Reduced Sialylation of Airway Mucin Impairs Mucus Transport by Altering the Biophysical Properties of Mucin

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Harris, Hannah J. McIntire, Marina Mazur, Hinnerk Schulz-Hildebrandt, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4421613/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 16 Jul, 2024 Read the published version in Scientific Reports → Version 1 posted 12 You are reading this latest preprint version Abstract Mucus stasis is a pathologic hallmark of muco-obstructive diseases, including cystic fibrosis (CF). Mucins, the principal component of mucus, are extensively modified with hydroxyl (O)-linked glycans, which are largely terminated by sialic acid. Sialic acid is a negatively charged monosaccharide and contributes to the biochemical/biophysical properties of mucins. Reports suggest that mucin sialylation may be altered in CF; however, the consequences of reduced sialylation on mucus clearance have not been fully determined. Here, we investigated the consequences of reduced sialylation on the charge state and conformation of the most prominent airway mucin, MUC5B, and defined the functional consequences of reduced sialylation on mucociliary transport (MCT). Reduced sialylation contributed to a lower charged MUC5B form and decreased polymer expansion. The inhibition of total mucin sialylation de novo impaired MCT in primary human bronchial epithelial cells and rat airways, and specific α-2,3 sialylation blockade was sufficient to recapitulate these findings. Finally, we show that ST3 beta-galactoside alpha-2,3-sialyltransferase (ST3Gal1) expression is downregulated in CF and partially restored by correcting CFTR via Elexacaftor/Tezacaftor/Ivacaftor treatment. Overall, this study demonstrates the importance of mucin sialylation in mucus clearance and identifies decreased sialylation by ST3Gal1 as a possible therapeutic target in CF and potentially other muco-obstructive diseases. Biological sciences/Biochemistry/Biophysical chemistry Biological sciences/Biochemistry/Glycobiology Biological sciences/Physiology/Respiration Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Cystic fibrosis (CF) is a genetic disease hallmarked by viscous and adhesive airway mucus in several organs ( 1 – 3 ). In the respiratory tract, CF mucus becomes static and leads to chronic infection, progressive organ decline, and early mortality ( 1 , 4 ). The rheological characteristics of the mucus gel are largely governed by the gel-forming mucins MUC5B and MUC5AC, the chief structural component of mucus ( 2 , 5 ). In CF, electrostatic driven abnormalities, stemming from impaired anion transport, alter mucin conformation and contribute to increased mucus viscosity and impaired mucociliary clearance ( 4 , 6 – 9 ). Gel-forming mucins are extensively modified with hydroxyl (O)-linked glycans that determine biophysical properties including normal mucin expansion and rheological characteristics ( 5 , 10 , 11 ). Mucin glycans are largely terminated by sialic acid attached in either an α-2,3 or α-2,6 linkage, facilitated by either ST3 beta-galactoside alpha-2,3-sialyltransferase (ST3Gal) or ST6 N-acetylgalactosaminide alpha-2,6-sialyltransferase (ST6GalNAc), respectively ( 12 , 13 ). Sialic acid is a negatively charged monosaccharide and exerts key electrostatic properties to mucins via its negative charge ( 14 ). The high anionic density of mucins is postulated to help stiffen the mucin polymer through charge repulsion and mediate interactions with cations to facilitate mucin granular packaging and post-secretory expansion ( 10 , 15 – 17 ). Within intracellular granules, calcium (Ca 2+ ) shields these negative charges to mitigate electrostatic repulsion and promote mucin condensation ( 11 , 18 ). Upon secretion, divalent sodium (Na + ) is exchanged with Ca 2+ to facilitate mucin expansion and hydration ( 5 , 6 ). Although anionic density is central to the mechanisms of mucin maturation and hydration, studies have historically centered around the altered ionic environment as the driver of aberrant mucin in CF, and the role of intrinsic mucin sialylation/charge in mucin biogenesis and MCC has remained understudied. Previous work has implicated lower charged mucin in several muco-obstructive diseases. The predominant gel-forming mucin of the airway, MUC5B, has been characterized to exist in two forms: ( 1 ) a more negative “high” charged form, and ( 2 ) a less negative “low” charged form (based on its migration following Agarose-PAGE) ( 19 ). Increased levels of the low charged form have been reported in CF, asthma, and COPD ( 5 , 20 , 21 ). In asthmatics, the low charged form was enriched within a viscous mucus plug and exhibited a compact and entangled conformation, linking decreased MUC5B charge with conformational and rheological defects ( 22 ). However, the etiology of this MUC5B form and the functional consequences of reduced/low mucin charge on polymer expansion and MCC have not been elucidated. Interestingly, changes in mucin sialylation have been documented in CF. Specifically, evidence supports that sialylation may be reduced as a direct consequence of defective CFTR, although the role of CFTR in regulating mucin sialylation/charge and its impact on CF mucus pathology remain unclear ( 23 – 26 ). In this study, we aimed to determine the role of mucin sialylation on the biophysical properties of mucin and overall mucus function in terms of charge state, compaction, and mucus transport. Furthermore, we aimed to determine the clinical relevance of aberrant mucin sialylation in CF. Here, we show that reduced sialylation of mucin contributed to a low charged form of MUC5B, increased MUC5B compaction, and ultimately impaired mucociliary transport in vitro and in vivo . Additionally, we provide evidence for a link between the defective CFTR and reduced sialylation of mucin in CF mucus stasis. Overall, this study demonstrates the importance of sialylation in mucus function and provides impetus to investigate the molecular mechanisms of mucin sialylation for treatment of mucociliary dysfunction in CF, as well as potentially other muco-obstructive diseases. Results Reducing the Sialylation of Contributes to a Low Charge Form of MUC5B Since sialic acid contributes to the overall charge of MUC5B, we hypothesized that reducing sialylation would result in a lower charged mucin, similar to the predominate forms in COPD and asthma ( 5 , 22 , 27 ). To test this, we collected and partially purified mucin from non-CF HBEC secretions, then treated this mucin with increasing concentrations of sialidase. Using a agarose polyacrylamide gel electrophoresis gel mobility assay to characterize mucin charge state ( 28 ), sialidase treated mucins were separated followed by sialylation analysis with WGA lectin blotting (Fig. 1 A). As sialylation was reduced by sialidase treatment, the faster migrating mucin forms were dose-dependently shifted to a slower mobility in the gel (Fig. 1 A) indicative of charge reduction. In parallel experiments, an incremental shift in gel mobility of MUC5B was observed, reflecting the dose-dependent loss of sialic acid following sialidase treatment (Fig. 1 B). Together, these results provide evidence that sialylation is important in the charge state of mucins and its reduction may contribute to the low charged MUC5B form previously implicated in muco-obstructive disease. Reducing Sialylation of Salivary MUC5B Impairs Mucin Linearization To determine the consequences of reduced sialylation on MUC5B conformation, we natively purified salivary MUC5B via CsCl gradient centrifugation as previously described, incubated it with either sialidase or vehicle, and subsequently evaluated the macromolecular conformations of MUC5B polymers via TEM ( 29 ). In parallel, we incubated untreated MUC5B with 10mM Ca 2+ in pH 5 to induce MUC5B condensation (Fig. 2 A) ( 8 ). We observed mostly linearized polymer chains under vehicle conditions, and condensed polymers that formed highly overlapping networks under high Ca 2+ / low pH (Fig. 2 A). MUC5B treated with sialidase primarily presented as highly entangled molecules, where the polymers frequently overlapped with themselves and did not take on a fully extended form, indicative of less electrostatic stiffening of the mucin (Fig. 2 A). A minimum of 72 polymers per condition were categorized into either linear, entangled, or condensed morphologies based on a previously established scoring method (Fig. 2 B) ( 6 , 8 ). Sialidase treated MUC5B was enriched with highly entangled molecules (43.0% linear, 44.4% entangled, 12.5% condensed; P < 0.0001) compared to vehicle treated MUC5B, which primarily exhibited linearized polymer chains (93.1% linear, 6.8% entangled, 0.0% condensed). MUC5B exposed to high Ca 2+ /low pH exhibited substantially more condensed polymers s (34.1% linear, 31.8% entangled, 34.1% condensed; P < 0.0001) (Fig. 2 A-B). These data suggest that reduced sialylation contributes to compaction of secreted MUC5B. To further define the relationship between mucin conformation and sialylation, we performed rate zonal centrifugation on purified salivary MUC5B using a linear 10–35% sucrose gradient to separate mucin by size and shape. Mucins that sediment faster during rate zonal centrifugation have been described as more compact and pathologic, while mucins that sediment slower exhibit an expanded (linear) conformation ( 6 , 9 ). We therefore hypothesized that slower sedimenting MUC5B would have a higher degree of sialylation, while faster sedimentation would be associated with reduced sialylation/charge. After rate zonal centrifugation, we collected fractions from the top of the gradient and subjected them to slot blotting for MUC5B and sialic acid. While MUC5B sedimented over fractions 3 to 11, the majority of sialylated MUC5B glycoforms were observed in the less dense (slower) sedimenting fractions (3 through 5) (Fig. 2 C-D). TEM images of pooled fractions from different sedimentation rates across the gradient show that the slowest sedimenting, highest sialylated MUC5B (Fractions 3–5) represent primarily linearized polymers, while faster sedimenting, less sialylated MUC5B (Fractions 11–14) represent more condensed and less expanded polymers (Fig. 2 E). Overall, these data indicate that mucin sialylation plays a major role in mucin linearization. Sialyltransferase Inhibition Impairs Mucociliary Transport in Primary HBECs To determine the consequences of reduced mucin sialylation on mucus transport, we treated non-CF HBECs with the sialyltransferase inhibitor 3Fax-Peracetyl Neu5Ac (STI) or DMSO vehicle for 24 hours and subsequently imaged them via µOCT ( 13 , 30 , 31 ). To ensure we studied only mucus biosynthesized under sialyltransferase inhibited conditions, we stimulated release of pre-existing mucin granules by purinergic stimulation with UTP prior to sialyltransferase treatment ( 32 ). HBECs treated with STI had significantly slower MCT rates (0.29 ± 0.05 mm/min; P < 0.001; Fig. 3 B&E, S1 and S2 Videos) compared to vehicle treated cells (1.0 ± 0.16 mm/min). In contrast to effects on MCT rate, treatment with STI had no effect of the hydration state of the mucus layer as indicated by ASL and PCL depths (Fig. 3 A-D) or CBF (SI Fig. 1 A). Together, these data provides preliminary evidence that adequate sialylation of secreted mucin is vital for normal mucus transport, and this phenomenon occurs independently from the hydration state of the mucus. Sialyltransferase Inhibition Impairs Mucociliary Transport in Rat Tracheae To better understand the consequences of inhibiting sialylation of mucins in vivo and in the context of mucin-rich gland secretions, we administered STI (500µM) to WT rat trachea by intratracheal instillation daily for 7 days. ( 33 , 34 ). The day before the last treatment, tracheae were excised and imaged via µOCT to evaluate the airway microanatomy. Consistent with the effects of STI on HBECs, tracheae treated with STI had significantly slower MCT (0.16 mm/min ± 0.05; P < 0.01; Fig. 4 B,E, S3 and S4 Videos) compared to vehicle treated tracheae (0.49 ± 0.12 mm/min). Additionally, there was no difference in ASL or PCL depth (Fig. 4 A,C-D), indicating that STI had no effect on airway hydration similar to our findings in HBE cells. CBF was also unaffected (SI Fig. 1 B). These data support that reduced sialylation impairs mucus transport independent from hydration in an in vivo mucus model and in the presence of mucus glands. α-2,3 Sialyltransferase Inhibition Alone is Sufficient to Impair Mucociliary Transport Since mucins contain mostly α-2,3 linked sialylated O-glycans ( 35 – 37 ) and evidence suggests that α-2,3 linked sialylation is selectively expressed in mucus producing goblet cells ( 38 ) we hypothesized that inhibition of α-2,3 sialylation alone would be sufficient to impair mucus transport. To determine the consequences of on mucus transport, we treated non-CF HBECs were treated with 120µM GA, an α-2,3 specific sialylation inhibitor, or vehicle for 24 hours and assessed for mucus physiology via µOCT ( 13 , 39 , 40 ). Similar to our findings with STI, cells treated with GA had significantly impaired MCT (0.35 ± 0.09 mm/min; P < 0.01; Fig. 5 B,E, S5 and S6 Videos) compared to vehicle treated cells (1.0 ± 0.18 mm/min), and GA had no effect on ASL or PCL (Fig. 5 A,C-D) or CBF (SI Fig. 1 C). Consistent with this, rat tracheae instilled with 300µM GA in WT daily for 7 days exhibited a significant impairment in MCT (0.18 ± 0.06 mm/min; P < 0.05; Fig. 6 B,E, S7 and S8 Videos) compared to those treated with vehicle (0.34 ± 0.07 mm/min). Furthermore, tracheae treated with GA showed no differences in ASL, PCL, or CBF (Fig. 6 A,C-D; SI Fig. 1 D). Altogether, these findings provide increasing evidence that normal sialylation, and specifically α-2,3 sialylation alone, may be vital for healthy mucus transport in vitro and in vivo , and that this mechanism is occurring independent from airway hydration. ST3Gal1 Expression is Decreased in CF and Increased with CFTR Modulation in HBECs Changes in terminal mucin sialylation have been documented in CF, but the clinical implications of this remain poorly understood ( 23 , 24 , 41 ). To determine a potential impact of mucin sialylation in CF airway disease, we next evaluated sialyltransferase expression of the predominant ST3Gal and ST6GalNAC isoforms, ST3Gal1 and ST6GalNAC1, expressed in mucus secreting epithelial cells ( 42 , 43 ).. We treated CF HBECs with the triple modulator combination, ETI, for 72hrs to restore CFTR function ( 44 , 45 ). Non-CF cells and paired CF cells were each treated with vehicle control. Prior to sialyltransferase evaluation, we measured mucus physiology via µOCT. As expected, CF HBECs had significantly depleted ASL and PCL depths (ASL 12.4 ± 1.0 µm; P < 0.01; PCL 6.2 ± 0.2 µm; P < 0.001; Fig. 7 A,C-D) compared to non-CF HBECs (ASL 56.2 ± 12.0 µm; PCL 7.4 ± 0.1 µm). CF HBECS also showed impaired MCT (0.02 ± 0.003 mm/min; P < 0.05; Fig. 7 B,E, S9 and S10 Videos) compared to non-CF HBECs (0.80 ± 0.3 mm/min). Furthermore, ETI treatment of CF HBECs significantly restored ASL and PCL depths (ASL 46.9 ± 8.1 µm; P < 0.01; PCL 7.4 ± 0.14 µm; P < 0.0001; Fig. 7 A,F-G), and improved MCT (0.75 ± 0.25 mm/min; P < 0.05; Fig. 7 B,H, S11 Video). These data showed expected phenotypic differences in mucus transport in the presence and absence of CFTR function, allowing us to interrogate how this relates to the forms of MUC5B present. Following µOCT evaluation, HBECs were collected and cell lysates immunoblotted for ST3Gal1 and ST6GalNAC1, the two primary siaylatransferases responsible for O-linked α-2,3 and α-2,6 sialylation, respectively ( 12 , 46 ). ST3Gal1 expression was significantly lower in CF HBECs (0.45 ± 0.1 ST3Gal1/βactin; P < 0.05; Fig. 7 I,K) than that of non-CF HBECs (0.76 ± 0.1 ST3Gal1/βactin). CF HBECs treated with ETI showed significantly increased expression of ST3Gal1 (0.56 ± 0.13 ST3Gal1/βactin; P < 0.05;Fig. 7 I,L) when compared to their paired, vehicle treated CF HBECs. There were no notable differences in ST6GalNAC1 expression when comparing non-CF, CF, or CF HBECs post ETI treatment (Fig. 7 J,M-N). In summary, these data provide evidence that sialylation is dysregulated in CF muco-obstructive disease, and that this finding is linked to CFTR function. Discussion In muco-obstructive diseases, such as CF, mucus stasis has largely been attributed to airway dehydration and mucus hyper-concentration ( 3 , 47 ), but recent evidence shows that electrostatic abnormalities of gel-forming mucins, MUC5B and MUC5AC, also contribute to aberrant mucus physiology ( 8 , 9 , 48 ). Although sialic acid highly contributes to mucin charge and electrostatics, the exact role of sialylation on the physiological and biophysical properties of mucin remains vastly understudied. Here, for the first time, we demonstrate the consequences of reduced mucin sialylation on the biophysical properties of MUC5B and the functional consequences of sialylation inhibition on mucus transport in vitro and in vivo . Furthermore, we identify decreased expression of ST3Gal1 in CF HBECs. Overall, our findings indicate that aberrant MUC5B sialyation occurs in CF lung disease, resulting in compact mucin forms that contribute to abnormally delayed mucociliary transport. The glycosylation profiles of mucins can be heterogeneous, resulting in multiple glycoforms. Previous reports evaluating the charge states of MUC5B and MUC5AC demonstrated that MUC5AC exists as a single major charge form, while MUC5B exists in two major charge forms, denoted as “high” and “low” charge forms ( 19 ). The high charged form predominates in healthy airway secretions, while the low charged form is more abundant in several muco-obstructive diseases including CF, COPD, and asthma. Furthermore, studies have demonstrated increased levels of the low charged MUC5B form within viscous mucus plugs ( 20 – 22 , 27 ). This suggests that decreased mucin charge may bear pathological significance in muco-obstructive diseases through increased mucus compaction and transport impairment. Here, using the same technique that initially identified the two charge variants of MUC5B, we show that the highest charged species of mucin also has the strongest sialic acid detection by WGA lectin blotting of MUC5B from cell secretions (Fig. 1 A). Furthermore, we show that sialylation reduction decreased the charge state of MUC5B and produced a low charged MUC5B similar to that observed in pathologic mucus (Fig. 1 B). Although other modifications, such as sulfation, are likely to also play a role in determining the charge state of MUC5B, reducing sialylation alone was sufficient to obtain the lower MUC5B charge form that is consistent with previously published reports in other muco-obstructive diseases ( 20 , 21 , 27 ). High negative charge density on mucins has been shown to promote stiffening of the mucin backbone through repulsion of neighboring charges, which is an important feature in the maturation of mucin polymers and the formation of the mucus gel ( 10 , 49 , 50 ). During packaging and prior to secretion, these anionic charges are stabilized by divalent Ca 2+ , allowing the mucin to condense for packaging and transport ( 6 , 18 , 51 ). Upon secretion into the airway, Ca 2+ is chelated by bicarbonate, freeing these charges to repel and extend the mucin backbone ( 7 , 16 , 52 ). Therefore, loss of these charges would be expected to weaken these repulsive forces and hinder mucin expansion after secretion. In support of this, we show a significantly increased occurrence of entangled polymers after sialidase treatment of salivary MUC5B (Fig. 2 A-B), suggesting that decreased MUC5B sialylation increases mucin compaction. Interestingly, the conformation of sialidase treated MUC5B, resembles the morphology of the previously reported low charged MUC5B from a mucus plug, which was also composed of mostly entangled and non-linear polymers (similar to our observations) ( 22 ). The low charge or reduced sialylated form of MUC5B may contribute to more compaction of the mucin and impaired expansion. Evidence for this was also demonstrated through rate zonal centrifugation, where the degree of sialylation strongly correlated with the sedimentation of MUC5B (Fig. 2 ). Here, we show that the slower sedimenting MUC5B forms that are mostly linear and mature contain higher amounts of sialic acid. Several studies are congruent with this finding and show slower sedimenting MUC5B during rate zonal centrifugation exhibits a more expanded conformation that is more fully extended and mature ( 6 , 8 ). Overall, these data provide evidence that higher levels of mucin sialylation facilitate linearization of secreted mucin, a feature imperative for clearance of mucus. We utilized sialyltransferase inhibitors to reduce the sialylation of secreted mucins de novo , which allowed us to evaluate the functional consequences of reduced mucin sialylation on MCT, where both MUC5B and MUC5AC are present and contribute to mucus clearance ( 13 , 30 , 40 , 53 ). We show that decreased sialylation of secreted mucin by STI significantly impaired MCT and had no effects on ASL, PCL, or CBF in both non-CF HBECs and WT rat tracheae (Fig. 3 – 4 ). In particular, inhibition of 2,3 sialyltransferase alone was sufficient to recapitulate this phenotype of impaired MCT without affecting hydration (Fig. 5 – 6 ). In addition to secreted mucins, ciliated epithelia are lined with membrane bound (tethered) mucins that are important for PCL hydration and ciliary beating ( 54 , 55 ). It is possible that reduced sialylation of tethered mucins may have contributed to the impaired MCT observed; however, it is more likely that the impaired MCT was due to reduced sialylation of secreted mucins, since we observed no changes in ASL or PCL depth or CBF. Additionally, previous work shows that ciliated epithelium primarily express α-2,6 linked sialylation while mucin secreting goblet cells selectively express α-2,3 linked ( 38 ). Our findings are likely consequence to reduced sialyation of secreted mucin, since we observed a decrease in MCT after α-2,3 specific inhibition. These findings not only underscore the importance of normal sialylation for mucociliary clearance but also show that the relationship between mucin sialylation state and MCT is most likely attributed to the abundance of 2,3 sialylation on O-linked glycans ( 37 – 39 ). CFTR has been suggested to regulate terminal glycosylation of mucins ( 23 , 24 , 41 ), but whether this is due to CFTR dependent anion transport is unknown. Some studies document changes in mucin sialylation as a result of infection in CF ( 56 , 57 ), while others have reported altered mucin sialylation in CFTR −/− newborn piglets before the onset of inflammation or infection ( 58 ). Interestingly, there is also evidence that sialylation may be altered as a direct consequence of the defective CFTR due to its role in organelle acidification or protein turnover both of which could affect glycosyltransferases including the sialyltransferases ( 25 , 59 , 60 ). Here, we measured sialyltransferase protein expression in non-CF, CF, and CF-ETI corrected HBECs. We found that ST3Gal1 protein was significantly lower in CF HBECs compared to non-CF (Fig. 7 ). Additionally, ST3Gal1 expression was significantly increased after 72hr ETI treatment suggesting that CFTR correction may augment ST3GAL1 expression. Future studies are required to determine the relationship between CFTR and ST3Gal1 expression. Nevertheless, our findings suggest that dysregulated sialylation of mucin may be a contributing factor and therapeutic target in CF muco-obstructive disease. In summary, we demonstrate the consequences of reduced mucin sialylation on mucin charge state, mucin confirmation, and mucus transport. Our data supports a novel model in which sialylation promotes normal MUC5B linearization and MCC by increasing mucin charge state (Fig. 8 A). Conversely, when sialylation/charge is reduced as observed in CF HBECs, MUC5B becomes entangled and MCC is impaired (Fig. 8 B). In addition, we posit that reduced expression of ST3GAL1 in CF, which can be corrected by ETI, may contribute to reduced mucin charge, expansion, and mucus clearance. Furthermore, these findings provide impetus for evaluating mucin sialylation and cognate transferases as therapeutic targets to combat mucus stasis in a plethora of muco-obstructive diseases. Methods Sex as a Biological Variable Our study examined male and female animals, and similar findings are reported for both sexes. Primary HBE Cell Culturing Primary human bronchial epithelial cells (HBECs) harvested from lung explants of previously healthy (Non-CF) or F508del-CFTR homozygous (CF) donors. First or second-passage cells, were seeded onto 6.5-mm-diameter permeable supports (Corning Inc., Corning, NY) coated with NIH 3T3 fibroblast conditioned media at a density of 0.5 × 10 6 cells per filter. Cells were grown in in PneumaCult™-ALI Medium (STEMCELL Technologies, Canada) to induce terminal differentiation at air liquid interface for at least 4 weeks ( 61 , 62 ). Prior to all studies, primary HBECs were treated apically with 100uM UTP in PBS for 40 minutes to induce granular mucin secretion to remove pre-existing intracellular mucin produced before experimental conditions ( 32 ). Rat Model All animal experiments at UAB were conducted in accordance with UAB Institutional Animal Care and Use Committee (IACUC) approved protocols. All animal experiments used wild-type (WT) Sprague-Dawley rats. Animals were bred and housed in standard cages with a 12-h light/dark cycle with ad libitum access to food and water and were routinely monitored. Rats of the same sex were co-housed from time of weaning to study conclusion. Weaned rats were maintained on a standard rodent diet. Animals were euthanized by intraperitoneal injection of 500µL pentobarbital sodium (390 mg/mL) followed by exsanguination of the hepatic portal vein. Animals used in this study were ≥ 6 months to allow maturation of submucosal glands ( 33 ). Male and Female rats were used and all experimental groups were matched by age and sex. Sialyltransferase Inhibition Terminal differentiated HBECs at ALI were treated with 200µM 3Fax-Peracetyl Neu5Ac (STI) to block sialylation, or 120µM glycolithocholic acid (GA) to specifically inhibit α-2,3 sialylation, in the basolateral compartment for 24 hours and then imaged via µOCT ( 13 ). Control cells were treated with DMSO vehicle. To inhibit sialylation in rat tracheae, 100µL of 500µM STI or 300µM GA diluted in PBS, was intratracheally instilled in WT rats daily for seven days following established methods ( 63 ). Control groups were treated with DMSO vehicle diluted in PBS. µOCT Imaging and Analysis Measurements of the functional microanatomy of primary HBECs or freshly excised rat tracheae were performed using micro-optical coherence tomography (µOCT), a high-speed, high-resolution microscopic imaging modality as previously described ( 64 ). The µOCT instrument provides cross-sectional images of the epithelium with a sub-cellular resolution sufficient to directly visualize and quantify micro-anatomic parameters including air surface liquid (ASL) depth, periciliary liquid (PCL) depth, mucociliary transport (MCT) rates, and ciliary beat frequency (CBF). Images were acquired at a rate of 40 frames per second and at 512 A-lines per frame. ASL and PCL depths were quantified by direct geometric measurement of the respective layers with a correction factor based the estimated refractive index of n = 1.33, using ImageJ (NIH) software ( 62 ). MCT rate was determined using time elapsed and distance traveled of native particles in the mucus layer over multiple frames. Ciliary beat frequency (CBF) and was investigated by Fourier analysis of the time-varying reflectance due to beating cilia using MATLAB. For consistency, HBECs were measured at four standardized locations for each culture. Tracheae were placed in the same proximal to distal orientation and the imaging beam was placed at six standardized locations along the ventral surface of the trachea as previously described ( 33 , 62 ). Native MUC5B Isolation Whole human saliva from a healthy donor was collected by chewing on Parafilm to stimulate secretion and collected into a 50-ml falcon tube. Saliva was centrifuged at 3000 x g for 25 minutes at 4°C to remove cells and debris. Clarified saliva was solubilized overnight in 0.1M NaCl 20mM/Tris pH 7.4 at 4°C with rotation. Following solubilization, cesium chloride (CsCl) was added to a starting density of 1.45g/mL, and saliva was fractionated by isopycnic density centrifugation in a Beckman SW41 Ti swinging bucket rotor at 118k x g for 72hrs at 15C ( 8 , 29 ). Following centrifugation, MUC5B containing fractions were pooled and buffer exchanged into 10mM NaCl/10mM Tris, pH 7.4. MUC5B Treatments To evaluate the role of sialylation on charge state of MUC5B, secreted mucus from non-CF HBECs was collected and solubilized in 4M guanidinium hydrochloride (GuHCl). Following solubilization, mucins were partially purified and subjected to a neuraminidase buffer exchange containing 50mM sodium acetate, 4mM CaCl 2 pH 6 using a 100kDa Amicon Ultra centrifugation column (Millipore Sigma, Burlington, MA). Partially purified mucin was split into equal (5µg) aliquots and treated with increasing amounts of neuraminidase from Vibrio Cholerae (Roche, Bavaria, Germany), ranging from 0mU/mL to 25mU/mL, for 2hrs at 37°C. Neuraminidase vehicle was added to equalize volumes. After incubation, a denaturing buffer containing 6M urea and 25mM dithioreitol (DTT) was added to inactivate sialidase before electrophoresis. To evaluate the consequences of reduced sialylation on MUC5B conformation via TEM, 50µg/mL of MUC5B purified by CsCl gradient centrifugation was treated with either neuraminidase vehicle or 12.5mU/mL neuraminidase for 2hrs at 37°C. CaCl 2 was added to a final of 1mM to enable neuraminidase activity. After treatment, 10mM EGTA, pH7.4 was added to vehicle and neuraminidase treated mucin to remove CaCl 2 . As a positive control, 10mM CaCl 2 was added to vehicle treated mucin and adjusted to pH 5. Samples were incubated overnight at 4°C prior to negative staining. Agarose-polyacrylamide Gel Electrophoresis Following partial purification and neuraminidase treatment, secreted mucus from non-CF HBECs was buffer exchanged into gel-loading buffer containing 6M Urea, 25mM DTT, and 0.1% SDS and heated at 95°C for 10 minutes. Mucins were separated by their inherent charge on an agarose-polyacrylamide-urea gel as previously described ( 65 ). Briefly, gels were prepared with 1% agarose, 1.5% polyacrylamide, and 4M urea. Sample (5µg) was loaded and electrophoresed at 80V for 2hrs, followed by transfer at 40V for 6 hours onto a 0.45µm nitrocellulose membrane. Separate, identical blots were incubated with either MUC5B primary antibody (4A10-H2, Novus Biologicals, Centennial, CO) at 1:500 dilution followed by mouse anti-IgG horse radish peroxidase (HRP) conjugated secondary (31340, Invitrogen, Waltham, MA) at 1:3000 dilution or 1µg/mL biotinylated WGA (B-1025-5, Vector Labs, Newark, CA) followed by Vectastain ABC-HRP reagent at 1:2000 dilution. HRP activity was detected using enhanced chemiluminescence detection solution (Bio-Rad, Hercules, CA) and imaged on a Bio-Rad Gel Doc XR Gel Documentation System. Transmission Electron Microscopy and Evaluation Samples were adjusted to 5µg/mL and incubated for 30 seconds on carbon coated CF400-Cu grids (EMS, Hatfield, PA) that had been glow discharged at 30 Volts for 30 seconds. Grids were washed in ddH2O for 10 seconds and then negative stained with 2% (w/v) uranyl acetate for 1 minute ( 8 ). TEM data were recorded using a JEOL JEM-1400Flash microscope (JEOL USA, Peabody, MA) at 120 Kv in a magnification range between 30,000 to 50,000x. MUC5B polymers from vehicle, sialidase, and 10mM calcium pH 5 conditions were counted and categorized, based on their appearance, into three groups: condensed, entangled, or linearized ( 8 ). A total of 30–35 images were collected for each condition, representing 72–85 polymers per condition. Images were blinded prior to polymer scoring. Rate Zonal Centrifugation Rate zonal centrifugation was performed on 11mL linear 10–35% sucrose gradients in PBS, pH 7.4. Gradients were prepared as 4-step discontinuous gradients and thawed at 4°C for 20 hours prior to centrifugation to form linear gradients ( 66 ). 500µl of sample was layered onto the top of the gradient and centrifuged in a SW41 Ti swinging bucket rotor at 210k x g for 90 minutes at 15°C ( 8 ). Following centrifugation, the gradient was fractioned from the top in 500µL increments, giving 23 total fractions. The fractions were evaluated for mucin and sialic acid content. Slot Blotting and Detection Equal volumes of samples were diluted in buffer containing 4M GuHCl, 25mM DTT, 0.1M Tris pH 7.4 and heated at 95°C prior to loading. Samples (200uL) were transferred onto a 0.45µm nitrocellulose membrane under gentle vacuum using Bio-Rad Bio-Dot SF microfiltration apparatus (Bio-Rad, Life Sciences, USA). MUC5B content was evaluated using anti-human MUC5B primary antibody (4A10-H2, Novus Biologicals, Centennial, CO) at 1:500 dilution followed by IRDye® 680RD Goat anti-Mouse IgG Secondary Antibody (Li-COR, Lincoln, NE) at a 1:10,000 dilution. Sialic acid content was evaluated using SIAFIND™ biotinylated Pan-Specific Lectenz (Lectenz Bio, Athens, GA) at 20µg/mL followed by IRDye® 800CW Streptavidin (Li-COR) at a 1:5000 dilution. Secondary antibodies were visualized, and densitometry performed on band intensities using a LI- COR Odyssey® CLx Infrared Imaging System (Li-COR Biosciences, Lincoln, USA). Elexacaftor tezacaftor ivacaftor (ETI) Treatment on CF HBECs Well differentiated CF HBECs, homozygous for F508del-CFTR, at ALI were treated with the three drug cystic fibrosis transmembrane conductance regulator (CFTR) modulator combination of 3µM Elexacaftor (E, VX-445), 3µM Tezacaftor (T, VX-661), and 1µM Ivacaftor (I, VX-770, Selleck Chemicals LLC, Houston, TX), or DMSO vehicle in the basolateral compartment for 72hrs. Drugs were refreshed every 24hrs. Non-CF HBECs were treated with DMSO vehicle. After 72 hours, cells were imaged via µOCT and subsequently collected for sialyltransferase protein evaluation. Sialyltransferase Western Blotting Following treatments, HBECs were lysed in RIPA buffer (RPI Corp, Mt. Prospect, Illinois) with Halt protease and phosphatase inhibitor cocktail (Thermo Scientific, Waltham, MA). Three filters per donor/condition were pooled and total protein concentration was determined by Bradford protein assay (Thermo Scientific, Waltham, MA). 15µg of protein per condition were loaded and separated by SDS-PAGE. Separate, identical blots were incubated with ST3Gal1 (PA5-21721, Invitrogen) or ST6GalNAc1 (PA5-31200, Invitrogen) primary antibody at 1:1000 dilution followed by rabbit anti-IgG horseradish peroxidase conjugated secondary antibody (31466, Invitrogen). HRP activity was detected using enhanced chemiluminescence detection solution (Bio-Rad, Hercules, CA) and imaged on a Bio-Rad Gel Doc XR Gel Documentation System for quantification. ImageJ (NIH) software was used to perform densitometry measurements ( 67 ). Statistics Statistical analysis was performed in GraphPad Prism version 9 or greater. Data were tested for normality using Shapiro-Wilk’s test followed by non-parametric or parametric analysis when appropriate. All µOCT data comparing two groups (Figs. 3 – 6 ) and immunoblotting data were subjected to either a two-tailed, unpaired T-test when parametric or Mann-Whitney test comparing mean ranks when non-parametric. µOCT data comparing three groups were analyzed by Kruskal-Wallis test with Dunn’s post hoc to compare groups. The categorical scoring of TEM imaged mucin polymers was analyzed by Chi-Square to test occurrence. A p-value of less than 0.05 was considered statistically significant. Statistics are presented as mean ± SEM. Study Approval Use of human bronchial epithelial cells was approved by the University of Alabama at Birmingham (UAB) Institutional Review Board (IRB) 300001383. Use of human saliva was approved by the UAB IRB 120523006. Written informed consent was received from all participants who provided sputum samples and for acquisition of airway tissues to procure primary human airway cells. All experiments were performed in accordance with relevant guidelines and regulations. Use of WT rats was approved by the UAB Institutional Animal Care and Use Committee (IACUC) IACUC-21806. All animal studies were reported in accordance with ARRIVE guidelines. Data Availability: Data is provided within the manuscript or supplementary information files. Declarations Data Availability: Data is provided within the manuscript or supplementary information files. Acknowledgments: We would like to thank Dr. Susan E Birket, PharmD, PhD and the CFTR Rat Models Core for providing all rats used in these studies. We would also like to thank Melissa Chimento and Ed Phillips of the UAB High Resolution Imaging Facility and James Kizziah of the UAB Cryo-EM Facility for their assistance and guidance with TEM preparation and imaging. Funding Disclosure: This work was supported by the NIH (www.nih.gov) F31HL164005-02 to ESH, 1R35HL135816-01 and P30DK072482 to SMR, R01HL152246 to JWB, and CF Foundation Grant (www.cff.org) ROWE19RO to SMR. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Author Contributions: ESH, HJM, SK, SMR and JWB conceived the experiments; ESH and HJM conducted the research; ESH, HJM, SMR, and JWB analyzed the data; ESH, SMR, and JWB wrote the manuscript; ESH, HJM, MM, HSH, HML, GJT, SK, SMR, and JWB edited the manuscript. MM, HSH, HML, and GJT provided resources. SMR and JWB supervised the project. Conflict of Interest: The authors have declared that no conflict of interest exists. References Rowe SM, Miller S, Sorscher EJ. Cystic fibrosis. N Engl J Med. 2005;352(19):1992–2001. Fahy JV, Dickey BF. Airway mucus function and dysfunction. N Engl J Med. 2010;363(23):2233–47. Boucher RC. Muco-Obstructive Lung Diseases. N Engl J Med. 2019;380(20):1941–53. Morrison CB, Markovetz MR, Ehre C. Mucus, mucins, and cystic fibrosis. Pediatr Pulmonol. 2019;54 Suppl 3(Suppl 3):S84-S96. Ridley C, Thornton DJ. 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Turnover of the cystic fibrosis transmembrane conductance regulator (CFTR): slow degradation of wild-type and delta F508 CFTR in surface membrane preparations of immortalized airway epithelial cells. J Cell Physiol. 1996;168(2):373–84. Liu J, Lu W, Guha S, Baltazar GC, Coffey EE, Laties AM, et al. Cystic fibrosis transmembrane conductance regulator contributes to reacidification of alkalinized lysosomes in RPE cells. Am J Physiol Cell Physiol. 2012;303(2):C160-9. Birket SE, Chu KK, Houser GH, Liu L, Fernandez CM, Solomon GM, et al. Combination therapy with cystic fibrosis transmembrane conductance regulator modulators augment the airway functional microanatomy. Am J Physiol Lung Cell Mol Physiol. 2016;310(10):L928-39. Birket SE, Chu KK, Liu L, Houser GH, Diephuis BJ, Wilsterman EJ, et al. A functional anatomic defect of the cystic fibrosis airway. Am J Respir Crit Care Med. 2014;190(4):421–32. Ortiz-Munoz G, Looney MR. Non-invasive Intratracheal Instillation in Mice. Bio Protoc. 2015;5(12). Liu L, Chu KK, Houser GH, Diephuis BJ, Li Y, Wilsterman EJ, et al. Method for quantitative study of airway functional microanatomy using micro-optical coherence tomography. PLoS One. 2013;8(1):e54473. Issa SM, Schulz BL, Packer NH, Karlsson NG. Analysis of mucosal mucins separated by SDS-urea agarose polyacrylamide composite gel electrophoresis. Electrophoresis. 2011;32(24):3554–63. Luthe DS. A simple technique for the preparation and storage of sucrose gradients. Anal Biochem. 1983;135(1):230–2. Schneider CA, Rasband WS, Eliceiri KW. NIH Image to ImageJ: 25 years of image analysis. Nat Methods. 2012;9(7):671–5. Additional Declarations No competing interests reported. Supplementary Files S1Video.avi S2Video.avi S3Video.avi S4Video.avi S5Video.avi S6Video.avi S7Video.avi S8Video.avi S9Video.avi S10Video.avi S11Video.avi SupplementalFigure1.jpg SI Figure 1. CBF of HBECs and tracheae after sialyltransferase inhibition. Quantification of CBF from (A) HBECs treated with either vehicle or 200uM STI, (B) excised WT tracheae from rats treated with either PBS vehicle or 500 μM STI, (C) HBECs treated with either vehicle or 120 μM GA, and (D) excised WT tracheae from rats treated with either PBS vehicle or 300 μM GA. N=9-18/condition. nsP>0.05 by unpaired T-test. 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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. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-4421613","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":308725821,"identity":"c8a57d7a-3420-4baa-b759-6b5081226d64","order_by":0,"name":"Elex S. 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Mucin was treated with increasing concentrations of neuraminidase, ranging from 0 to 25mu/mL, to remove sialic acid and separated by \u0026nbsp;gel electrophoresis before being probed for \u003cstrong\u003e(A)\u003c/strong\u003e sialic acid (WGA) and \u003cstrong\u003e(B)\u003c/strong\u003eMUC5B. The faster migrating/highest charged species (red bar) disappears as sialic acid is increasingly removed. The gel mobility of MUC5B is decreased as sialic acid is removed indicating a decrease in charge.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-4421613/v1/c7b59bada661052c0069ded9.png"},{"id":57491532,"identity":"430a2709-e962-4a24-bdf4-5777eb5b9f4e","added_by":"auto","created_at":"2024-05-31 11:31:37","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":13026250,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eReducing Sialylation of MUC5B Impairs Mucin Linearization\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A-B)\u003c/strong\u003e Natively purified MUC5B was treated with either vehicle or sialidase at pH 7.4 or 10mM CaCl\u003csub\u003e2\u003c/sub\u003e at a pH of 5. 10mM EGTA at pH 7.4 was added to vehicle and sialidase groups prior to ON incubation at 4°C. MUC5B polymers were subsequently visualized by negative stain TEM. \u003cstrong\u003e(A)\u003c/strong\u003e representative TEM images of MUC5B polymers treated with either vehicle, sialidase, or calcium CaCl\u003csub\u003e2\u003c/sub\u003e/pH 5. Imaging was performed to capture 72-85 polymers per group (vehicle, N=73; sialidase, N=72; and 10mM CaCl\u003csub\u003e2\u003c/sub\u003e/pH 5, N=85). After blinding, polymers were counted and categorized into 3 groups for each condition: linear, entangled, or condensed. White arrows indicate a linearized polymer in the vehicle group, an entangled polymer in the sialidase group, and a condensed polymer in the CaCl\u003csub\u003e2\u003c/sub\u003e/pH 5 group. \u003cstrong\u003e(B)\u003c/strong\u003e Quantification showing the percentage total polymers that were linear, entangled, or condensed for each group. N=72-85 per group. **P\u0026lt;0.01, ****P\u0026lt;0.0001 by Chi Square. Scale bars, 200 nm. \u003cstrong\u003e(C-D)\u003c/strong\u003e Untreated MUC5B was separated on 10-35% Sucrose gradient by rate zonal centrifugation to separate MUC5B by shape; more compact mucins sediment faster. Gradient fractions were slot blotted and probed for MUC5B and sialic acid content. \u003cstrong\u003e(C)\u003c/strong\u003e Intensities were quantified as a percentage of whole for both MUC5B and sialic acid. \u003cstrong\u003e(D)\u003c/strong\u003e Representative slot blots of MUC5B and sialic acid, showing a\u0026nbsp; higher percentage of sialic acid content compared to MUC5B in the early (more linear) fractions. (E) Fractions were pooled to represent 4 different sedimentation rates across the gradient and imaged via TEM. Scale bars, 200 nm.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-4421613/v1/3c8dd3fbec78f82f34af6520.png"},{"id":57491553,"identity":"54ebe3ed-237f-4a6b-888f-a022a991f5cc","added_by":"auto","created_at":"2024-05-31 11:31:37","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2694901,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSialyltransferase inhibition impairs mucociliary transport in primary HBECs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Representative μOCT images of HBECs treated with either vehicle or 200 μM STI demonstrate no differences in ASL (yellow bar) or PCL (red bar). \u003cstrong\u003e(B)\u003c/strong\u003e Reprocessed M-mode (e.g., x vs time) µOCT images show tracks of mucus particles above the epithelial surface of HBECs treated with vehicle or 200 μM STI; the more horizontal direction of particle streaks (yellow arrow) indicates more rapid transport. Summary data shows the effect of STI on \u003cstrong\u003e(C)\u003c/strong\u003e ASL and \u003cstrong\u003e(D)\u003c/strong\u003e PCL depths and \u003cstrong\u003e(E)\u003c/strong\u003eMCT. Regions of interest were measured and averaged for each HBEC filter. N=18-19/condition, representing 3 donors. Measurements were normalized to vehicle for each donor. nsP\u0026gt;0.05, ***P\u0026lt;0.001 by unpaired T-test or Mann-Whitney. Scale bars, 20μm.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-4421613/v1/4d3daad391f08db2ce4ca5b0.png"},{"id":57491975,"identity":"46e16b2d-d1ef-4f9f-828e-98355352af22","added_by":"auto","created_at":"2024-05-31 11:39:37","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1862320,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSialyltransferase inhibition impairs mucociliary transport in rat tracheae\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Representative μOCT images of excised WT rat tracheae from rats, treated with either PBS vehicle or 500 μM STI by intratracheal instillation daily for 7 days, demonstrate no differences in ASL (yellow bar) or PCL (red bar). \u003cstrong\u003e(B)\u003c/strong\u003e Reprocessed M-mode (e.g., x vs time) µOCT images show tracks of mucus particles above the epithelial surface of rat tracheae treated with vehicle or 500 μM STI; the more horizontal direction of particle streaks (yellow arrow) indicates more rapid transport. Summary data shows the effect of STI on \u003cstrong\u003e(C)\u003c/strong\u003e ASL and \u003cstrong\u003e(D)\u003c/strong\u003ePCL depths and \u003cstrong\u003e(E)\u003c/strong\u003e MCT. Regions of interest were measured and averaged for each trachea. N=9/condition. nsP\u0026gt;0.05, **P\u0026lt;0.01 by unpaired T-test or Mann-Whitney. Scale bars, 20μm.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-4421613/v1/0eb04d7d3a6629a31ba7041c.png"},{"id":57491561,"identity":"72a8c6d6-da44-4c19-b231-1201fe907797","added_by":"auto","created_at":"2024-05-31 11:31:38","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1946959,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eα-2,3 sialyltransferase inhibition alone is sufficient to impair mucociliary transport in HBECs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Representative μOCT images of HBECs treated with either vehicle or 120 μM GA demonstrate no differences in ASL (yellow bar) or PCL (red bar). \u003cstrong\u003e(B)\u003c/strong\u003e Reprocessed M-mode (e.g., x vs time) µOCT images show tracks of mucus particles above the epithelial surface of HBECs treated with vehicle or 120 μM GA; the more horizontal direction of particle streaks (yellow arrow) indicates more rapid transport. Summary data shows the effect of GA on \u003cstrong\u003e(C)\u003c/strong\u003e ASL and \u003cstrong\u003e(D)\u003c/strong\u003e PCL depths and \u003cstrong\u003e(E)\u003c/strong\u003eMCT. Regions of interest were measured and averaged for each HBEC filter. N=17/condition, representing 3 donors. Measurements were normalized to vehicle for each donor. nsP\u0026gt;0.05, **P\u0026lt;0.01 by unpaired T-test or Mann-Whitney. Scale bars, 20μm.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-4421613/v1/656804b58bed22fff593e08c.png"},{"id":57491533,"identity":"8e24d611-1bf6-43a3-bf20-15890dbdc7c9","added_by":"auto","created_at":"2024-05-31 11:31:37","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1873109,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eα-2,3 sialyltransferase inhibition alone is sufficient to impair mucociliary transport in trachea\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Representative μOCT images of excised WT rat tracheae from rats, treated with either PBS vehicle or 300 μM GA by intratracheal instillation daily for 7 days, demonstrate no differences in ASL (yellow bar) or PCL (red bar). \u003cstrong\u003e(B)\u003c/strong\u003e Reprocessed M-mode (e.g., x vs time) µOCT images show tracks of mucus particles above the epithelial surface of rat tracheae treated with vehicle or 300 μM GA; the more horizontal direction of particle streaks (yellow arrow) indicates more rapid transport. Summary data shows the effect of GA on \u003cstrong\u003e(C)\u003c/strong\u003e ASL and \u003cstrong\u003e(D)\u003c/strong\u003e PCL depths and \u003cstrong\u003e(E)\u003c/strong\u003eMCT. Regions of interest were measured and averaged for each trachea. N=15-17/condition. nsP\u0026gt;0.05, *P\u0026lt;0.05 by unpaired T-test or Mann-Whitney. Scale bars, 20μm.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-4421613/v1/992c399d62b9d46c3d6961f3.png"},{"id":57491974,"identity":"e0481828-fa52-4bf6-99cf-5795af66ce15","added_by":"auto","created_at":"2024-05-31 11:39:37","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":3300437,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMCT and ST3Gal1 expression are decreased in CF and increased with CFTR modulation in HBECs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Representative μOCT images of non-CF HBECs treated with vehicle and CF HBECs treated with either vehicle or ETI triple modulators for 72hrs demonstrate decreased ASL (yellow bar) and PCL (red bar) in CF HBECs that were increased with ETI treatment. \u003cstrong\u003e(B)\u003c/strong\u003e Reprocessed M-mode (e.g., x vs time) µOCT images show tracks of mucus particles above the epithelial surface of non-CF HBECs treated with vehicle and CF HBECs treated with vehicle or ETI modulators; the more horizontal direction of particle streaks (yellow arrow) indicates more rapid transport. Summary data shows the differences in \u003cstrong\u003e(C)\u003c/strong\u003e ASL and \u003cstrong\u003e(D)\u003c/strong\u003e PCL depths and \u003cstrong\u003e(E)\u003c/strong\u003e MCT between non-CF and CF and \u003cstrong\u003e(F-H)\u003c/strong\u003e CF after ETI correction. Regions of interest from 4 different filters were measured and averaged for each HBEC donor. \u003cstrong\u003e(I)\u003c/strong\u003e Representative western blot of cell lysates probed for ST3Gal1 from non-CF HBECs treated with vehicle and CF HBECs treated with either vehicle or ETI triple modulators for 72hrs, demonstrate decreased expression of ST3Gal1 in CF HBECs that was partially restored after ETI treatment. \u003cstrong\u003e(J)\u003c/strong\u003eRepresentative western blot of cell lysates probed for ST6GalNAC1 from non-CF HBECs treated with vehicle and CF HBECs treated with either vehicle or ETI triple modulators for 72hrs, demonstrate no differences in expression of ST6GalNAC1. Quantification by densitometry of ST3Gal1 expression showing (\u003cstrong\u003eK)\u003c/strong\u003ea significant decrease in ST3Gal1 expression in CF HBECs compared to non-CF and \u003cstrong\u003e(L)\u003c/strong\u003e an increase in paired ETI treated CF HBECs. Quantification by densitometry of ST6GalNAC1 expression showing no differences in ST6GalNAC1 expression between \u003cstrong\u003e(M)\u003c/strong\u003e non-CF and CF or \u003cstrong\u003e(N)\u003c/strong\u003e paired ETI treated CF HBECs. N=5-6 donors/ condition. nsP\u0026gt;0.05, *P\u0026lt;0.05, **P\u0026lt;0.01 by unpaired or paired T-test as appropriate. Scale bars, 20μm. Densitometry is presented normalized to βactin.\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-4421613/v1/79bde139d294343c8e1fa7af.png"},{"id":57491559,"identity":"bb92e97e-d9d5-4128-b9df-04006739f4b1","added_by":"auto","created_at":"2024-05-31 11:31:37","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":1530181,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe consequences of reduced sialylation on mucus physiology\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Under normal conditions, gel-forming mucins are heavily sialylated on the terminus of their glycan chains. This sialylation produces a high negative charge density across the mucin backbone. These negative charges promote mucin expansion and linearization through charge repulsion of neighboring sialic acids within and across mucin polymers. This promotes the formation of a normal mucin network and facilitates mucus transport. \u003cstrong\u003e(B)\u003c/strong\u003e When the level of sialylation is reduced, the net charge across the mucin backbone is also reduced. The charge deficit ameliorates the repulsive forces within and between mucin polymers and results in less linear, more entangled mucins. This results in an aberrant mucin network and ultimately impairs mucus transport.\u003c/p\u003e","description":"","filename":"Figure8.png","url":"https://assets-eu.researchsquare.com/files/rs-4421613/v1/1cf3995070dbb7e6496fd73c.png"},{"id":60536796,"identity":"cb00fbbb-d872-4ea3-88f5-c4c075bcc2f1","added_by":"auto","created_at":"2024-07-18 00:19:12","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":35473427,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4421613/v1/cd46e0ae-3334-4eb2-88d8-bbcf1c1fb24c.pdf"},{"id":57491530,"identity":"437e21dd-b85d-4a6c-8002-63d320dbc836","added_by":"auto","created_at":"2024-05-31 11:31:37","extension":"avi","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":12701958,"visible":true,"origin":"","legend":"","description":"","filename":"S1Video.avi","url":"https://assets-eu.researchsquare.com/files/rs-4421613/v1/e031b064ce5992265bda1cb3.avi"},{"id":57491528,"identity":"9880c07f-0f32-48a3-b41c-8e4bda1702db","added_by":"auto","created_at":"2024-05-31 11:31:36","extension":"avi","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":4108,"visible":true,"origin":"","legend":"","description":"","filename":"S2Video.avi","url":"https://assets-eu.researchsquare.com/files/rs-4421613/v1/650fc218191f9bb30997e670.avi"},{"id":57491531,"identity":"21bf354c-6ea5-442e-9d10-4fabb2a00c90","added_by":"auto","created_at":"2024-05-31 11:31:37","extension":"avi","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":9208572,"visible":true,"origin":"","legend":"","description":"","filename":"S3Video.avi","url":"https://assets-eu.researchsquare.com/files/rs-4421613/v1/ad9d5bf6fa7d7a07de0c500f.avi"},{"id":57491562,"identity":"062f5aa0-16cf-4049-85be-199d46a7d17c","added_by":"auto","created_at":"2024-05-31 11:31:38","extension":"avi","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":8829764,"visible":true,"origin":"","legend":"","description":"","filename":"S4Video.avi","url":"https://assets-eu.researchsquare.com/files/rs-4421613/v1/f0aab405579c2dd372dedd5f.avi"},{"id":57491554,"identity":"2cdeb561-0c69-4d82-afdb-7f3a74aa9216","added_by":"auto","created_at":"2024-05-31 11:31:37","extension":"avi","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":10274884,"visible":true,"origin":"","legend":"","description":"","filename":"S5Video.avi","url":"https://assets-eu.researchsquare.com/files/rs-4421613/v1/7c5b3ca45a9cc6f1d86cbeba.avi"},{"id":57491564,"identity":"81886a15-96f7-47cb-b2d9-2a2c1dab1aba","added_by":"auto","created_at":"2024-05-31 11:31:38","extension":"avi","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":11546070,"visible":true,"origin":"","legend":"","description":"","filename":"S6Video.avi","url":"https://assets-eu.researchsquare.com/files/rs-4421613/v1/e72f44011f04c9fdb44a2bc2.avi"},{"id":57491558,"identity":"01be5d28-f5ea-4023-8572-3edb3c5b7a81","added_by":"auto","created_at":"2024-05-31 11:31:37","extension":"avi","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":9295190,"visible":true,"origin":"","legend":"","description":"","filename":"S7Video.avi","url":"https://assets-eu.researchsquare.com/files/rs-4421613/v1/5dc5b9434007447c382fda02.avi"},{"id":57491556,"identity":"b958b110-e482-47bc-8dbe-1fbeebc2fda6","added_by":"auto","created_at":"2024-05-31 11:31:37","extension":"avi","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":9959194,"visible":true,"origin":"","legend":"","description":"","filename":"S8Video.avi","url":"https://assets-eu.researchsquare.com/files/rs-4421613/v1/facb185f7059818e52e258f5.avi"},{"id":57491565,"identity":"acc56332-876f-456c-b5d6-df13a119d05a","added_by":"auto","created_at":"2024-05-31 11:31:38","extension":"avi","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":10910254,"visible":true,"origin":"","legend":"","description":"","filename":"S9Video.avi","url":"https://assets-eu.researchsquare.com/files/rs-4421613/v1/27f6bb9bfc09052350252e76.avi"},{"id":57491566,"identity":"c0d76281-1c43-4c51-93c2-18689d01162c","added_by":"auto","created_at":"2024-05-31 11:31:38","extension":"avi","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":9672578,"visible":true,"origin":"","legend":"","description":"","filename":"S10Video.avi","url":"https://assets-eu.researchsquare.com/files/rs-4421613/v1/99803feb14def3951db0a5d9.avi"},{"id":57491976,"identity":"8d187d90-fbe5-4096-8dbc-88cb99784082","added_by":"auto","created_at":"2024-05-31 11:39:39","extension":"avi","order_by":11,"title":"","display":"","copyAsset":false,"role":"supplement","size":10943500,"visible":true,"origin":"","legend":"","description":"","filename":"S11Video.avi","url":"https://assets-eu.researchsquare.com/files/rs-4421613/v1/1d257a248394b1eaf8cb9dac.avi"},{"id":57491563,"identity":"f9ff82be-17e9-4771-9b62-fda21a106144","added_by":"auto","created_at":"2024-05-31 11:31:38","extension":"jpg","order_by":12,"title":"","display":"","copyAsset":false,"role":"supplement","size":217150,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSI Figure 1.\u003c/strong\u003e \u003cstrong\u003eCBF of HBECs and tracheae after sialyltransferase inhibition.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eQuantification of CBF from (A) HBECs treated with either vehicle or 200uM STI, (B) excised WT tracheae from rats treated with either PBS vehicle or 500 μM STI, (C) HBECs treated with either vehicle or 120 μM GA, and (D) excised WT tracheae from rats treated with either PBS vehicle or 300 μM GA. N=9-18/condition. nsP\u0026gt;0.05 by unpaired T-test.\u003c/p\u003e","description":"","filename":"SupplementalFigure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4421613/v1/8d1b3805c09c95846b63dd10.jpg"},{"id":57491567,"identity":"dcb5ae02-d033-4eda-9f5f-2da4f01d3c03","added_by":"auto","created_at":"2024-05-31 11:31:39","extension":"pdf","order_by":13,"title":"","display":"","copyAsset":false,"role":"supplement","size":227100,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementalMaterialSciReports.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4421613/v1/4d05c1ff6866ff283c881dc7.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Reduced Sialylation of Airway Mucin Impairs Mucus Transport by Altering the Biophysical Properties of Mucin","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCystic fibrosis (CF) is a genetic disease hallmarked by viscous and adhesive airway mucus in several organs (\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). In the respiratory tract, CF mucus becomes static and leads to chronic infection, progressive organ decline, and early mortality (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). The rheological characteristics of the mucus gel are largely governed by the gel-forming mucins MUC5B and MUC5AC, the chief structural component of mucus (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). In CF, electrostatic driven abnormalities, stemming from impaired anion transport, alter mucin conformation and contribute to increased mucus viscosity and impaired mucociliary clearance (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan additionalcitationids=\"CR7 CR8\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eGel-forming mucins are extensively modified with hydroxyl (O)-linked glycans that determine biophysical properties including normal mucin expansion and rheological characteristics (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). Mucin glycans are largely terminated by sialic acid attached in either an α-2,3 or α-2,6 linkage, facilitated by either ST3 beta-galactoside alpha-2,3-sialyltransferase (ST3Gal) or ST6 N-acetylgalactosaminide alpha-2,6-sialyltransferase (ST6GalNAc), respectively (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). Sialic acid is a negatively charged monosaccharide and exerts key electrostatic properties to mucins via its negative charge (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). The high anionic density of mucins is postulated to help stiffen the mucin polymer through charge repulsion and mediate interactions with cations to facilitate mucin granular packaging and post-secretory expansion (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). Within intracellular granules, calcium (Ca\u003csup\u003e2+\u003c/sup\u003e) shields these negative charges to mitigate electrostatic repulsion and promote mucin condensation (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). Upon secretion, divalent sodium (Na\u003csup\u003e+\u003c/sup\u003e) is exchanged with Ca\u003csup\u003e2+\u003c/sup\u003e to facilitate mucin expansion and hydration (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). Although anionic density is central to the mechanisms of mucin maturation and hydration, studies have historically centered around the altered ionic environment as the driver of aberrant mucin in CF, and the role of intrinsic mucin sialylation/charge in mucin biogenesis and MCC has remained understudied.\u003c/p\u003e \u003cp\u003ePrevious work has implicated lower charged mucin in several muco-obstructive diseases. The predominant gel-forming mucin of the airway, MUC5B, has been characterized to exist in two forms: (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e) a more negative \u0026ldquo;high\u0026rdquo; charged form, and (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e) a less negative \u0026ldquo;low\u0026rdquo; charged form (based on its migration following Agarose-PAGE) (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). Increased levels of the low charged form have been reported in CF, asthma, and COPD (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). In asthmatics, the low charged form was enriched within a viscous mucus plug and exhibited a compact and entangled conformation, linking decreased MUC5B charge with conformational and rheological defects (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). However, the etiology of this MUC5B form and the functional consequences of reduced/low mucin charge on polymer expansion and MCC have not been elucidated. Interestingly, changes in mucin sialylation have been documented in CF. Specifically, evidence supports that sialylation may be reduced as a direct consequence of defective CFTR, although the role of CFTR in regulating mucin sialylation/charge and its impact on CF mucus pathology remain unclear (\u003cspan additionalcitationids=\"CR24 CR25\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn this study, we aimed to determine the role of mucin sialylation on the biophysical properties of mucin and overall mucus function in terms of charge state, compaction, and mucus transport. Furthermore, we aimed to determine the clinical relevance of aberrant mucin sialylation in CF. Here, we show that reduced sialylation of mucin contributed to a low charged form of MUC5B, increased MUC5B compaction, and ultimately impaired mucociliary transport \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e. Additionally, we provide evidence for a link between the defective CFTR and reduced sialylation of mucin in CF mucus stasis. Overall, this study demonstrates the importance of sialylation in mucus function and provides impetus to investigate the molecular mechanisms of mucin sialylation for treatment of mucociliary dysfunction in CF, as well as potentially other muco-obstructive diseases.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eReducing the Sialylation of Contributes to a Low Charge Form of MUC5B\u003c/h2\u003e \u003cp\u003eSince sialic acid contributes to the overall charge of MUC5B, we hypothesized that reducing sialylation would result in a lower charged mucin, similar to the predominate forms in COPD and asthma (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). To test this, we collected and partially purified mucin from non-CF HBEC secretions, then treated this mucin with increasing concentrations of sialidase. Using a agarose polyacrylamide gel electrophoresis gel mobility assay to characterize mucin charge state (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e), sialidase treated mucins were separated followed by sialylation analysis with WGA lectin blotting (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). As sialylation was reduced by sialidase treatment, the faster migrating mucin forms were dose-dependently shifted to a slower mobility in the gel (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA) indicative of charge reduction. In parallel experiments, an incremental shift in gel mobility of MUC5B was observed, reflecting the dose-dependent loss of sialic acid following sialidase treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Together, these results provide evidence that sialylation is important in the charge state of mucins and its reduction may contribute to the low charged MUC5B form previously implicated in muco-obstructive disease.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eReducing Sialylation of Salivary MUC5B Impairs Mucin Linearization\u003c/h2\u003e \u003cp\u003eTo determine the consequences of reduced sialylation on MUC5B conformation, we natively purified salivary MUC5B via CsCl gradient centrifugation as previously described, incubated it with either sialidase or vehicle, and subsequently evaluated the macromolecular conformations of MUC5B polymers via TEM (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). In parallel, we incubated untreated MUC5B with 10mM Ca\u003csup\u003e2+\u003c/sup\u003e in pH 5 to induce MUC5B condensation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA) (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). We observed mostly linearized polymer chains under vehicle conditions, and condensed polymers that formed highly overlapping networks under high Ca\u003csup\u003e2+\u003c/sup\u003e/ low pH (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). MUC5B treated with sialidase primarily presented as highly entangled molecules, where the polymers frequently overlapped with themselves and did not take on a fully extended form, indicative of less electrostatic stiffening of the mucin (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). A minimum of 72 polymers per condition were categorized into either linear, entangled, or condensed morphologies based on a previously established scoring method (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB) (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). Sialidase treated MUC5B was enriched with highly entangled molecules (43.0% linear, 44.4% entangled, 12.5% condensed; P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) compared to vehicle treated MUC5B, which primarily exhibited linearized polymer chains (93.1% linear, 6.8% entangled, 0.0% condensed). MUC5B exposed to high Ca\u003csup\u003e2+\u003c/sup\u003e/low pH exhibited substantially more condensed polymers s (34.1% linear, 31.8% entangled, 34.1% condensed; P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-B). These data suggest that reduced sialylation contributes to compaction of secreted MUC5B.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo further define the relationship between mucin conformation and sialylation, we performed rate zonal centrifugation on purified salivary MUC5B using a linear 10\u0026ndash;35% sucrose gradient to separate mucin by size and shape. Mucins that sediment faster during rate zonal centrifugation have been described as more compact and pathologic, while mucins that sediment slower exhibit an expanded (linear) conformation (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). We therefore hypothesized that slower sedimenting MUC5B would have a higher degree of sialylation, while faster sedimentation would be associated with reduced sialylation/charge. After rate zonal centrifugation, we collected fractions from the top of the gradient and subjected them to slot blotting for MUC5B and sialic acid. While MUC5B sedimented over fractions 3 to 11, the majority of sialylated MUC5B glycoforms were observed in the less dense (slower) sedimenting fractions (3 through 5) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC-D). TEM images of pooled fractions from different sedimentation rates across the gradient show that the slowest sedimenting, highest sialylated MUC5B (Fractions 3\u0026ndash;5) represent primarily linearized polymers, while faster sedimenting, less sialylated MUC5B (Fractions 11\u0026ndash;14) represent more condensed and less expanded polymers (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). Overall, these data indicate that mucin sialylation plays a major role in mucin linearization.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eSialyltransferase Inhibition Impairs Mucociliary Transport in Primary HBECs\u003c/h2\u003e \u003cp\u003eTo determine the consequences of reduced mucin sialylation on mucus transport, we treated non-CF HBECs with the sialyltransferase inhibitor 3Fax-Peracetyl Neu5Ac (STI) or DMSO vehicle for 24 hours and subsequently imaged them via \u0026micro;OCT (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). To ensure we studied only mucus biosynthesized under sialyltransferase inhibited conditions, we stimulated release of pre-existing mucin granules by purinergic stimulation with UTP prior to sialyltransferase treatment (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). HBECs treated with STI had significantly slower MCT rates (0.29\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05 mm/min; P\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB\u0026amp;E, S1 and S2 Videos) compared to vehicle treated cells (1.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16 mm/min). In contrast to effects on MCT rate, treatment with STI had no effect of the hydration state of the mucus layer as indicated by ASL and PCL depths (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-D) or CBF (SI Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Together, these data provides preliminary evidence that adequate sialylation of secreted mucin is vital for normal mucus transport, and this phenomenon occurs independently from the hydration state of the mucus.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eSialyltransferase Inhibition Impairs Mucociliary Transport in Rat Tracheae\u003c/h2\u003e \u003cp\u003eTo better understand the consequences of inhibiting sialylation of mucins \u003cem\u003ein vivo\u003c/em\u003e and in the context of mucin-rich gland secretions, we administered STI (500\u0026micro;M) to WT rat trachea by intratracheal instillation daily for 7 days. (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e). The day before the last treatment, tracheae were excised and imaged via \u0026micro;OCT to evaluate the airway microanatomy. Consistent with the effects of STI on HBECs, tracheae treated with STI had significantly slower MCT (0.16 mm/min\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05; P\u0026thinsp;\u0026lt;\u0026thinsp;0.01; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB,E, S3 and S4 Videos) compared to vehicle treated tracheae (0.49\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12 mm/min). Additionally, there was no difference in ASL or PCL depth (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA,C-D), indicating that STI had no effect on airway hydration similar to our findings in HBE cells. CBF was also unaffected (SI Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). These data support that reduced sialylation impairs mucus transport independent from hydration in an \u003cem\u003ein vivo\u003c/em\u003e mucus model and in the presence of mucus glands.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eα-2,3 Sialyltransferase Inhibition Alone is Sufficient to Impair Mucociliary Transport\u003c/h2\u003e \u003cp\u003eSince mucins contain mostly α-2,3 linked sialylated O-glycans (\u003cspan additionalcitationids=\"CR36\" citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e) and evidence suggests that α-2,3 linked sialylation is selectively expressed in mucus producing goblet cells (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e) we hypothesized that inhibition of α-2,3 sialylation alone would be sufficient to impair mucus transport. To determine the consequences of on mucus transport, we treated non-CF HBECs were treated with 120\u0026micro;M GA, an α-2,3 specific sialylation inhibitor, or vehicle for 24 hours and assessed for mucus physiology via \u0026micro;OCT (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e). Similar to our findings with STI, cells treated with GA had significantly impaired MCT (0.35\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09 mm/min; P\u0026thinsp;\u0026lt;\u0026thinsp;0.01; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB,E, S5 and S6 Videos) compared to vehicle treated cells (1.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18 mm/min), and GA had no effect on ASL or PCL (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA,C-D) or CBF (SI Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Consistent with this, rat tracheae instilled with 300\u0026micro;M GA in WT daily for 7 days exhibited a significant impairment in MCT (0.18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06 mm/min; P\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB,E, S7 and S8 Videos) compared to those treated with vehicle (0.34\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07 mm/min). Furthermore, tracheae treated with GA showed no differences in ASL, PCL, or CBF (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA,C-D; SI Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). Altogether, these findings provide increasing evidence that normal sialylation, and specifically α-2,3 sialylation alone, may be vital for healthy mucus transport \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e, and that this mechanism is occurring independent from airway hydration.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003eST3Gal1 Expression is Decreased in CF and Increased with CFTR Modulation in HBECs\u003c/h2\u003e \u003cp\u003eChanges in terminal mucin sialylation have been documented in CF, but the clinical implications of this remain poorly understood (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e). To determine a potential impact of mucin sialylation in CF airway disease, we next evaluated sialyltransferase expression of the predominant ST3Gal and ST6GalNAC isoforms, ST3Gal1 and ST6GalNAC1, expressed in mucus secreting epithelial cells (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e).. We treated CF HBECs with the triple modulator combination, ETI, for 72hrs to restore CFTR function (\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e). Non-CF cells and paired CF cells were each treated with vehicle control. Prior to sialyltransferase evaluation, we measured mucus physiology via \u0026micro;OCT. As expected, CF HBECs had significantly depleted ASL and PCL depths (ASL 12.4\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0 \u0026micro;m; P\u0026thinsp;\u0026lt;\u0026thinsp;0.01; PCL 6.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2 \u0026micro;m; P\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA,C-D) compared to non-CF HBECs (ASL 56.2\u0026thinsp;\u0026plusmn;\u0026thinsp;12.0 \u0026micro;m; PCL 7.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 \u0026micro;m). CF HBECS also showed impaired MCT (0.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.003 mm/min; P\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB,E, S9 and S10 Videos) compared to non-CF HBECs (0.80\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3 mm/min). Furthermore, ETI treatment of CF HBECs significantly restored ASL and PCL depths (ASL 46.9\u0026thinsp;\u0026plusmn;\u0026thinsp;8.1 \u0026micro;m; P\u0026thinsp;\u0026lt;\u0026thinsp;0.01; PCL 7.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14 \u0026micro;m; P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA,F-G), and improved MCT (0.75\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25 mm/min; P\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB,H, S11 Video). These data showed expected phenotypic differences in mucus transport in the presence and absence of CFTR function, allowing us to interrogate how this relates to the forms of MUC5B present.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFollowing \u0026micro;OCT evaluation, HBECs were collected and cell lysates immunoblotted for ST3Gal1 and ST6GalNAC1, the two primary siaylatransferases responsible for O-linked α-2,3 and α-2,6 sialylation, respectively (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e). ST3Gal1 expression was significantly lower in CF HBECs (0.45\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 ST3Gal1/βactin; P\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eI,K) than that of non-CF HBECs (0.76\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 ST3Gal1/βactin). CF HBECs treated with ETI showed significantly increased expression of ST3Gal1 (0.56\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13 ST3Gal1/βactin; P\u0026thinsp;\u0026lt;\u0026thinsp;0.05;Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eI,L) when compared to their paired, vehicle treated CF HBECs. There were no notable differences in ST6GalNAC1 expression when comparing non-CF, CF, or CF HBECs post ETI treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eJ,M-N). In summary, these data provide evidence that sialylation is dysregulated in CF muco-obstructive disease, and that this finding is linked to CFTR function.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn muco-obstructive diseases, such as CF, mucus stasis has largely been attributed to airway dehydration and mucus hyper-concentration (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e), but recent evidence shows that electrostatic abnormalities of gel-forming mucins, MUC5B and MUC5AC, also contribute to aberrant mucus physiology (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e). Although sialic acid highly contributes to mucin charge and electrostatics, the exact role of sialylation on the physiological and biophysical properties of mucin remains vastly understudied. Here, for the first time, we demonstrate the consequences of reduced mucin sialylation on the biophysical properties of MUC5B and the functional consequences of sialylation inhibition on mucus transport \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e. Furthermore, we identify decreased expression of ST3Gal1 in CF HBECs. Overall, our findings indicate that aberrant MUC5B sialyation occurs in CF lung disease, resulting in compact mucin forms that contribute to abnormally delayed mucociliary transport.\u003c/p\u003e \u003cp\u003eThe glycosylation profiles of mucins can be heterogeneous, resulting in multiple glycoforms. Previous reports evaluating the charge states of MUC5B and MUC5AC demonstrated that MUC5AC exists as a single major charge form, while MUC5B exists in two major charge forms, denoted as \u0026ldquo;high\u0026rdquo; and \u0026ldquo;low\u0026rdquo; charge forms (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). The high charged form predominates in healthy airway secretions, while the low charged form is more abundant in several muco-obstructive diseases including CF, COPD, and asthma. Furthermore, studies have demonstrated increased levels of the low charged MUC5B form within viscous mucus plugs (\u003cspan additionalcitationids=\"CR21\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). This suggests that decreased mucin charge may bear pathological significance in muco-obstructive diseases through increased mucus compaction and transport impairment. Here, using the same technique that initially identified the two charge variants of MUC5B, we show that the highest charged species of mucin also has the strongest sialic acid detection by WGA lectin blotting of MUC5B from cell secretions (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Furthermore, we show that sialylation reduction decreased the charge state of MUC5B and produced a low charged MUC5B similar to that observed in pathologic mucus (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Although other modifications, such as sulfation, are likely to also play a role in determining the charge state of MUC5B, reducing sialylation alone was sufficient to obtain the lower MUC5B charge form that is consistent with previously published reports in other muco-obstructive diseases (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eHigh negative charge density on mucins has been shown to promote stiffening of the mucin backbone through repulsion of neighboring charges, which is an important feature in the maturation of mucin polymers and the formation of the mucus gel (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e). During packaging and prior to secretion, these anionic charges are stabilized by divalent Ca\u003csup\u003e2+\u003c/sup\u003e, allowing the mucin to condense for packaging and transport (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e). Upon secretion into the airway, Ca\u003csup\u003e2+\u003c/sup\u003e is chelated by bicarbonate, freeing these charges to repel and extend the mucin backbone (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e). Therefore, loss of these charges would be expected to weaken these repulsive forces and hinder mucin expansion after secretion. In support of this, we show a significantly increased occurrence of entangled polymers after sialidase treatment of salivary MUC5B (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-B), suggesting that decreased MUC5B sialylation increases mucin compaction. Interestingly, the conformation of sialidase treated MUC5B, resembles the morphology of the previously reported low charged MUC5B from a mucus plug, which was also composed of mostly entangled and non-linear polymers (similar to our observations) (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). The low charge or reduced sialylated form of MUC5B may contribute to more compaction of the mucin and impaired expansion. Evidence for this was also demonstrated through rate zonal centrifugation, where the degree of sialylation strongly correlated with the sedimentation of MUC5B (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Here, we show that the slower sedimenting MUC5B forms that are mostly linear and mature contain higher amounts of sialic acid. Several studies are congruent with this finding and show slower sedimenting MUC5B during rate zonal centrifugation exhibits a more expanded conformation that is more fully extended and mature (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). Overall, these data provide evidence that higher levels of mucin sialylation facilitate linearization of secreted mucin, a feature imperative for clearance of mucus.\u003c/p\u003e \u003cp\u003eWe utilized sialyltransferase inhibitors to reduce the sialylation of secreted mucins \u003cem\u003ede novo\u003c/em\u003e, which allowed us to evaluate the functional consequences of reduced mucin sialylation on MCT, where both MUC5B and MUC5AC are present and contribute to mucus clearance (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e). We show that decreased sialylation of secreted mucin by STI significantly impaired MCT and had no effects on ASL, PCL, or CBF in both non-CF HBECs and WT rat tracheae (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). In particular, inhibition of 2,3 sialyltransferase alone was sufficient to recapitulate this phenotype of impaired MCT without affecting hydration (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). In addition to secreted mucins, ciliated epithelia are lined with membrane bound (tethered) mucins that are important for PCL hydration and ciliary beating (\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e). It is possible that reduced sialylation of tethered mucins may have contributed to the impaired MCT observed; however, it is more likely that the impaired MCT was due to reduced sialylation of secreted mucins, since we observed no changes in ASL or PCL depth or CBF. Additionally, previous work shows that ciliated epithelium primarily express α-2,6 linked sialylation while mucin secreting goblet cells selectively express α-2,3 linked (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e). Our findings are likely consequence to reduced sialyation of secreted mucin, since we observed a decrease in MCT after α-2,3 specific inhibition. These findings not only underscore the importance of normal sialylation for mucociliary clearance but also show that the relationship between mucin sialylation state and MCT is most likely attributed to the abundance of 2,3 sialylation on O-linked glycans (\u003cspan additionalcitationids=\"CR38\" citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eCFTR has been suggested to regulate terminal glycosylation of mucins (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e), but whether this is due to CFTR dependent anion transport is unknown. Some studies document changes in mucin sialylation as a result of infection in CF (\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e), while others have reported altered mucin sialylation in CFTR\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e newborn piglets before the onset of inflammation or infection (\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e). Interestingly, there is also evidence that sialylation may be altered as a direct consequence of the defective CFTR due to its role in organelle acidification or protein turnover both of which could affect glycosyltransferases including the sialyltransferases (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e, \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e). Here, we measured sialyltransferase protein expression in non-CF, CF, and CF-ETI corrected HBECs. We found that ST3Gal1 protein was significantly lower in CF HBECs compared to non-CF (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). Additionally, ST3Gal1 expression was significantly increased after 72hr ETI treatment suggesting that CFTR correction may augment ST3GAL1 expression. Future studies are required to determine the relationship between CFTR and ST3Gal1 expression. Nevertheless, our findings suggest that dysregulated sialylation of mucin may be a contributing factor and therapeutic target in CF muco-obstructive disease.\u003c/p\u003e \u003cp\u003eIn summary, we demonstrate the consequences of reduced mucin sialylation on mucin charge state, mucin confirmation, and mucus transport. Our data supports a novel model in which sialylation promotes normal MUC5B linearization and MCC by increasing mucin charge state (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA). Conversely, when sialylation/charge is reduced as observed in CF HBECs, MUC5B becomes entangled and MCC is impaired (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB). In addition, we posit that reduced expression of ST3GAL1 in CF, which can be corrected by ETI, may contribute to reduced mucin charge, expansion, and mucus clearance. Furthermore, these findings provide impetus for evaluating mucin sialylation and cognate transferases as therapeutic targets to combat mucus stasis in a plethora of muco-obstructive diseases.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eSex as a Biological Variable\u003c/h2\u003e \u003cp\u003eOur study examined male and female animals, and similar findings are reported for both sexes.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003ePrimary HBE Cell Culturing\u003c/h2\u003e \u003cp\u003ePrimary human bronchial epithelial cells (HBECs) harvested from lung explants of previously healthy (Non-CF) or F508del-CFTR homozygous (CF) donors. First or second-passage cells, were seeded onto 6.5-mm-diameter permeable supports (Corning Inc., Corning, NY) coated with NIH 3T3 fibroblast conditioned media at a density of 0.5 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells per filter. Cells were grown in in PneumaCult\u0026trade;-ALI Medium (STEMCELL Technologies, Canada) to induce terminal differentiation at air liquid interface for at least 4 weeks (\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e, \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e). Prior to all studies, primary HBECs were treated apically with 100uM UTP in PBS for 40 minutes to induce granular mucin secretion to remove pre-existing intracellular mucin produced before experimental conditions (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eRat Model\u003c/h2\u003e \u003cp\u003e All animal experiments at UAB were conducted in accordance with UAB Institutional Animal Care and Use Committee (IACUC) approved protocols. All animal experiments used wild-type (WT) Sprague-Dawley rats. Animals were bred and housed in standard cages with a 12-h light/dark cycle with ad libitum access to food and water and were routinely monitored. Rats of the same sex were co-housed from time of weaning to study conclusion. Weaned rats were maintained on a standard rodent diet. Animals were euthanized by intraperitoneal injection of 500\u0026micro;L pentobarbital sodium (390 mg/mL) followed by exsanguination of the hepatic portal vein. Animals used in this study were \u0026ge;\u0026thinsp;6 months to allow maturation of submucosal glands (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e). Male and Female rats were used and all experimental groups were matched by age and sex.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eSialyltransferase Inhibition\u003c/h2\u003e \u003cp\u003eTerminal differentiated HBECs at ALI were treated with 200\u0026micro;M 3Fax-Peracetyl Neu5Ac (STI) to block sialylation, or 120\u0026micro;M glycolithocholic acid (GA) to specifically inhibit α-2,3 sialylation, in the basolateral compartment for 24 hours and then imaged via \u0026micro;OCT (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). Control cells were treated with DMSO vehicle. To inhibit sialylation in rat tracheae, 100\u0026micro;L of 500\u0026micro;M STI or 300\u0026micro;M GA diluted in PBS, was intratracheally instilled in WT rats daily for seven days following established methods (\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e). Control groups were treated with DMSO vehicle diluted in PBS.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e\u0026micro;OCT Imaging and Analysis\u003c/h2\u003e \u003cp\u003eMeasurements of the functional microanatomy of primary HBECs or freshly excised rat tracheae were performed using micro-optical coherence tomography (\u0026micro;OCT), a high-speed, high-resolution microscopic imaging modality as previously described (\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e). The \u0026micro;OCT instrument provides cross-sectional images of the epithelium with a sub-cellular resolution sufficient to directly visualize and quantify micro-anatomic parameters including air surface liquid (ASL) depth, periciliary liquid (PCL) depth, mucociliary transport (MCT) rates, and ciliary beat frequency (CBF). Images were acquired at a rate of 40 frames per second and at 512 A-lines per frame. ASL and PCL depths were quantified by direct geometric measurement of the respective layers with a correction factor based the estimated refractive index of n\u0026thinsp;=\u0026thinsp;1.33, using ImageJ (NIH) software (\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e). MCT rate was determined using time elapsed and distance traveled of native particles in the mucus layer over multiple frames. Ciliary beat frequency (CBF) and was investigated by Fourier analysis of the time-varying reflectance due to beating cilia using MATLAB. For consistency, HBECs were measured at four standardized locations for each culture. Tracheae were placed in the same proximal to distal orientation and the imaging beam was placed at six standardized locations along the ventral surface of the trachea as previously described (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eNative MUC5B Isolation\u003c/h2\u003e \u003cp\u003eWhole human saliva from a healthy donor was collected by chewing on Parafilm to stimulate secretion and collected into a 50-ml falcon tube. Saliva was centrifuged at 3000 x g for 25 minutes at 4\u0026deg;C to remove cells and debris. Clarified saliva was solubilized overnight in 0.1M NaCl 20mM/Tris pH 7.4 at 4\u0026deg;C with rotation. Following solubilization, cesium chloride (CsCl) was added to a starting density of 1.45g/mL, and saliva was fractionated by isopycnic density centrifugation in a Beckman SW41 Ti swinging bucket rotor at 118k x g for 72hrs at 15C (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). Following centrifugation, MUC5B containing fractions were pooled and buffer exchanged into 10mM NaCl/10mM Tris, pH 7.4.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eMUC5B Treatments\u003c/h2\u003e \u003cp\u003eTo evaluate the role of sialylation on charge state of MUC5B, secreted mucus from non-CF HBECs was collected and solubilized in 4M guanidinium hydrochloride (GuHCl). Following solubilization, mucins were partially purified and subjected to a neuraminidase buffer exchange containing 50mM sodium acetate, 4mM CaCl\u003csub\u003e2\u003c/sub\u003e pH 6 using a 100kDa Amicon Ultra centrifugation column (Millipore Sigma, Burlington, MA). Partially purified mucin was split into equal (5\u0026micro;g) aliquots and treated with increasing amounts of neuraminidase from \u003cem\u003eVibrio Cholerae\u003c/em\u003e (Roche, Bavaria, Germany), ranging from 0mU/mL to 25mU/mL, for 2hrs at 37\u0026deg;C. Neuraminidase vehicle was added to equalize volumes. After incubation, a denaturing buffer containing 6M urea and 25mM dithioreitol (DTT) was added to inactivate sialidase before electrophoresis.\u003c/p\u003e \u003cp\u003eTo evaluate the consequences of reduced sialylation on MUC5B conformation via TEM, 50\u0026micro;g/mL of MUC5B purified by CsCl gradient centrifugation was treated with either neuraminidase vehicle or 12.5mU/mL neuraminidase for 2hrs at 37\u0026deg;C. CaCl\u003csub\u003e2\u003c/sub\u003e was added to a final of 1mM to enable neuraminidase activity. After treatment, 10mM EGTA, pH7.4 was added to vehicle and neuraminidase treated mucin to remove CaCl\u003csub\u003e2\u003c/sub\u003e. As a positive control, 10mM CaCl\u003csub\u003e2\u003c/sub\u003e was added to vehicle treated mucin and adjusted to pH 5. Samples were incubated overnight at 4\u0026deg;C prior to negative staining.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eAgarose-polyacrylamide Gel Electrophoresis\u003c/h2\u003e \u003cp\u003eFollowing partial purification and neuraminidase treatment, secreted mucus from non-CF HBECs was buffer exchanged into gel-loading buffer containing 6M Urea, 25mM DTT, and 0.1% SDS and heated at 95\u0026deg;C for 10 minutes. Mucins were separated by their inherent charge on an agarose-polyacrylamide-urea gel as previously described (\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e). Briefly, gels were prepared with 1% agarose, 1.5% polyacrylamide, and 4M urea. Sample (5\u0026micro;g) was loaded and electrophoresed at 80V for 2hrs, followed by transfer at 40V for 6 hours onto a 0.45\u0026micro;m nitrocellulose membrane. Separate, identical blots were incubated with either MUC5B primary antibody (4A10-H2, Novus Biologicals, Centennial, CO) at 1:500 dilution followed by mouse anti-IgG horse radish peroxidase (HRP) conjugated secondary (31340, Invitrogen, Waltham, MA) at 1:3000 dilution or 1\u0026micro;g/mL biotinylated WGA (B-1025-5, Vector Labs, Newark, CA) followed by Vectastain ABC-HRP reagent at 1:2000 dilution. HRP activity was detected using enhanced chemiluminescence detection solution (Bio-Rad, Hercules, CA) and imaged on a Bio-Rad Gel Doc XR Gel Documentation System.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eTransmission Electron Microscopy and Evaluation\u003c/h2\u003e \u003cp\u003eSamples were adjusted to 5\u0026micro;g/mL and incubated for 30 seconds on carbon coated CF400-Cu grids (EMS, Hatfield, PA) that had been glow discharged at 30 Volts for 30 seconds. Grids were washed in ddH2O for 10 seconds and then negative stained with 2% (w/v) uranyl acetate for 1 minute (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). TEM data were recorded using a JEOL JEM-1400Flash microscope (JEOL USA, Peabody, MA) at 120 Kv in a magnification range between 30,000 to 50,000x. MUC5B polymers from vehicle, sialidase, and 10mM calcium pH 5 conditions were counted and categorized, based on their appearance, into three groups: condensed, entangled, or linearized (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). A total of 30\u0026ndash;35 images were collected for each condition, representing 72\u0026ndash;85 polymers per condition. Images were blinded prior to polymer scoring.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eRate Zonal Centrifugation\u003c/h2\u003e \u003cp\u003eRate zonal centrifugation was performed on 11mL linear 10\u0026ndash;35% sucrose gradients in PBS, pH 7.4. Gradients were prepared as 4-step discontinuous gradients and thawed at 4\u0026deg;C for 20 hours prior to centrifugation to form linear gradients (\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e). 500\u0026micro;l of sample was layered onto the top of the gradient and centrifuged in a SW41 Ti swinging bucket rotor at 210k x g for 90 minutes at 15\u0026deg;C (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). Following centrifugation, the gradient was fractioned from the top in 500\u0026micro;L increments, giving 23 total fractions. The fractions were evaluated for mucin and sialic acid content.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eSlot Blotting and Detection\u003c/h2\u003e \u003cp\u003eEqual volumes of samples were diluted in buffer containing 4M GuHCl, 25mM DTT, 0.1M Tris pH 7.4 and heated at 95\u0026deg;C prior to loading. Samples (200uL) were transferred onto a 0.45\u0026micro;m nitrocellulose membrane under gentle vacuum using Bio-Rad Bio-Dot SF microfiltration apparatus (Bio-Rad, Life Sciences, USA). MUC5B content was evaluated using anti-human MUC5B primary antibody (4A10-H2, Novus Biologicals, Centennial, CO) at 1:500 dilution followed by IRDye\u0026reg; 680RD Goat anti-Mouse IgG Secondary Antibody (Li-COR, Lincoln, NE) at a 1:10,000 dilution. Sialic acid content was evaluated using SIAFIND\u0026trade; biotinylated Pan-Specific Lectenz (Lectenz Bio, Athens, GA) at 20\u0026micro;g/mL followed by IRDye\u0026reg; 800CW Streptavidin (Li-COR) at a 1:5000 dilution. Secondary antibodies were visualized, and densitometry performed on band intensities using a LI- COR Odyssey\u0026reg; CLx Infrared Imaging System (Li-COR Biosciences, Lincoln, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eElexacaftor tezacaftor ivacaftor (ETI) Treatment on CF HBECs\u003c/h2\u003e \u003cp\u003eWell differentiated CF HBECs, homozygous for F508del-CFTR, at ALI were treated with the three drug cystic fibrosis transmembrane conductance regulator (CFTR) modulator combination of 3\u0026micro;M Elexacaftor (E, VX-445), 3\u0026micro;M Tezacaftor (T, VX-661), and 1\u0026micro;M Ivacaftor (I, VX-770, Selleck Chemicals LLC, Houston, TX), or DMSO vehicle in the basolateral compartment for 72hrs. Drugs were refreshed every 24hrs. Non-CF HBECs were treated with DMSO vehicle. After 72 hours, cells were imaged via \u0026micro;OCT and subsequently collected for sialyltransferase protein evaluation.\u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eSialyltransferase Western Blotting\u003c/h2\u003e \u003cp\u003eFollowing treatments, HBECs were lysed in RIPA buffer (RPI Corp, Mt. Prospect, Illinois) with Halt protease and phosphatase inhibitor cocktail (Thermo Scientific, Waltham, MA). Three filters per donor/condition were pooled and total protein concentration was determined by Bradford protein assay (Thermo Scientific, Waltham, MA). 15\u0026micro;g of protein per condition were loaded and separated by SDS-PAGE. Separate, identical blots were incubated with ST3Gal1 (PA5-21721, Invitrogen) or ST6GalNAc1 (PA5-31200, Invitrogen) primary antibody at 1:1000 dilution followed by rabbit anti-IgG horseradish peroxidase conjugated secondary antibody (31466, Invitrogen). HRP activity was detected using enhanced chemiluminescence detection solution (Bio-Rad, Hercules, CA) and imaged on a Bio-Rad Gel Doc XR Gel Documentation System for quantification. ImageJ (NIH) software was used to perform densitometry measurements (\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eStatistics\u003c/h2\u003e \u003cp\u003eStatistical analysis was performed in GraphPad Prism version 9 or greater. Data were tested for normality using Shapiro-Wilk\u0026rsquo;s test followed by non-parametric or parametric analysis when appropriate. All \u0026micro;OCT data comparing two groups (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e) and immunoblotting data were subjected to either a two-tailed, unpaired T-test when parametric or Mann-Whitney test comparing mean ranks when non-parametric. \u0026micro;OCT data comparing three groups were analyzed by Kruskal-Wallis test with Dunn\u0026rsquo;s post hoc to compare groups. The categorical scoring of TEM imaged mucin polymers was analyzed by Chi-Square to test occurrence. A p-value of less than 0.05 was considered statistically significant. Statistics are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM.\u003c/p\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003eStudy Approval\u003c/h2\u003e \u003cp\u003e Use of human bronchial epithelial cells was approved by the University of Alabama at Birmingham (UAB) Institutional Review Board (IRB) 300001383. Use of human saliva was approved by the UAB IRB 120523006. Written informed consent was received from all participants who provided sputum samples and for acquisition of airway tissues to procure primary human airway cells. All experiments were performed in accordance with relevant guidelines and regulations. Use of WT rats was approved by the UAB Institutional Animal Care and Use Committee (IACUC) IACUC-21806. All animal studies were reported in accordance with ARRIVE guidelines.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section3\"\u003e \u003ch2\u003eData Availability:\u003c/h2\u003e \u003cp\u003eData is provided within the manuscript or supplementary information files.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData Availability:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData is provided within the manuscript or supplementary information files.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to thank Dr. Susan E Birket, PharmD, PhD and the CFTR Rat Models Core for providing all rats used in these studies. We would also like to thank Melissa Chimento and Ed Phillips of the UAB High Resolution Imaging Facility and James Kizziah of the UAB Cryo-EM Facility for their assistance and guidance with TEM preparation and imaging. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding Disclosure:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the NIH (www.nih.gov) F31HL164005-02 to ESH, 1R35HL135816-01 and P30DK072482 to SMR, R01HL152246 to JWB, and CF Foundation Grant (www.cff.org) ROWE19RO to SMR. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eESH, HJM, SK, SMR and JWB conceived the experiments; ESH and HJM conducted the research; ESH, HJM, SMR, and JWB analyzed the data; ESH, SMR, and JWB wrote the manuscript; ESH, HJM, MM, HSH, HML, GJT, SK, SMR, and JWB edited the manuscript. MM, HSH, HML, and GJT provided resources. SMR and JWB supervised the project. \u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConflict of Interest: \u0026nbsp;\u003c/strong\u003eThe authors have declared that no conflict of interest exists.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eRowe SM, Miller S, Sorscher EJ. Cystic fibrosis. 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Anal Biochem. 1983;135(1):230\u0026ndash;2.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchneider CA, Rasband WS, Eliceiri KW. NIH Image to ImageJ: 25 years of image analysis. Nat Methods. 2012;9(7):671\u0026ndash;5.\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":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-4421613/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4421613/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMucus stasis is a pathologic hallmark of muco-obstructive diseases, including cystic fibrosis (CF). Mucins, the principal component of mucus, are extensively modified with hydroxyl (O)-linked glycans, which are largely terminated by sialic acid. Sialic acid is a negatively charged monosaccharide and contributes to the biochemical/biophysical properties of mucins. Reports suggest that mucin sialylation may be altered in CF; however, the consequences of reduced sialylation on mucus clearance have not been fully determined. Here, we investigated the consequences of reduced sialylation on the charge state and conformation of the most prominent airway mucin, MUC5B, and defined the functional consequences of reduced sialylation on mucociliary transport (MCT). Reduced sialylation contributed to a lower charged MUC5B form and decreased polymer expansion. The inhibition of total mucin sialylation \u003cem\u003ede novo\u003c/em\u003e impaired MCT in primary human bronchial epithelial cells and rat airways, and specific α-2,3 sialylation blockade was sufficient to recapitulate these findings. Finally, we show that ST3 beta-galactoside alpha-2,3-sialyltransferase (ST3Gal1) expression is downregulated in CF and partially restored by correcting CFTR via Elexacaftor/Tezacaftor/Ivacaftor treatment. Overall, this study demonstrates the importance of mucin sialylation in mucus clearance and identifies decreased sialylation by ST3Gal1 as a possible therapeutic target in CF and potentially other muco-obstructive diseases.\u003c/p\u003e","manuscriptTitle":"Reduced Sialylation of Airway Mucin Impairs Mucus Transport by Altering the Biophysical Properties of Mucin","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-05-31 11:31:31","doi":"10.21203/rs.3.rs-4421613/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-06-05T07:23:56+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-06-04T08:49:20+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-05-31T11:21:10+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-05-30T14:55:26+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"204603621321235273137077861659027255094","date":"2024-05-25T06:57:18+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"274343647887785415639493381104102516037","date":"2024-05-24T19:07:29+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"116192427026379127330835889539620963134","date":"2024-05-24T01:04:35+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-05-23T15:18:53+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-05-23T15:12:53+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-05-22T17:45:38+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-05-21T05:30:21+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2024-05-14T22:57:33+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"66d2e56b-25e3-4cc3-bbec-9ead13c4d9c4","owner":[],"postedDate":"May 31st, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":32616516,"name":"Biological sciences/Biochemistry/Biophysical chemistry"},{"id":32616517,"name":"Biological sciences/Biochemistry/Glycobiology"},{"id":32616518,"name":"Biological sciences/Physiology/Respiration"}],"tags":[],"updatedAt":"2024-07-18T00:18:46+00:00","versionOfRecord":{"articleIdentity":"rs-4421613","link":"https://doi.org/10.1038/s41598-024-66510-2","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2024-07-17 00:18:46","publishedOnDateReadable":"July 17th, 2024"},"versionCreatedAt":"2024-05-31 11:31:31","video":"","vorDoi":"10.1038/s41598-024-66510-2","vorDoiUrl":"https://doi.org/10.1038/s41598-024-66510-2","workflowStages":[]},"version":"v1","identity":"rs-4421613","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4421613","identity":"rs-4421613","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}