,"identity":"85095fa7-92be-4eb4-a2b9-21536704d455","added_by":"auto","created_at":"2026-02-20 11:28:31","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":48652722,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary figures1-7\u003c/p\u003e","description":"","filename":"SjLFSupplementaryFigures.docx","url":"https://assets-eu.researchsquare.com/files/rs-8453779/v1/658ff934ec48bd53d9bdf795.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Site-specific N-glycosylation of alpha-2-macroglobulin reflects the progression of Schistosoma japonicum-induced liver fibrosis","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSchistosoma infection continues to impose a significant global health burden as a persistent neglected tropical disease, with an estimated 230\u0026nbsp;million individuals at risk worldwide, particularly in the endemic regions of Asia and the Western Pacific\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. The central pathological mechanism of schistosomiasis is characterized by the host's immune response to the parasite's eggs, which are highly immunogenic and capable of inducing vigorous circulating and local immune reactions\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eSchistosomes are dioecious trematodes that reside in the host's blood vessels as paired male and female adults. When schistosome eggs become trapped in the portal venules of the liver, they elicit a T-cell-mediated granulomatous inflammatory response, initiating a cascade of immunopathological events. In the early stages, this inflammatory process is reversible; however, with disease progression, excessive collagen deposition ensues, culminating in portal fibrosis, portal hypertension, and potentially irreversible organ damage\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Advanced liver fibrosis occurs in 5\u0026ndash;10% untreated individuals, manifesting as life-threatening complications including ascites and esophageal varices\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Notably, elimination of adult worm burdens from the host fails to mitigate established granuloma-mediated liver damage\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Although praziquantel remains the cornerstone of schistosomiasis management, the persistence of reinfection cycles and the presence of irreversible fibrotic sequelae in endemic populations highlight critical gaps in our understanding of fibrogenesis and its regulatory checkpoints\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Therefore, effective prevention strategies, coupled with precise diagnosis and timely intervention, represent the most efficacious approaches for the control and management of this disease.\u003c/p\u003e \u003cp\u003eAs the final common pathway of all chronic inflammatory injuries, liver fibrosis precise diagnosis was established with the fibrosis scores and non-invasive serum biomarkers. The Fibrosis-4 (FIB-4) index, AST-to-platelet ratio index (APRI), NAFLD fibrosis score, and BARD score were the most commonly employed with alanine aminotransferase (ALT) and aspartate transferase (AST) level serving as central components\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. However, the diagnosis of schistosome-induced liver fibrosis remains challenging because the level of ALT and AST do not change significantly during the progression\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. It was found that soluble egg antigen (SEA) and \u003cem\u003eS. japonicum\u003c/em\u003e eggs release exosomal miR-33 play critical role in the schistosomiasis japonica but not enough for the diagnosis\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Concurrently, glycosylation, one of the key post-translational modifications, play an important role in schistosome infection and liver fibrosis, which include the mediating host-parasite interactions and host immunity\u003csup\u003e\u003cspan additionalcitationids=\"CR15 CR16\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. As a powerful tool, glycoproteomics can systematically unveil the protein glycosylation modifications during the liver fibrosis progression. Clinical glycoproteomics studies of virus-induced and MASLD-related liver fibrosis have revealed stage-specific glycosylation changes, including the increasing core fucosylation of haptoglobin, sialic acid truncation on α-1-antitrypsin, and bisected N-glycan accumulation on IgA\u003csup\u003e\u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Moreover, N-glycosylation heterogeneity of Mac-2 binding protein serves as a superior prognostic marker for portal hypertension risk stratification in liver fibrosis, outperforming conventional biomarkers\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Yet, the glycosylation alteration of schistosome-induced liver fibrosis is largely unclear.\u003c/p\u003e \u003cp\u003eIn this study, a well-characterized rabbit model was established for studying the entire progression of \u003cem\u003eS. japonicum\u003c/em\u003e-induced liver fibrosis (SjLF). We utilized a multi-omics approach, combining lectin microarrays and high-resolution mass spectrometry (MS), to comprehensively profile glycopatterns and identify intact glycopeptides (IGPs) of the host serum across distinct stages of the SjLF. Our findings reveal dynamic changes in branched sialylated and fucosylated glycopatterns as well as the site-specific N-glycosylation heterogeneity of serum proteins. Particularly, the alpha-2-macroglobulin (A2M) was found to exhibit significant alterations in site-specific glycosylation, with specific glycan structures emerging as candidate biomarkers for the progression of SjLF. Targeted parallel reaction monitoring (PRM)-MS in clinical cohorts confirmed the diagnostic utility of these A2M glycosylation. Our study highlights the role of glycosylation in schistosome-host interplay and offers a tool for clinically diagnosing the progression of SjLF.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eEstablishment of a rabbit model for the study of\u003c/b\u003e \u003cb\u003eSchistosoma japonicum\u003c/b\u003e\u003cb\u003e-induced liver fibrosis (SjLF)\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo systematically investigate the glycosylation landscape during \u003cem\u003eSchistosoma japonicum\u003c/em\u003e-induced liver fibrosis (SjLF), a rabbit model was established to faithfully recapitulate the entire process, including the infection and liver fibrosis phases (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). New Zealand rabbits were percutaneously infected with \u003cem\u003eS. japonicum\u003c/em\u003e cercariae via abdominal application, following a standardized protocol\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Infection was confirmed by detection of ciliated miracidia in fecal samples at four weeks post-infection. At seven weeks post-infection, rabbits were treated with praziquantel (PZQ) for two consecutive days and then maintained for an additional seven weeks to allow progression of liver fibrosis. Serum samples were collected at multiple time points throughout the SjLF timeline: three days before infection served as the healthy control (Control); The one week and six weeks post-infection represented the early (I-1w) and late (I-6w) stages of infection, respectively; The twelve weeks and fourteen weeks post-infection corresponded to the early (I-12w) and late (I-14w) stages of liver fibrosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). Noteworthy, twelfth week post-infection (I-12w) is also the fifth week after the PZQ treatment, reflecting the cure effect. These longitudinal serum samples were utilized to analyze dynamic changes in glycosylation profiles associated with the progression of SjLF, which includes the infection of \u003cem\u003eS. japonicum\u003c/em\u003e (Sj infection) and liver fibrosis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe model rabbits were euthanized in the fourteenth week, and liver samples were collected for histopathological analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). The H\u0026amp;E and Masson's trichrome staining revealed that liver fibrosis was characterized by egg-induced granulomas, inflammatory tissue damage, and subsequent reparative fibrosis resulting from the deposition of \u003cem\u003eS. japonicum\u003c/em\u003e eggs in the hepatic vasculature (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). Additionally, elevated expression levels of α-smooth muscle actin (α-SMA) in the SjLF samples indicated activation of hepatic stellate cells and progressive fibrogenesis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). These findings demonstrated marked structural alterations, including increased collagen deposition and tissue stiffening in the livers of SjLF rabbits compared to healthy controls, thereby validating the reliability of the rabbit model for recapitulating human-like SjLF.\u003c/p\u003e \u003cp\u003e \u003cb\u003eLectin microarray analysis reveals alterations in glycosylation patterns during the progression of SjLF.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo explore the alterations in glycosylation patterns in the serum of the SjLF rabbit model, lectin microarray analysis was utilized to profile and quantify glycopatterns based on normalized relative fluorescence intensities (NFIs, Supplementary Table\u0026nbsp;1). As a reference, serum glycopatterns from the \u0026ldquo;Control\u0026rdquo; were initially examined, which exhibited high levels of T-antigen (Gal β1,3 GalNAc α-Ser/Thr, detected by Jacalin), oligomannosylation (α-D-Man, recognized by ConA), core fucosylation (Fuc α1,6 GlcNAc, bound by LCA), and terminal Gal β1,4 GlcNAc structures (identified by RCA120) (Supplementary Fig.\u0026nbsp;1).\u003c/p\u003e \u003cp\u003eFrom a comprehensive perspective, our lectin microarray analysis unveiled substantial alterations in glycopatterns during the progression of SjLF. Hierarchical clustering indicated that glycosylation profiles from the early (I-1w) and late (I-6w) stages of infection, as well as the late liver fibrosis stage (I-14w), were significantly distinct from those of the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea), suggesting dynamic changes associated with Sj infection and liver fibrosis phases. Noteworthy, serum samples collected at I-12w (the fifth week after PZQ treatment, see Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea) exhibited a glycosylation profile highly similar to that of the \u0026ldquo;Control\u0026rdquo;, implying functional recovery likely due to the therapeutic effects of drug treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn detail, a total of 17 lectin-recognized glycopatterns exhibited statistically significant alterations during SjLF (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). Glycopatterns with terminal fucosylation, branched (LacNAc)n structures, and sialylation (Neu5Ac), recognized by EEL, LTL, UEA-1 and MAL-I respectively, showed marked upregulation at the late stage of liver fibrosis (I-14w). Meanwhile, higher levels of branched (LacNAc)n and H(O) antigen (Fuc α1,2 Gal β1,4 GlcNAc) were detected by PWM and UEA-I at the early infection stage (I-1w). Additionally, the WFA-recognized epitope GalNAc α/β 1,3/6 Gal was elevated at the late stage of infection (I-6w) but rapidly normalized following PZQ treatment, indicating its potential as a prognostic biomarker for infection status and therapeutic response (Supplementary Fig.\u0026nbsp;2). Collectively, these findings underscore the crucial role of branched, sialylated, and fucosylated glycopatterns in the process of SjLF, highlighting their potential as candidates for early diagnosis of SjLF.\u003c/p\u003e \u003cp\u003e \u003cb\u003eComprehensive glycoproteomics delineates N-glycosylation features throughout the entire process of SjLF.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo achieve a deeper comprehension of the alterations in N-glycan structures throughout the SjLF process, we employed a high-resolution glycoproteomics technique, utilizing liquid chromatography-tandem mass spectrometry (LC-MS/MS) to directly investigate intact glycopeptides (IGPs) within the serum samples of model rabbits. A total of 2,987 unique intact glycopeptides (IGPs) were identified. The Venn diagram analysis revealed that approximately 458 N-linked IGPs were consistently present across all stages (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). Conversely, a substantial number of SjLF-specific IGPs, which only emerge during the infection and liver fibrosis phases, were also observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). Hierarchical clustering of the normalized IGP abundances demonstrated distinct group separations driven by site-specific N-linked glycosylation features (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eBased on the dynamic changes occurring during the infection and liver fibrosis phases, IGPs can be categorized into nine clusters (Supplementary Fig.\u0026nbsp;3). Among these clusters, glycopeptides in clusters 1 and 2 exhibited a rapid response to Sj infection, whereas clusters 3 and 4 showed a delayed response. Notably, IGPs in cluster 2, which increased in both the early and late stages of infection (I-1w and I-6w, Supplementary Fig.\u0026nbsp;3), garnered our attention. It was discovered that, among the 231 IGPs in cluster 2, 33 glycopeptides originated from alpha-2-macroglobulin (A2M) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec), indicating significant alterations in site-specific glycosylation on A2M. Similar unique IGPs were also identified as cluster 3, which increased during the late infection stage and subsequently returned to a low level during the early liver fibrosis stage (I-12w) following treatment with praziquantel (PZQ) (Supplementary Fig.\u0026nbsp;3). Among the 556 IGPs in cluster 3, 37 glycopeptides were derived from ceruloplasmin (CP), and 19 were from A2M (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed).\u003c/p\u003e \u003cp\u003eIn comparison to the IGPs that responded to the Sj infection, we were particularly intrigued by the IGPs that exhibited increased intensity during the liver fibrosis phase (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, Supplementary Fig.\u0026nbsp;3 and Supplementary Table\u0026nbsp;2). Specifically, IGPs in cluster 5 remained unchanged during the stages of Sj infection (I-1w and I-6w) and early liver fibrosis (I-12w) but showed an increase in the late stage of liver fibrosis (I-14w). According to our Venn diagram and hierarchical clustering analyses, the 250 IGPs in cluster 5, which were exclusive to the late stage of liver fibrosis (I-14w), were predominantly fucosylated and sialylated (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb), highlighting the role of site-specific glycosylation in liver fibrosis phase. Among these, 11 IGPs were derived from A2M, and 8 were from immunoglobulin heavy constant mu (IGHM) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee). Additionally, IGPs in cluster 4 also showed a significant increase in the late stage of liver fibrosis, despite their distinct response to Sj infection and subsequent restoration in the early stage of liver fibrosis following PZQ treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee, and Supplementary Fig.\u0026nbsp;3). The main contributors to this cluster of IGPs included IGHM (15 IGPs), haptoglobin (HP, 11 IGPs), and A2M (8 IGPs) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef).\u003c/p\u003e \u003cp\u003eIn summary, IGPs with distinct enhancements were identified throughout the SjLF process, many of which originated from proteins related to humoral immune responses (e.g., CP, IGHM, HP, and A2M, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Notably, among these proteins, the glycosylation of A2M changed markedly across all stages, representing the interactions between its glycosylation remodeling and the progression of SjLF.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003eSite-specific N-glycosylation on alpha-2-macroglobulin (A2M) reflects the progression of SjLF\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eThe preceding analysis unveiled dynamic alterations in alpha-2-macroglobulin (A2M)-derived IGPs across the phases of Sj infection and liver fibrosis. To elucidate the causes of these changes in IGP intensities, we initially examined the global glycosylation profile of A2M. As depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea, the glycopeptide spectrum matches (GPSM) of IGPs from A2M remained relatively stable during the progression of SjLF, including the early and late stages of Sj infection and liver fibrosis. Additionally, a previous study has demonstrated consistent protein expression levels of A2M across these stages \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Based on these findings, we confirmed that the global N-glycosylation and protein expression of A2M are not the primary factors driving the dynamic changes observed in A2M-derived IGPs.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo gain insights into the site-specific glycosylation of A2M and assess its potential clinical applications, we further analyzed the mass spectrometry data of A2M-derived IGPs. We identified 10 glycosylation sites with 110 unique IGPs and 49 N-linked glycan structures in host serum, representing the site-specific glycosylation heterogeneity of this protein across all stages (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). Overall, 84% of the glycans on A2M were fucosylated and sialylated. Regarding site-specific glycosylation subtypes, nearly all glycosylation sites exhibited high sialylation, except for the N\u003csub\u003e1399\u003c/sub\u003eRT site, which was predominantly occupied by high mannose-type glycans (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). Moreover, the N\u003csub\u003e381\u003c/sub\u003eKT motif was identified as the most N-glycosylated site, harboring approximately 25 glycan structures (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). From a diagnostic perspective, IGPs such as N\u003csub\u003e55\u003c/sub\u003eET-N4H5S2, N\u003csub\u003e247\u003c/sub\u003eVS-N4H5F2, N\u003csub\u003e381\u003c/sub\u003eKT-N5H5F1S1, N\u003csub\u003e410\u003c/sub\u003eTT-N4H5S1, N\u003csub\u003e410\u003c/sub\u003eTT-N4H5F2, and N\u003csub\u003e940\u003c/sub\u003eES-N4H5S1 (highlighted in red, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec) exhibited increased levels in both Sj infection stages (I-1w and I-6w) and the late stage of liver fibrosis phase (I-14w). In contrast, IGPs like N\u003csub\u003e247\u003c/sub\u003eVS-N3H3F1, N\u003csub\u003e381\u003c/sub\u003eKT-N5H5S2, and N\u003csub\u003e1417\u003c/sub\u003eQT-N2H5 (highlighted in orange, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec) were specifically elevated in the late stage of liver fibrosis (I-14w, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). These novel IGPs reflect the development of SjLF. Additionally, IGPs such as N\u003csub\u003e1359\u003c/sub\u003eLS-N5H5F1 and N\u003csub\u003e1399\u003c/sub\u003eRT-N2H7 (highlighted in blue, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec) were exclusively detected in the infection stages, indicating the presence of \u003cem\u003eS. japonicum\u003c/em\u003e infection. Collectively, our results suggest that the site-specific glycosylation of A2M may serve as potential biomarkers for the progression of SjLF.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eClinical validation of the site-specific glycosylation on human A2M identified diagnostic biomarkers for the SjLF\u003c/h3\u003e\n\u003cp\u003eTo determine whether the site-specific glycosylation of A2M can serve as a biomarker for diagnosing SjLF, a cohort study was conducted using serum samples from SjLF patients. The clinical serum cohort included 12 \u003cem\u003eS. japonicum\u003c/em\u003e-infected patients who underwent three follow-up sessions, as well as 12 healthy volunteers as controls. The baseline characteristics of the patients are detailed in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, where the severity of SjLF patients were categorized into three stages (normal, mild, and severe) based on liver parenchyma grading. The ALT and AST levels in serum samples from patients were not significantly elevated during the three follow-up detections (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and Supplementary Fig.\u0026nbsp;4). It is important to note that, as classic biomarkers of liver fibrosis, the standard ALT and AST levels for healthy individuals in women/men are typically\u0026thinsp;\u0026le;\u0026thinsp;35/\u0026le;50 U/L and \u0026le;\u0026thinsp;40/\u0026le;60 U/L, respectively\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Most of the ALT and AST values of our patients fall within the normal range (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and Supplementary Fig.\u0026nbsp;4). However, liver fibrosis progression, as determined by liver parenchyma grading, was detected in 58% of patients during the second follow-up and in 100% of patients during the third follow-up (Supplementary Fig.\u0026nbsp;4). This discrepancy suggests that the classic ALT and AST biomarkers may not be suitable for diagnosing liver fibrosis induced by \u003cem\u003eS. japonicum.\u003c/em\u003e\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe clinical information and diagnostic performance of SjLF patients\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCase Name\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGender\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFollow-up\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eLiver parenchyma grading*\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eFibrosis stage\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eALT(U/L)**\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eAST(U/L)***\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eP1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003enormal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e34.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e28.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003enormal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e22.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e29.8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eII\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003esevere\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e77.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e82.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eP2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003enormal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e40.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e27.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003enormal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e39.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e33.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMild\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e34.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e44.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eP3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003enormal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e29.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e19.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003emild\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e20.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e23.9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003emild\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e15.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e31.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eP4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003enormal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e23.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e29.7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003enormal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e32.9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003emild\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e38.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e55.7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eP5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003emild\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e25.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e31.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eII\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003esevere\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e21.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e23.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eIII\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003esevere\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eP6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eII\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003esevere\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e21.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e27.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003emild\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e23\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eIII\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003esevere\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eP7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003emild\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e26.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e24.8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eII\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003esevere\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e14.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e17.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eII\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003esevere\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eP8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003emild\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e14.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e21.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eII\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003esevere\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e10.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e12.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eII\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003esevere\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e17\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eP9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003enormal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e22.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e19.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003enormal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e24.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e21.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003emild\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e8.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e30.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eP10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003enormal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e42.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003enormal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e30.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e27.8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003emild\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e46.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e35.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eP11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003emild\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e16.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e32.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eII\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003esevere\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e22.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e13.7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eII\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003esevere\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eP12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003emild\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e15.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e22.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eII\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003esevere\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e37.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e25.7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eII\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003esevere\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003e\u003csup\u003e*\u003c/sup\u003e The liver parenchyma grading of SjLF patients. \u003cb\u003eGrade 0\u003c/b\u003e: Normal. Uniform echo pattern with moderate brightness and homogeneous distribution. \u003cb\u003eGrade I\u003c/b\u003e: Coarse echo texture. Characterized by thickened and hyperechoic foci with heterogeneous distribution. \u003cb\u003eGrade II\u003c/b\u003e: Prominent hyperechoic bands forming a \"fish-scale\" pattern, with mesh diameters mostly\u0026thinsp;\u0026lt;\u0026thinsp;2 cm. \u003cb\u003eGrade III\u003c/b\u003e: Dense hyperechoic bands creating a reticular framework, resembling \"tortoise shell\" or \"map-like\" morphology, with mesh diameters mostly\u0026thinsp;\u0026gt;\u0026thinsp;2 cm.\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003e\u003csup\u003e* *\u003c/sup\u003e The levels of alanine aminotransferase (ALT) in patients with SjLF are presented. For healthy individuals, the standard ALT concentrations are usually\u0026thinsp;\u0026le;\u0026thinsp;35 U/L for females and \u0026le;\u0026thinsp;40 U/L for males. Values exceeding 35 U/L in female or 40 U/L in male are indicated in red.\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003e\u003csup\u003e* * *\u003c/sup\u003eThe levels of aspartate aminotransferase (AST) in patients with SjLF are presented. For healthy individuals, the standard AST concentrations are usually\u0026thinsp;\u0026le;\u0026thinsp;50 U/L for females and \u0026le;\u0026thinsp;60 U/L for males. Elevated values above 50 U/L in females or 60 U/L in males are highlighted in blue.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eTo validate the site-specific glycosylation of A2M in the patient cohort, an integrated MS strategy was developed for targeted detection of A2M-derived IGPs, instead of the conventional assay using bifunctional antibodies. Initially, non-targeted site-specific glycosylation analysis of A2M was performed using parallel accumulation-serial fragmentation (PASEF) MS/MS to create a spectral library for selecting A2M-derived IGPs. Subsequently, the selected IGPs were quantified using parallel reaction monitoring (PRM) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn the non-targeted analysis, 39 IGPs and 5 glycosylation sites from human A2M were identified (Supplementary Tables\u0026nbsp;3). Glycopeptides were selected for PRM quantification based on four criteria: (i) detection in all samples from rabbit infection and liver fibrosis phases, (ii) significant alteration during the progression of SjLF, (iii) identification in clinical cohort samples via DDA analysis, and (iv) detection in over 97% of clinical cohort samples in PRM analysis (Supplementary Table\u0026nbsp;4). Consequently, two glycosylation sites (N\u003csub\u003e55\u003c/sub\u003eET and N\u003csub\u003e1424\u003c/sub\u003eQT) of human A2M, which matched with the N\u003csub\u003e55\u003c/sub\u003eET and N\u003csub\u003e1417\u003c/sub\u003eQT sties of rabbit serum (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec), were identified through this selection process. The human peptide containing the N\u003csub\u003e1424\u003c/sub\u003eQT site, VSN\u003csub\u003e1424\u003c/sub\u003eQTLSLFFTVLQDVPVR, was modified with 19 glycan structures, while the N55ET site only carried 6 glycans (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). Ultimately, three N\u003csub\u003e1424\u003c/sub\u003eQT-containing IGPs, featuring sialylated bi-antennary glycans N4H5S1 and N4H5S2, and a sialo-fucosylated bi-antennary glycan N4H5F1S1, were selected for further PRM quantification (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec).\u003c/p\u003e \u003cp\u003eIn the PRM analysis, oxonium ions (204.0866, 138.0550, 292.1019, and 274.0920), b/y and B/Y ions were used for glycopeptide identification, while Y1 ions were used for target quantification (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed). The three selected IGPs (N\u003csub\u003e1424\u003c/sub\u003eQT-N4H5S1, N\u003csub\u003e1424\u003c/sub\u003eQT-N4H5F1S1, and N\u003csub\u003e1424\u003c/sub\u003eQT-N4H5S2) exhibited differential abundance across normal, mild, and severe SjLF (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee and Supplementary Fig.\u0026nbsp;5). Importantly, PRM was able to distinguish glycan structures, such as core- and terminal-fucosylation, and also differentiate glycan isomers, like α2,3 and α2,6 linked sialic acid, using MS/MS spectra and retention time drifts\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. In our study, the sialylated glycosylation of SGP was confirmed with three isomers (Supplementary Fig.\u0026nbsp;6). The isomers of A2M-N\u003csub\u003e1424\u003c/sub\u003eQT-N4H5F1S1 were identified as core-fucosylated with α2,6 (isomer 1) and α2,3 (isomer 2) sialic acid, showing a 4s gradient shift and a core-fucosylated peak (Y3-Fuc). These isomers also differed between SjLF patients and healthy volunteers (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ef). The three individual A2M IGPs demonstrated strong diagnostic discrimination (AUC\u0026thinsp;\u0026gt;\u0026thinsp;0.6), with combined analysis showing higher discrimination (AUC\u0026thinsp;\u0026gt;\u0026thinsp;0.7) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eg).\u003c/p\u003e \u003cp\u003eIt should be noted that, prior to analyzing the glycopeptides of A2M, the protein expression and global glycosylation of A2M in clinical serums were examined and compared with those of healthy volunteers. Western blotting results indicated no significant changes in A2M protein expression in mild and severe SjLF patients (Supplementary Fig.\u0026nbsp;7a). For global glycosylation, lectin blotting with Con A and AAL demonstrated that the N-linked glycosylation of A2M was also not significantly different between healthy volunteers and patients (Supplementary Fig.\u0026nbsp;7b). These findings ruled out the possibility that global N-glycosylation and protein expression of A2M caused the alteration of glycosylation at the A2M-N\u003csub\u003e1424\u003c/sub\u003eQT site. Overall, our studies demonstrated that the site-specific glycosylation panel of A2M-N\u003csub\u003e1424\u003c/sub\u003eQT enhances the diagnostic accuracy of SjLF, indicating that PRM can serve as a targeted detection tool for the clinical diagnosis of site-specific glycosylation.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eSchistosomiasis affects over 300\u0026nbsp;million people globally, with a significant proportion at risk of developing liver fibrosis and cirrhosis. Currently, there are no molecular diagnostic markers available for the early detection of schistosome-induced liver fibrosis. Given the well-established association between protein glycosylation and liver fibrosis, we hypothesized that a systematic analysis of glycosylation changes during infection and disease progression could identify novel biomarkers for early diagnosis. Here, we developed a rabbit model of \u003cem\u003eS. japonicum\u003c/em\u003e\u0026ndash;induced liver fibrosis (SjLF), which recapitulates key features of human SjLF. Through the comprehensive analysis of N-glycosylation in this model, a set of potential glycoprotein biomarkers was discovered for the early detection of SjLF.\u003c/p\u003e \u003cp\u003eOur lectin microarrays analysis unveiled a significant increase in branched, sialylated, and fucosylated glycan structures during the progression of SjLF (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). This finding was further validated and explored through site-specific glycoproteomics, which directly analyzes IGPs while preserving glycan structural integrity and enables precise mapping of glycosylation events to specific proteins and sites\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. We identified distinct enhancements in site-specific sialylated and fucosylated glycosylation during the progression of SjLF, including the Sj infection and liver fibrosis phases, particularly on humoral immune response-related proteins such as A2M, HP, and IGHM (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Previous studies on parasitic infections and the immunopathology of schistosomiasis have highlighted the physiological significance of humoral immunity in host defense against parasites\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Consistent with these observations, our analysis revealed alterations in N-glycan modifications on these proteins. Among them, the site-specific glycosylation of A2M exhibited the most unique features, showing dynamic alterations throughout the entire SjLF process (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eA2M, an extracellular macromolecule primarily synthesized in the liver, is well-known as a broad-spectrum protease inhibitor and plays a crucial role in immune responses and the removal of damaged extracellular proteins\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. Alterations in site-specific N-glycosylation on A2M during the progression of SjLF were confirmed in both rabbit models and human clinical cohorts (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). The high-abundance glycans N4H5S1, N4H5S2, and N4H5F1S1 on the N\u003csub\u003e1424\u003c/sub\u003eQT site of human A2M could clearly distinguish normal, mild, and severe SjLF stages, with a combined AUC exceeding 0.7 as a panel (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e), highlighting the clinical relevance of A2M's site-specific glycosylation and glycan structures. Notably, A2M has been proposed as a serum fibromarker for chronic hepatitis C infection and liver fibrosis due to its upregulated protein levels in liver biopsies and serum of chronic hepatitis C patients\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e,\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. However, A2M protein expression did not change across the progression of Sj infection and liver fibrosis\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e, indicating that its expression level is not suitable as a serologic biomarker for \u003cem\u003eS. japonicum\u003c/em\u003e-induced liver fibrosis. In contrast, our findings on the alterations of site-specific N-glycosylation on A2M, especially IGPs containing the N\u003csub\u003e1424\u003c/sub\u003eQT site, could fill the gap for early SjLF diagnosis, an area not previously emphasized in clinical determinations.\u003c/p\u003e \u003cp\u003eSeveral other potentially valuable discoveries were made from our comprehensive analysis of serum glycosylation. In the rabbit model, only the N\u003csub\u003e1399\u003c/sub\u003eRT glycosylation site of A2M was specifically modified with high mannose-type glycans, with IGPs containing N2H7 significantly increasing during infection stages (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). It has been reported that mannose-binding protein (MBP) directly binds to Man5-7 high mannose of A2M to opsonize and activate the lectin pathway of the complement cascade, eliminating infection. This binding triggers the lectin pathway via MASP-2, cleaving complement components C4 and C2 to activate the C3 convertase C4b2a\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Thus, the N\u003csub\u003e1399\u003c/sub\u003eRT site may contribute to activating the lectin pathway of the complement cascade through its binding to MBP. It is conceivable that similar N-glycosylation sites with specific high-mannose modifications also exist in human A2M. Moreover, we detected several glycans typically considered schistosome-specific (data not shown), such as xylose-containing pentose glycans\u003csup\u003e\u003cspan additionalcitationids=\"CR35 CR36\" citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. This raises the possibility of glycan transfer from the parasite to host proteins, a phenomenon that may contribute to immune modulation or pathogenesis.\u003c/p\u003e \u003cp\u003eIn conclusion, our study has comprehensively delineated and confirmed the site-specific glycosylation alterations induced by \u003cem\u003eS. japonicum\u003c/em\u003e infection and the development of liver fibrosis. Notably, the glycosylation of human A2M at the N\u003csub\u003e1424\u003c/sub\u003eQT site emerges as a promising biomarker for the diagnosis of schistosomiasis infection and the early detection of SjLF. Further investigation into the molecular mechanisms underlying how site-specific glycosylation on A2M responds to SjLF may pave the way for the development of a vaccine against the \u003cem\u003eS. japonicum\u003c/em\u003e infection.\u003c/p\u003e"},{"header":"Material and Methods","content":"\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eEthics statement\u003c/h2\u003e \u003cp\u003e The animal experiments were performed in strict accordance with the Laboratory Animal Guideline for Ethical Review of Animal Welfare (GB 14925\u0026thinsp;\u0026minus;\u0026thinsp;2010). The experimental protocol was reviewed and approved by the Ethics Review Committee of the Jiangsu Institute of Parasitic Diseases, under the approval number JIPD \u0026minus;\u0026thinsp;2020\u0026ndash;013. The human serum samples utilized in this study were sourced from individuals participating in a prospective cohort study involving exposure to \u003cem\u003eS. japonicum\u003c/em\u003e. This cohort study was granted approval by the Ethics Committee of the Jiangsu Institute of Parasitic Diseases (approval number JIPD \u0026minus;\u0026thinsp;2019\u0026ndash;008) \u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. Prior to their involvement, written informed consent was obtained from all participants.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003ePretreatment and protein preparation of sera from the rabbits\u003c/h2\u003e \u003cp\u003eNew Zealand rabbits (weighing 2.0\u0026ndash;2.5 kg, n\u0026thinsp;=\u0026thinsp;4) were experimentally infected with 1000 cercariae of \u003cem\u003eS. japonicum\u003c/em\u003e sourced from the Jiangsu Institute of Parasitic Diseases in China using the abdominal patch method. To verify the success of infection, fecal samples from these rabbits were subjected to the miracidium hatching test \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Sera were collected from \u003cem\u003eS. japonicum\u003c/em\u003e-infected rabbits at 1 week post-infection (I-1w, early infection) and 6 weeks post-infection (I-6w, late infection) to represent the infection group. Following 7 weeks of \u003cem\u003eS. japonicum\u003c/em\u003e infection, rabbits were orally administered praziquantel (PZQ, 150 mg/kg) for two consecutive days. Sera were further collected from rabbits at 12 weeks post-infection (I-12w, early liver fibrosis) and 14 weeks post-infection (I-14w, late liver fibrosis) to investigate liver fibrosis progression (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). Additionally, sera were also obtained from uninfected New Zealand rabbits to serve as a healthy control group (Control). Finally, all 20 rabbits were euthanized for morphological examination.\u003c/p\u003e \u003cp\u003eAfter incubation at 37℃ for 4 h, sera were isolated by centrifugation at 200 \u0026times; g for 5 min and subsequently stored at \u0026minus;\u0026thinsp;80℃. Equal volumes of serum protein from each sample were mixed and combined \u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. Albumin and IgG were depleted from the sera to simplify the protein composition, following the instructions provided with the ProteoExtract albumin/IgG removal kit (Merck Millipore, Billerica, MA, USA) \u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. The serum proteins were then concentrated using 10 kDa centrifuge ultrafiltration tubes (Amicon ultra \u0026minus;\u0026thinsp;0.5, Millipore, MA, USA) by centrifugation at 13,000 \u0026times; g for 5 min.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePatients and sera sample collection\u003c/h3\u003e\n\u003cp\u003eThe study cohort consisted of 12 healthy controls and 12 patients with confirmed \u003cem\u003eS. japonicum\u003c/em\u003e infection. All serum samples were derived from participants enrolled in the prospective cohort study of \u003cem\u003eS. japonicum\u003c/em\u003e exposure\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. The cohort-related follow-up procedures, including questionnaires, physical examinations, and biospecimen collection, were conducted in strict adherence to the established work manual. The patients were recruited from Yangzhou, Jiangsu, China, an area endemic for \u003cem\u003eS. japonicum\u003c/em\u003e, between August 2017 and July 2019. Each participant provided three serum samples collected during follow-ups in 2019, 2021, and 2025. Additionally, 12 control serum samples were collected in 2025 from age- and gender-matched healthy individuals with no history of \u003cem\u003eS. japonicum\u003c/em\u003e infection from the same region. All patients had been treated with praziquantel (PZQ) upon diagnosis of \u003cem\u003eS. japonicum\u003c/em\u003e infection during the 1960s to 1970s. Patients with the following conditions were excluded: mixed liver disease; coinfection with other viruses, such as hepatitis A virus (HAV), HBV, HCV, or HDV; decompensated liver disease; primary liver cancer or other malignant tumors; autoimmune or immunologically mediated diseases; organ transplantation; or severe cardiac, hepatic, or renal dysfunction. Basic physical examination information was documented. Liver parenchyma and size, the width of the main portal vein, spleen size, and the presence of ascites were assessed via ultrasound examinations. Hepatic parenchymal status (Grade 0 - III) and stages (normal, mild, and severe) were determined by community physicians. Relevant clinical information of the patients is summarized in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and supplementary Table\u0026nbsp;5.\u003c/p\u003e\n\u003ch3\u003eLectin microarray determination and data analysis\u003c/h3\u003e\n\u003cp\u003eBriefly, the lectin microarray procedure was conducted as follows: 37 lectins were spotted in triplicate using the CapitalBio SmartArrayer 48 Microarray Spotter (CapitalBio, China). The microarrays were incubated and dried at 37\u0026deg;C, followed by storage at 4\u0026deg;C in the dark. Approximately 100 \u0026micro;g of serum proteins were labeled with Cy3 fluorescent dye at room temperature and purified using a G-25 column. The microarray was blocked in an LF-IIIA hybridization oven (SCIENTZ, Ningbo, China), and then 6 \u0026micro;g of the fluorescence-labeled proteins were applied. The lectin microarray was subsequently read using a Genepix 4000B confocal microarray scanner (Axon Instruments, USA). The median values of all lectins were normalized. One-way ANOVA was performed on the five groups of lectin-normalized fluorescence intensities (NFIs), with post-hoc analysis conducted using Tukey\u0026rsquo;s method to adjust p-values. A lectin NFI was considered significantly elevated if the NFI ratio between groups was \u0026gt;\u0026thinsp;1.5 and p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, and significantly decreased if the NFI ratio was \u0026lt;\u0026thinsp;0.67 and p\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eSDS-PAGE and lectin blotting\u003c/h2\u003e \u003cp\u003eA 10% separating gel and a 5% stacking gel were prepared, and samples containing 20 \u0026micro;g of protein were loaded onto the gel. The gel was subsequently stained with Coomassie brilliant blue. For lectin staining, the proteins were transferred to a PVDF membrane (0.45 \u0026micro;m) using transfer buffer. To minimize non-specific binding, the membrane was blocked with Carbo-free blocking solution for 1 hour at room temperature. Subsequently, 2 \u0026micro;g/mL solutions of biotinylated-PWM (pokeweed mitogen), biotinylated-UEA-Ⅰ (Ulex europaeus agglutinin I), biotinylated-WFA (Wisteria floribunda agglutinin), biotinylated-ConA (concanavalin A), and biotinylated-AAL (Aleuria aurantia lectin) were prepared by diluting with blocking solution and incubated with the membrane overnight at 4\u0026deg;C. After washing the membrane, streptavidin-HRP (Horseradish Peroxidase, diluted 1:10,000) was added and incubated for 1 hour at room temperature. Following electrochemiluminescence (ECL) development for 1\u0026ndash;2 minutes, the bands were visualized using the Molecular Imager ChemiDoc XRS\u0026thinsp;+\u0026thinsp;System and Image Lab Version 3.0. The results were analyzed using ImageJ software, with the relative intensity of the bands, which was calculated as the ratio of the grayscale value sum of the bands to the sum of the corresponding Coomassie brilliant blue (CB) staining \u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eSerum protein digestion and glycopeptide enrichment\u003c/h2\u003e \u003cp\u003eThe serum proteins from rabbits and patients were pretreated and denatured in an 8 mM urea/40 mM NH4HCO3 buffer. They were then treated with 10 mM dithiothreitol (DTT) at room temperature, followed by 20 mM iodoacetamide (IAM) for 45 minutes in the dark. Sequencing-grade modified trypsin (Promega, Wisconsin, USA) was added, and the proteins were digested using filter-aided protein digestion in a 10 kDa centrifugation ultrafiltration tube. The reaction was terminated with 50% formic acid (FA) (v/v), and the peptide concentration was measured using NanoDrop. The peptides were purified using a combination of C18 and mixed anion exchange (MAX) solid - phase extraction columns \u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. Specifically, 600 \u0026micro;g of the peptide solution was transferred to the prepared column. The mixed column was washed sequentially with acetonitrile (ACN), 100 mM triethylammonium acetate buffer (TEAB), 1% trifluoroacetic acid (TFA) (v/v), and 95% ACN (v/v). After injection, the sample was washed three times with 1% TFA (v/v) in 95% ACN (v/v) and finally eluted with 50% ACN (v/v) in 1% FA to collect the enriched glycopeptide samples. The samples were then lyophilized using a freeze dryer (Labconco, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eThe intact glycopeptide (IGP) analysis by high-resolution liquid chromatography-tandem mass spectrometry (LC-MS/MS)\u003c/h2\u003e \u003cp\u003eIntact glycopeptides (IGPs) were resuspended in a 0.1% FA with 2% ACN solution at a concentration of 0.5 \u0026micro;g/\u0026micro;L. The rabbit serum samples were analyzed using an EASY - nLC 1200 system (Thermo Fisher Scientific, USA) coupled to a high - resolution Orbitrap Fusion Lumos Mass Spectrometer (Thermo Fisher Scientific, USA), with an injection volume of 3 \u0026micro;L per sample.\u003c/p\u003e \u003cp\u003eThe samples were initially separated on the EASY-nLC 1200 system using mobile phase buffer A (water) and buffer B (90% ACN) at a flow rate of 550 nL/min. The liquid phase gradient was programmed as follows: 0\u0026ndash;1 min, buffer B linear gradient from 2% to 6%; 1\u0026ndash;91 min, buffer B linear gradient from 6% to 30%; 91\u0026ndash;113 min, buffer B linear gradient from 30% to 38%; 113\u0026ndash;118 min, buffer B linear gradient from 38% to 80%; 118\u0026ndash;123 min, buffer B maintained at 80%. Data-dependent high energy collision dissociation (HCD) was performed on the 20 most abundant ions. The AGC target was set at 4.0\u0026times;10\u003csup\u003e5\u003c/sup\u003e, the maximum injection time was 250 ms, and the acquisition was conducted within the resolution range of m/z 350\u0026ndash;2000.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eMS data analysis\u003c/h2\u003e \u003cp\u003eThe protein databases of \u003cem\u003eS. japonicum\u003c/em\u003e species in Uniprot database (downloaded on 09/23/2020) were downloaded and searched using Byonic software \u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e, and the combined N-glycan databases of New Zealand rabbits\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e and \u003cem\u003eS. japonicum\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e,\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e were constructed, containing a total of 121 N-glycans. Byonic software parameters are set as follows: restriction enzyme digestion site is R/K, maximum missed cleavage is 2, precursor mass tolerance is 10 ppm, fragment type is HCD, fragment mass tolerance is 20 ppm, cysteine alkylate is set to fixed modification, the false discovery rate (FDR)\u0026thinsp;\u0026lt;\u0026thinsp;1%, and cut-off score was set to 300. Student's t-test (two-tailed, unpaired, and equal variance) was performed and the significant increased (adjusted p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, FC\u0026thinsp;\u0026gt;\u0026thinsp;2) or decreased (adjusted p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, FC\u0026thinsp;\u0026lt;\u0026thinsp;0.5) protein glycosylation in each experimental group compared to the control group was set.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eClinical serum IGPs analysis using LC-MS/MS\u003c/h2\u003e \u003cp\u003eThe IGPs from clinical sera DDA based on parallel accumulation-serial fragmentation (PASEF) model was carried out using a timsTOF Pro2 (Bruker Daltics, Bremen, Germany) coupled to a nanoElute UPLC system (Bruker Daltics, Bremen, Germany). Water with 0.1% formic acid (mobile phase A) and ACN with 0.1% formic acid (mobile phase B) were used for LC separation. One microliter was injected onto a C18 trap column (Bruker Daltic; particle size 1.9 \u0026micro;m, 70 \u0026micro;m i.d. \u0026times; 150 mm) and subjected to a gradient elution over 0\u0026ndash;45 min, which ACN with 0.1% formic acid (2\u0026ndash;22%), wash step (45\u0026ndash;50 min) with ACN with 0.1% formic acid (22\u0026ndash;37%), and equilibration with 80% acetonitrile with 0.1% formic acid (55\u0026ndash;60 min). The column temperature was set at 50\u0026deg;C. LC-PRM only performs MS/MS on precursors shown on the preloaded target list.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003ePRM glycopeptide quantification\u003c/h2\u003e \u003cp\u003eThe IGPs analysis with DDA strategy were analyzed using MSFragger-Glyco\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. The PRM analysis were performed using Skyline software (MacCoss Lab Software) \u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e, which requires peptide and transition settings to generate a spectral library for patient sample quantification. A library was generated by uploading raw data and a list of glycopeptides identified by MSFragger-Glyco from the same data set. The spectral library was generated by oxonium ions, Y1, B, and b/y ions. The *.ssl format (.txt-based) list included raw data file names, scan numbers, charge states, peptide sequences with [+\u0026thinsp;glycan mass] at modified Asn, and retention times. Digestion parameters and peptide length filters were configured accordingly, with all possible glycan side chains added as structural modifications. For glycopeptide quantification, Skyline utilized Y1 ions (peptide\u0026thinsp;+\u0026thinsp;GlcNAc), oxonium ions (138.055, 204.087), and b/y ions for identification, with Y1 ions specifically used for quantification. Transitions were set for b, y, and special ions (oxonium 138.055, 204.087, and Y1 ions), with a 0.05 Da tolerance for parent ions and transitions. Area under receiver operating characteristic curve (AUC) values were obtained using pROC package (version 1.18.5).\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by One Hundred Person Project of the Chinese Academy of Sciences (Project No. 292024000225). This work was carried out using the facilities at Huairou Interdisciplinary Research Center for Engineering Mesoscience, CAS-IPE.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eG. X.-D., Y. G.,\u0026nbsp;H. N.,\u0026nbsp;and Z. S. conceptualized and designed the study. With assistance from G. X.-D. and Y.G., Z. S., L. H., L.G., N.B. and X. L. conducted the experiments. Z. S., G.Y. and G. X.-D. wrote the manuscript with input from all authors. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThere are no conflicts of interest to declare.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe raw data and identification results have been deposited at the GlycoPOST (announced ID: GPST000660; https://glycopost.glycosmos.org/preview/6695917376946047b66aa9)\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eMcManus, D.P. et al. Schistosomiasis. \u003cem\u003eNat Rev Dis Primers\u003c/em\u003e 4, 13 (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLoVerde, P.T. Schistosomiasis. \u003cem\u003eAdv Exp Med Biol\u003c/em\u003e 1454, 75\u0026ndash;105 (2024).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi, Q.-F. et al. 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Skyline: an open source document editor for creating and analyzing targeted proteomics experiments. \u003cem\u003eBioinformatics\u003c/em\u003e 26, 966\u0026ndash;968 (2010).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Schistosomiasis, liver fibrosis, alpha-2-macroglobulin, intact glycopeptide, site-specific glycosylation","lastPublishedDoi":"10.21203/rs.3.rs-8453779/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8453779/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSchistosomiasis leads to liver fibrosis through an egg-induced granulomatous inflammatory response in the host. Although parasite glycosylation is known to contribute to immune evasion and pathogenesis, alterations in host protein glycosylation during infection progression and liver fibrosis development remain poorly understood. Using a rabbit model specifically established to study \u003cem\u003eSchistosoma japonicum\u003c/em\u003e-induced liver fibrosis (SjLF), we performed comprehensive glycoprofiling of serum glycomes and glycoproteomes via lectin microarrays and high-resolution mass spectrometry. Our analysis revealed dynamic changes in N-glycosylation of proteins involved in the humoral immune response, with particular emphasis on site-specific glycosylation of alpha-2-macroglobulin (A2M). Ten glycosylation sites on A2M were identified, exhibiting significant variations across different stages of infection and liver fibrosis in SjLF, suggesting their potential as indicators for monitoring SjLF progression. Further validation demonstrated that the N\u003csub\u003e1424\u003c/sub\u003eQT glycosylation site of human A2M in clinical serum samples serves as a promising diagnostic biomarker for SjLF, underscoring the value of targeted glycoproteomics in precise disease monitoring. This study provides insights into host glycosylation remodeling during SjLF and highlights its critical role in schistosome\u0026ndash;host interactions.\u003c/p\u003e","manuscriptTitle":"Site-specific N-glycosylation of alpha-2-macroglobulin reflects the progression of Schistosoma japonicum-induced liver fibrosis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-20 11:28:25","doi":"10.21203/rs.3.rs-8453779/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"communications-medicine","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"commsmed","sideBox":"Learn more about [Communications Medicine](http://www.nature.com/commsmed)","snPcode":"43856","submissionUrl":"https://mts-commsmed.nature.com/cgi-bin/main.plex","title":"Communications Medicine","twitterHandle":"@commsmedicine","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Communications Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"c5ef29a1-4be0-46e2-9015-3711baacc212","owner":[],"postedDate":"February 20th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":60881186,"name":"Biological sciences/Immunology/Infectious diseases/Parasitic infection"},{"id":60881187,"name":"Biological sciences/Cell biology/Glycobiology"}],"tags":[],"updatedAt":"2026-02-20T11:28:25+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-20 11:28:25","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8453779","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8453779","identity":"rs-8453779","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}