Roles of the structural units, glycotopes / mammalian N-glycans for Con A - Glycan interactions, their codes, and their recognition Factors

Roles of the structural units, glycotopes / mammalian N-glycans for Con A - Glycan interactions, their codes, and their recognition Factors | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Roles of the structural units, glycotopes / mammalian N-glycans for Con A - Glycan interactions, their codes, and their recognition Factors Albert M. Wu This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2533417/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 11 Sep, 2023 Read the published version in Glycoconjugate Journal → Version 1 posted 7 You are reading this latest preprint version Abstract The binding property of Con A has been studied intensively and applied widely to glycoconjugates / glycobiology for over 80 years. However, its role and functional relationship of Con A with these mammalian structural units, glycotopes, N -glycan chains, as well as their polyvalent forms in N -glycoproteins involved in the Con A- glycan interactions have not been all defined and organzied. In this study, the recognition factors involved in these inteactions were azalyzed by our well developed method- the enzyme linked lectinosorbent (ELLSA) and inhibition assay. Based on all data obtained, it is concluded that Con A, as previous report, has a relatively broad and wide recognition ability with the Manα1→ and Glcα1→ related glycans. In addition to it reacted strongly with yeast mannan and glycogens, it also bound well with a large number of mammalian N -glycans, including the N -glycans of rat sublingual gp (RSL), human Tamm-Horsfall glycoprotein (THGP), thyroglobin and lactosferrin. The recognition specificity of Con A towards ligands, expressed by M olar R elative P otency (Molar R.P.), in a decreasing order is as follows: a1→3, a1→6 Mannopentaose ( M 5 ) and Biantennary N -linked core pentasaccharide ( M Di ) ≥ a1→3, a1→6 Mannotriose ( M 3 ) > Mana1→3Man (α1→3Mannobiose), Mana1→2Man (α1→2Mannobiose), Mana1→6Man (α1→6Mannobiose), Mana1→4Man (α1→4Mannobiose) > GlcNAcb1→2 Man (b1→2 N-Acetyl glucosamine-mannose) > Mana1→/Glcα1→ > Man > Glc, while Gal / GalNAc were inactive. Furthermore, the Man related code system, in this study, is proposed to express by both numbers of Man and GlcNAcb1→ branches ( M 3 to M 9 / M Mono to Penta etc. ) and a table of three Mana1→ and Glca1→ related biomasses of six recognition factors involved in the Con A-glycan interactions has also been demonstrated. These themes should be one of the most valuable advances since 1980s. Con A Plant lectins Glycoprotein binding proteins Carbohydrate specificities Recognition Factors Man structural units glycotopes N-Glycan and Man / N-Glycan Codes. Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Lectins are an important class of proteins or glycoproteins of non-immune origin that bind non-covalently to characteristic carbohydrate structures with specificity or selectivity [ 1 – 4 ]. They have been widely used as tools to probe the structural and functional roles of cell surface carbohydrates and to fractionate soluble or membrane glycoproteins of diverse origins and defined as applied lectins [ 1 – 7 ]. Based on lectin-reactive monosaccharides with the highest affinity, the applied lectins have been divided in five specificity groups [ 1 , 5 , 6 ]. These are: (i) the mannose/glucose-binding lectins, (ii) the N -acetylgalactosamine/galactose-binding lectins, (iii) the acetylglucosamine-lectins, (iv) the l-fucose-binding lectins and (v) sialic acid-binding lectins [ 1 , 5 ]. The mannose/glucose-binding lectins comprise a large group of agglutinins present in the family Leguminosae, primarily in seeds. Prominent among these lectins are those from the jack bean (Con A, Canavalia ensiformis ; concanavalin A), the lentil ( Lens culinaris ), the pea (PSA, Pisum sativum ), the fava bean ( Vicia faba ) and the common vetch ( Vicia cracca ). All these agglutinins are metalloproteins requiring metal ions e.g. Ca 2+ and/or Mn 2+ for their carbohydrate-binding activity [ 1 – 3 , 5 ]. After late 1980s, many other related lectins were continuously reported [ 6 , 8 – 11 ], these are including a novel lectin (Morniga M) from Mulberry ( Morus nigra ) Bark [ 8 ], Garlic ( Allium sativum ), ramsons ( Allium ursinum ) blubs [ 9 ], Clivia miniata [ 10 ], and Galanthus nivalis agglutinins (GNA, Snowdrop bulb) [ 11 ]. Concanavalin A (Con A) has been recognized as one of the most well established and useful lectins from plant seeds [ 1 – 4 ]. Its physical chemical properties and carbohydrate-bnding properties have been intensively documented. It was first isolated crystallized and found to require metal ions for its activity; it was also shown to precipitate glycogen; to agglutinate various erythrocytes; to precipitate many glycoproteins, such as blood group substances / immunoglobulins, and to react with a variety of bacterial and animal cells [ 1 – 3 ]. During the past five decades, it has been applied to initiate cell differentiation and division [ 12 , 13 ]; to stimulate T-cells to produce IL 1-like factors [ 14 ]; and to demonstrate macrophage histiocytes in pathological specimens [ 15 ]. Recently, Con A has been further demonstrated to have a potential to prevent cell death in experimental acute pancreatitis [ 16 ]. The native Con A is composed of four identical subunits of Mr 2.65⋅10 4 daltons each. Each of the four subunit of lectin is a compactly folded, dome-shaped structure [ 1 – 4 , 17 ]. Each subunit binds Ca + 2 and/or Mn + 2 metal atoms and one saccharide. [ 18 , 19 ]. Under pH 5.6, Con A is in a dimer form, the succinylated Con A is one of these dimer forms and has the same glycan specificity. This succinylated Con A is more soluble and stable than these of natural one and can stimulate DNA synthesis in mouse splenocytes [ 1 ], but it loses its ability to precipitate polysaccharides [ 1 , 2 ]. Their differences among recognition capacities and ranges are being investingated by our established approaches- ELLSA and inhibition assay. Although the recognition mechanism and factors of many Manα1→ / Glcα1→ specific lectins have been investigated by a great number of methods. These include calorimetric titration, quantitative precipitin assays, surface plasmon resonance, crystal structure etc. [ 1 – 4 , 20 – 30 ]. But, the concept of the Man related code was still ignored by the most investigators. Furthermore, their roles among the structural units, glycotopes and their polyvalency of glycotopes have not clearly described [ 1 – 4 , 24 , 31 ]. In this study, the recognition roles of Con A were analyzed by our well established method- the enzyme linked lectinosorbent (ELLSA) and inhibition assays [ 32 – 35 ], using a panel of natural polyvalent glycotopes (glycotope containing glycan masses) as well as an array of mono-, di-, structural units and glycotopes. Based on all the data provided, a Manα1→ related code for Con A- mammalian N -glycoprotein interactions was constructed and documented. Furthermore, a table of three biomasses of six Recognition Factors (RFs) was proposed. These two themes should provide one of most constructive advances in this field since 1980s. Materials And Methods Mammalian N-glycoproteins – Most mammalian N -glycoproteins, shown in the Table 1 and Fig. 1 [ 36 – 44 ], α- acid gp, bovine lactoferrin, porcine thyrogobulin, and laminin etc. were purchased from Sigma. THGP, Tamm-Horsfall glycoproteins provided by the late Dr. W.M. Watkins (University of London, Royal Postgraduate Medical School, Hammersmith Hospital, London, UK), were isolated with 0.58 M NaCl from the urine of donors with the Sd (a+) or Sd (a-) blood group by the method of Tamm and Horsfall [ 45 , 46 ]. The precipitated material was lyophilized, and its lipid content was removed with 9:1, 2:1, and 1:2 chloroform-methanol treatment and further purified as described. Polysaccharides – Yeast mannan [ 47 – 49 ], Glycogen [ 50 ], and colominic acid (poly-2,8- N -acetylneuraminic acid capsular polysaccharide, [Neu5Acα2-8Neu5Ac] n ) from E. coli [ 51 ] were also ordered from Sigma (Fig. 2 ). Mammalian N-/O- glycoprotein – RSL, Rat sublingual glycoprotein- Rat salivary glands of the adult Sprague–Dawleys obtained from Pel Freeze Biologicals, Inc., Rogers, AR, was a mixture of sublingual and submandibular glands. The RSL-major was isolated according to a modified method used for the preparation of bovine and ovine submandibular mucin [ 52 – 54 ]. Its N -glycans of RSL-major were determined to be non-bisected hybrid types similarly carrying a sialylated type II chain (Fig. 1 ) [ 55 ], while the predominant O -glycans carried on the rat sublingual mucin were defined as sialylated core 3 and 4 types (Fig. 4 ). Human Cyst O-glycoproteins (HOC) – Human blood group ABH active cyst gps (HOC), such as Cyst MSS, Mcdon, Beach, and Tighe etc, were prepared from human ovarian cyst fluid and their structures and recognition properties have been investigated for over 70 years [ 56 – 60 ]. The carbohydrate chains of HOC consist of multiple saccharide branches attached by O -glycosidic linkages at their internal reducing ends to serine or threonine residues of the polypeptide backbone [ 58 – 60 ]. The over all structures were mapped and constructed shown in Figs. 3 a, b, it has a linear structure of tetra monosaccharides with four branches of one Galβ1→3GlcNAcβ1→, I β , and three Galβ1→4GlcNAcβ1→, II β , to which GalNAcα1→3, LFucα1→2 and/or, LFucα1→3 or 4 key sugars are attached at the appropriate locations to form glycotope A h and/or A h - Le b.y . This linear tetrasaccharide is composed of one unit of Type I precursor unit at the nonreducing end and Type III precursor (Galβ1→3GalNAcα1→ O , T α , Core 1 determinant) at the other end, linked to Ser or Thr of the protein moiety. The Branch 1 , Type I precursor and Branch 2 , Type II precursor are linked to Gal at the nonreducing end of this linear tetra-structure. In Branch 3 , two kinds of linkages between GlcNAc of Branch 3 and crypto Tn glycotope were identified. These are GlcNAcβ 1.6 and GlcNAcβ 1.3 to the carbon-6 of the GalNAc of Tn glycotope at the reducing end. The length of Branch 3 is often increased by adding more saccharides to a combination of Type I and/or Type II chains. The Branch 4 is made up of Type II precursor unit, which in turn is β 1.6 linked to the Gal end of the T α glycotopes (Type III precursor sequence) determinant. Mammalian Salivary O-glycans – All salivary- O -glycans- ovine salivary mucin (OSM), porcine salivary mucin (PSM), bovine sub-mandibular gps (BSM) and armadillo submandibular gp etc. were purified according to the method of Tettamanti and Pigman (1968) and its modifications [ 52 – 55 ]. Bird nest O-glycoprotein – The crude bird nest-cementing substance from the salivary gland of Chinese swiftlets (genus Collocalia ) was extracted with distilled H 2 O at 60°C for 20 min from the commercial bird nest substance (Kim Hing Co., Singapore) [ 61 ]. Ligands used for the inhibition assay – Mono-, di-, and oligosaccharides (structural units/glycotopes) illustrated in Tables 3 /4 were purchased from Sigma (St. Louis, Mo, USA) and Dextra (Reading, Berkshire, UK). Con A and Biotinylation – Concanavalin A (Con A) was purchased from Vector (Burlingame, Ca, USA). Biotinylation of ConA was performed as the procedures described by Duk et al . [ 32 – 35 ], in which the biotinamidocaproate- N -hydroxy-succinimide ester (biotin ester; from Sigma Chemical, St. Louis, Mo., USA), the lectin [200 µg/250 µl phosphate-buffered saline (PBS; 0.14 M NaCl, 0.027 M KCl, 0.081 M Na 2 HPO 4 , 0.0014 M KH 2 PO 4 , pH 8.0)] was mixed with 400 µl of the biotin ester solution (100 µg biotin ester/200 µg lectin) for 30 min at room temperature. The biotinylated lectin was dialyzed for 2–3 h against ddH 2 O and overnight against Tris-buffered saline (TBS; 0.05 M Tris-HCl, 0.15 M NaCl, pH 8.0). After dialysis, the sample volume was adjusted to 1 ml with TBS, and 20 µl of 5% sodium azide was added (yielding a 200 µg/ml solution of Con A in 0.1% NaN 3 ) [ 32 , 33 ]. Enzyme-linked lectin sorbent assay (ELLSA) ELLSA was performed according to the conditions and procedures described by Duk et al. (1994) [ 32 , 33 ]. The volume of each reagent applied to the to the 96-well microtiter plate was 50 µl/well, and all incubations, except for coating, were performed at room temperature (20–25°C). All reagents were diluted with TBS containing 0.05% Tween 20 (TBS-T; 0.05 M Tris-HCl, 0.15 M NaCl, pH 7.35), unless otherwise indicated. Plates were washed with TBS-T between incubations. Inhibition assay of Con A-glycan interactions [ ] As suggested by Duk et al. in 1994 [ 32 , 33 ], serially diluted ligands were mixed with an equal volume of a fixed amount of Con A. The control lectin sample was diluted two-fold with TBS-T. After 1h at room temperature the samples were read in the binding assay, as described above. The inhibitory activity was estimated from the inhibition curve and is expressed as the amount of inhibitor (ng or nmol per well) giving 50% inhibition of the control lectin binding. All experiments were performed in duplicates or triplicates, and the data represent mean values of the results. The standard deviation did not exceed 10% and in most experiments was less than 5% of the mean value. The blank wells, where coating or addition of Con A was omitted, gave low absorbance values (below 0.1) after 2h and 4h incubation with the substrate at room temperature in the dark. It showed that blocking the wells before lectin addition was not necessary when Tween 20 was present in the TBS. Results I. Con A – glycan interactions to define their intensities and avidities (RF-6). In order to understand the roles of the mammalian N -glycans and their glycotopes and structural units in the Con A – glycan interactions, the avidities of Con A for selected glycans were analyzed by the ELLSA. The profiles of the results obtained with N -glycans / O -glycans and polysaccharides are summarized in Table 1 . Among the glycan biomasses tested, Con A reacted strongly with all N -glycans tested (#1 to #12) with binding avidity over 8+ (A 405 > 4.0). It also reacted strongly with yeast mannan and glycogen (#16 and #17), but not with most human blood group ABH active cyst O -glycans and salivary O -glycans tested (#19 to #36). These results were further confirmed by inhibition assay and shown in the next section (II). II. Inhibition of Con A – asialo bovin α 1 -acid gp ( N -glycoprotein) interaction by various N - and O - glycoproteins and natural polysaccharides to demonstrate their intensities (RF-6). The abilities of various gps to inhibit the binding of Con A to asialo bovin α 1 -acid gp (the poly oligomannosyl residues, M glycotope containing N -glycans) were analyzed by inhibition of ELLSA. A summary of the results is given in Table 2 . Among the gps and polysaccharides tested, Yeast mannan is the most potent one, which expressed by Mass R.P., is 1.7⋅10 3 more potent than monomeric Man. All other N -glycoproteins and glycogen tested were also active. But, all O -glycoproteins tested were poor or inactive. III. Inhibition of Con A – N -glycan Interactions by the mammalian N -glycans, glycotopes, structural units and mono/oligosaccharides to establish their Recognition Factors of #1 to 5. The ablilty of various glycan ligands to block the binding of Con A to N -glyco protein (asialo bovine α 1 -acid gp) was analyzed by the inhibition of ELLSA. The amount required for 50% inhibition of the binding of Con A to asialo bovinα 1 -acid gp are listed in Tables 3 and 4 . Among over 40 ligands tested, two Man containing penta-saccharide (#1 and #2 in Table 3 ), expressed by M olar R elative P otency (Molar R.P.), were the best, and 1.0⋅10 3 more potent the monomeric Man. M ( M 3 ), Man α1→3, α1→6, Mannotriose) is also the active one, and about 2/3 as potent as penta-one (#1 and #2). The recognition specificity of Con A towards ligands in decreasing order as follows: α1→3, α1→6 Mannopentaose ( M 5 ) and Biantennary N -linked core pentasaccharide ( M Di ) ≥ α1→3, α1→6 Mannotriose ( M Di ) > Manα1→3 Man (α1→3Mannobiose), Manα1→2 Man (α1→2Mannobiose), Manα1→6 Man (α1→6Mannobiose) > Manα1→4 Man (α1→4Mannobiose) > GlcNAcβ1→2 Man (β1→2 N-Acetyl glucosamine-mannose) > Manα1→/Glcα1→ > Man > > Glc and Gal / GalNAc were inactive. Discussion 1. Enzyme-linked lectinosorbent assay (ELLSA) and inhibition as one of the most useful and powerful tools to establish the glycan code and Recognition Factors of Lectin (GBP)- glycan interactions. Based on practical and economic considerations, the speed and accuracy of the assay, the amount of glycans, lectins, ligands required, and the range of binding intensities, ELLSA should be one of the best approaches to estimate the Recognition Factors involoved in the lectin-glycan interactions and to analyze the recognition sites of the lectins [ 32 – 35 ]. This approach has been applied to our Gal and GalNAc related glycan assay for over two decades and provided another angle to look at the mechanism of lectin-glycan interactions. Because of limitation of the technical approaches and availability of reagents, previous studies on the powers of recognition factors of Con A were restricted to plant/microbial glycans and some of N -glycans. The knowledge of the code system and the contribution of the polyvalent forms of the glycotopes in the mammalian glycans- Con A interactions were still incomplete. In this report, a Manα1→ related code system was constructed and a concept of three different glycan biomasses / six recognition factors of the interaction processes were illustrated (Tables 5 a, b). These two themes should be one of the most valuable contributions in glycoconjugates since 1980s. 2. Establishment of the glycan code for Manα1→ / Glcα1→ specific lectin (Con A)-glycan interactions. The glycan codes for Gal/GalNAc specific lectin-glycan interactions has been defined since early 1990 [ 7 , 31 ]. It is one of the best and easy ways to elucidate the structures and functional roles of Gal/GalNAc related glycan complexes. But, the glycan codes for Manα1→ and Glcα1→ specific lectins and their interactions with mammalian N -glycans have not been well illustrated. Combination of the results of this study and the previous data, a Man related code has been documented. In this code system (Tables 5 a, b), M 3 (M) , Manα1→6(Manα1→3) Mantriose is suggested as an essential and core element for all mammalian N -glycan chains. When they are expanded to various derivatives and/or the complex structures of N -glycans to perform their biological functions, two different Man related codes, as shown in Fig. 5a, was suggested to express by both the number of Man and the number of GlcNAcβ1→ branches, i.e: M : The number of Man present in the N -glycan chain is expressed by the subscript of M with 3 or 5 as M 3 ( M ) or M 5 . Branches from M : The number of GlcNAcα1→ or I β / II β (Galβ1→3/4GlcNAcβ1→) branches present in the N -glycan chain is illustrated by the subscript of Di to Penta of M , i.e M Di to M Penta or more . 3. Expressions of the intensities and avidities of Recognition Factors in the lectins - glycan interactions by the Molar Relative Potency [RFs- (i) to (v)] and the Mass Relative Potency [RF-(vi)]. The Factors involved in the recognition process of the lectin-glycan interactions are defined as R ecognition F actors (RFs) [ 31 ]. During the past 70 years, six of them have been selected and applied to demonstrate the mammalian Gal/GalNAc related glycoconjugate- glycan interactions [ 7 , 31 , 35 , 60 ]. They are: (i) Sub-monosaccharide specificity: anomers and epimers in the pyranose form of monosaccharides; (ii) seven biomedical impotence of monosaccharide: Gal, GalNAc, GlcNAc, Man,Glc, lFuc, and NeuNAc; (iii) Reactivities toward mammalian structural units (di), oligo –saccharides, and Tn glycotope in a decreasing order; (iv) The most active ligand (Glycotope) among the structural units and related oligo glycans; (v) Cluster forms of glycan structural units and glycotopes and/or Tn in glycopeptides; (vi) P olyvalent structural units/glycotopes and their resulting configuration / conformation present in the natural marco-complex carbohydrates. Although RFs of the most (ii)-(iv) [i.e RF-2 to RF-5] are the weak ones, while they are the essential and core elements in the recognition process, which are similar to amino acids peptide sequence in a protein. They have also been used to classify the applied lectins [ 7 , 31 , 35 ]. As the intensities and avidities of the polyvalency of the Gal/GalNAc related glycotopes and their resulting conformational features [RF-(vi)] has been found to be the critical factor for the most mechanism of lectin-glycan interactions. Their intensities can be increased 1.0×10 6 times more than their monomeric status [ 31 , 35 ]. Therefore, the RF-(vi), in the Gal/GalNAc system was coined as super recognition factor for the mechanism of lectin-glycan interactions. In order to demonstrate all possible intensities and powers of Recognition Factors (RFs) involved in the most Manα1→ and Glcα1→ specific-glycan interactions, two kind of relative potencies have been applied to express their powers and intensities. These two are: (a) M olar R ecognition F actors ( M olar R.P. ) for RFs of (i) to (v); (b) M ass R ecognition F actors ( M ass R.P. ) for RFs of (v) and (vi). At the present time, the intensity and avidity of the recognition factor- (vi) has to be expressed by Mass R.P. only. This is due to the number of the glycotope residues and their resulting conformation features in the most of natural N -glycan complexes have not been identified and estimated. It may take a great number of decades or centuries to reach to the Molar level. At the age of the data of RF-6 become available to express by the Molar R.P., it is sure that the actural data should be much higher (stronger) than these of Mass unit illustrated in this report. 4. Classification of three glycan masses and six Recognition Factors for the Con A- natural macro glycan interactions. Con A has been found to have a wide range of recognition capacity among the natural Manα1→, Glcα1→ and GlcNAc containing macro-glycans (glycan biomasses). In this report, three glycan biomasses were selected (Table 5 b). These are: (1) Mammalian N -glycans (Manα1→), (2) Yeast mannan (Manα1→) and (3) Glycogen (Glcα1→4 and/or Glcα1→6). Both yeast mannan and glycogen have their own structural advantages with the poly forms of structural units and/or glycotopes at their terminal ends. While most structural units and/or glycotope in the mammalian N -glycans are in the crypto forms with β1→4 linked to chitin disaccharide (GlcNAcβ1→4GlcNAcβ1→Asn in Fig. 1 ) at reducing end. Thus, their intensities may be affected by shielding (masking) effects in the recognition process. Furthermore, the number of N -glycan chains are much less than these in O -glycoporteins. However, Con A still has an ability to overcome these weakness and to keep as one of the most powerful, useful and economic reagents to detect the mammalian N -glycans [ 1 – 3 ]. 5. Accommodation (Recognition) sizes and sites of Con A for mammalian N -glycan interactions. The loci on a lectin molecule to accommodate the structures of RF-(i) to (iv) [RF-1 to RF-4] is defined as the combining sites of a lectin and glycan, in which the most active structural unit(s), RF-(iv) has been chosen as the glycotope of a lectin. From all the data obtained from the profile of lectin-glycan interactions and inhibitory potencies of the ligands (RFs), the shape and size of recognition site of a lectin can be plotted. As shown in Table 3 , four potential structural units (RF-3) were chosen for Con A- i.e #4, Manα1→3Man (α1, 3Mannobiose); #5, Manα1→2Man (α1, 2Mannobiose); #6, Manα1→6Man (α1, 6Mannobiose); #7, Manα1→4Man (α1, 4Mannobiose). They are 18 to 33 more potent than monomeric Man. Three glycotopes (RF-4) were selected [#1 to #3, i.e M 3 (α1→3, α1→6 Mannotriose); M 5 (α1→3, α1→6 Mannopentaose), and M Di , (Biantennary GlcNAcβ1→4M or Biantennary N -linked core pentasaccharide) in Table 3 ]. They are 6.7×10 2 to 1.0×10 3 more potent than monomeric Man. 6. Construction of Manα1→ code and its Recognition Factors (iii) to (vi) from the recognition intensities and structural profiles of both Con A and Morniga M. Morniga M is a jacalin-related and mannose-specific lectin isolated from the bark of the mulberry (Morus nigra) [ 8 ]. It is the first Manα1→ specific lectin, as shown in Tables 6 a to c, analyzed by our established method- ELLSA / inhibition assay [ 8 ]. It was shown that the binding affinity of Morniga M for ligands, expressed by Mass R.P., can be ranked in decreasing order as follows: mammalian glycoproteins carrying multiple N -glycan chains > > N -glycan chain with a single trimannosyl core, [ M or M 3 , Manα1→6(Manα1→3) Man], Penta-Man oligomer [ M 5 , Manα1→6(Manα1→3)Manα1→6(Manα1→3)Man] ≥ Manα1→2, 3 or 6 Man > Man > GlcNAc/Glc, while Gal, GalNAc and LFuc were inactive. Mapping the interaction profiles of both lectins, it is concluded that (1) both Manα1→ specific lectins can recognize many the cryptic forms of Man related structural units and glycotopes in the N -glycans (Fig. 1 and Tables 5 a, 6 c); (2) the presence of multiple N -glycan chains in the most glycoproteins enhances their activity (Tables 6 a,b); (3) the other mammalian structural units, such as Gal/GalNAc related codes (structural units)and glycotopes were poor or inactive; (4) as the Con A also reacted well with most mammalian N -glycans, it is provided more constructive evidences to establish the concept of the Manα1→ related codes, the structural units, and glycotopes (Tables 5 a/b); (5) The data of Con A reacted well yeast mannan and glycogens (Tables 1 and 6 a) vs Morniga M was inactive [ 8 ] provide an additional evidence to support the concept of most lectins have their own binding characters [ 5 , 6 ]. Conclusion And Highlights The recognition mechanism of Con A has been studied intensively and applied to glycoconjugates and glycobiology for over 80 years. However, the roles and the relationships among mammalian structural units, glycotopes, N -glycan chains, and their polyvalent forms in N -glycoproteins involved in the Con A- glycan interactions have not been well defined and organzied. In this study, they were azalyzed by our well developed method- the enzyme linked lectinosorbent (ELLSA) and inhibition assay. Based on all data obtained, Con A has a relatively broad and wide recognition range for both Manα1→ and Glcα1→ related glycans. It reacted strongly with yeast mannan, and glycogen, and a wide range of mammalian N -glycans, including the rat sublingual gp (RSL), human Tamm-Horsfall glycoprotein (THGP), thyroglobin and lactosferrin. Furthermore, the Man code system, expressed by both numbers of Man and GlcNAcβ1→ branches ( M 3 to M 9 / M Mono to Penta etc .), and a table of three Manα1→ and Glcα1→ related biomasses of six recognition factors involved in the Con A-glycan interactions should be two of the valuable advances in the field of glycosciences since 1980s. Recognition Factors of several more Manα1→ specific lectins are being studied in our lab. Abbreviations Lectin: C(G)BP, Carbohydrate (Glycan) Binding Protein; Con A, Concanavalin A lectin; Morniga-M, Morus nigra lectin; Lentil, Lens culinaris lectin; PSA, Pea ( Pisum sativum ) lectin. Glycoproteins: THGP, Tamm-Horsfall glycoprotein; HOC, cyst gps: glycoproteins isolated from human ovarian cyst fluid; RSL, rat sublingual gp-major; OSM, ovine submandibular gp-major; PSM, porcine salivary mucin-major; BSM, bovine submandibular gp-major. Mammalian monosaccharide: Man, D-manno pyranose; Glc, D-glucopyranose; GlcNAc, N -acetylglucosamine; Gal, D-galactopyranose; GalNAc, N -acetylgalactosamine; GalN, Galactosamine; LFuc, 6-deoxy-L-galactopyranose; Neu5Ac, SA, NeuAc, sialic acid; LacNAc, N -acetyllactosamine. For Man codes, two kinds of abbreviations ( M 3 to 9 and Mono - Penta ) are used to expresses the structural units and the glycotopes in the N -Glycan chains. M 3 / M 5 : The No. of subscript in M an indicates the No. of Man in the structures of the mammalian N -glycans; M or M 3 , Manα1→6(Manα1→3)Man, or α1→3, α1→6 Mannotriose; M 5 , Manα1→6(Manα1→3)Manα1→6(Manα1→3)Man, or α1→3, α1→6 Mannopentaose; ii. M Mono to Tetra : The Mono to Tetra or more of subscript in M an indicates the No. of GlcNAcβ1→ or II b branches in the structures of the mammalian N -glycan chain; The structural units, glycotopes and codes and the structures of human blood group antigens in the mammalian O -glycans. Tn , GalNAca1→Ser/Thr; T , Thomsen-Friedenreich disaccharide; T a , Galb1→3GalNAca1→; A , GalNAcα1→3Gal; A h , GalNAcα1→3(LFucα1→2)Gal (Human blood group A glycotope); B , Galα1→3Gal; B h , Galα1→3(LFucα1→2)Gal (Human blood group B glycotope); H , LFucα1→2Gal (Human blood group O (H) glycotope); h , crypto LFucα1→2Gal; I β , Galβ1→3GlcNAcβ1→, human blood group type I precursor sequence; II β , Galβ1→4GlcNAcβ, human blood group type II precursor sequence; Le a , Lewis a , Galβ1→3(Fucα1→4)GlcNAc; Le b , Lewis b , Fucα1→2Galβ1→3(Fucα1→4)GlcNAc; Le x , Lewis x , Galβ1→4(Fucα1→3)GlcNAc; Le y , Lewis y , Fucα1→2Galβ1→4(Fucα1→3)GlcNAc; sLe a , sialyl Lewis a , NeuAcα2→3Galβ1→3(Fucα1→4)GlcNAc; sLe x , sialyl Lewis x , NeuAcα2→3Galβ1→4(Fucα1→3)GlcNAc; C , GlcNAcβ1→4GlcNAc, chitin disaccharide; C f , GlcNAcβ1→4(LFucα1→6)GlcNAc. RFs: Six Recognition Factors have been selected and defined- RF-1 to RF-6 or RF-(i) to (vi). Molar R.P.: Molar Relative Potency for the avidities and/or intensities of RF-1 to RF-5 or RF-(i) to RF-(v); Mass R.P.: Mass Relative Potency for the avidity and/or intensity of RF-6 or RF-(vi). ELLSA, Enzyme linked lectinosorbent assay; ELLISA, inhibition assay. Others: TBS, Tris-HCl buffered saline; TBS-T, TBS with Tween 20; PBS, phosphate-buffered saline; MMCO, molecular mass cut off. Declarations Acknowledgments This work was supported by CGU research grant, BMRP 008, BMRP 530, and MICCs Forever Fund (in process), Kwei-San, Tao-yuan, Taiwan. The author would like to thanks Dr. Ming-Sung Tsai and Ms. Chien-Ching Wu for their immunochemical analyses, and Ms. Ko ’s typing assistance.\ Authors’ contribution s It is only one author. Funding It has been indicated at the end of our manuscript as this work was supported by CGU research grant, BMRP 008, BMRP 530, and MICCs Forever Fund (in process), Kwei-San, Tao-yuan, Taiwan. Availability of data and materials It has been described in the tables of our manuscript. Ethical approval This article does not contain any studies with human participants or animals performed by the author. Competing interests The author has no competing interests to declare that are relevant to the content of this article. References Goldstein, I.J., Poretz, R.D.: Isolation, physicochemical characterization, and carbohydrate-binding specificity of lectins. In: Liener, I.E., Sharon, N., Goldstein, I.J. (eds.) The lectins: properties, functions, and applications in biology and medicine. Chapter 2, pp. 35–244. Orlando Academic Press (1986) Goldstein, I.J., Winter, H.C., Poretz, R.D.: Plant lectins: tools for the study of complex carbohydrates. In: Montreuil, J., Vliegenthart, J.F.G. and Schachter, H. (eds.) Glycoproteins II . New comprehensive biochemistry, Vol. 29b, Chap. 12, pp. 403–470. Elsevier Science B. V., Amsterdam (1997) Sharon, N., Lis, H.: Specificity and affinity. In: Sharon, N., Lis, H. (eds.) Lectins, pp. 63–103. Kluwer Academic Publishers, Dordrecht, The Netherlands (2003). Chap. 4 Sharon, N., Lis, H.: Lectins as Cell Recognition Molecules. Science. 246 , 227–234 (1989) Wu, A.M., Sugii, S.J., Herp, A.: A Guide for Carbohydrate specificities of Lectins. In: Wu, A.M. (ed.) The Molecular Immunology of Complex Carbohydrates. Adv. Exp. Med. Biol. 228 , the appendix, pp. 817–853. Plenum Press, New York and London (1988) Wu, A.M., Song, S.C., Tsai, M.S., Herp, A.: A guide to the carbohydrate specificities of applied lectins-2 (updated in 2000). In: Wu, A.M. (ed.) The Molecular Immunology of Complex Carbohydrates-2. Adv. Exp. Med. Biol. 491 , pp. 551–585. Kluwer Acadmeic/Plenum Publishers (2001) Wu, A.M., Liu, J.H., Singh, T., Yang, Z.: Recognition Roles of mammalian structural units and polyvalency in lectin-glycan interactions. In: Wu, A.M. (ed.) The Molecular Immunology of Complex Carbohydrates-3 , Chap. 6, Adv. Exp. Med. Biol., 705 : pp. 99–116. Springer New York Dordrecht Heidelberg London (2011) Wu, A.M., Wu, J.H., Singh, T., Chu, K.C., Peumans, W.J., Rougé, P., Van Damme, E.J.M.: A Novel Lectin (Morniga M) from Mulberry ( Morus nigra ) Bark Recognizes Oligomannosyl Residues in N-Glycans. J. Biomed. Sci. 11 , 874–885 (2004) Kaku, H., Goldstein, I.J., Van Damme, E.J.M., Peumans, W.J.: New mannose-specific lectins from garlic ( Allium sativum ) and ramsons ( Allium ursinum ) blubs. Carbohydr. Res. 229 , 347–353 (1992) Van Damme, E.J.M., Smeets, K., Van Leuven, F., Peumans, W.J.: Molecular cloning of mannose-binding lectins from Clivia miniata . Plant. Mol. Biol. 24 , 825–830 (1994) Sybuya, N., Goldstein, I.J., Van Damme, E.J.M., Peumans, W.J.: Binding properties of a mannose-specific lectin from the snowdrop (Galanthus nivalis) bulb. J. Biol. Chem. 263 , 728–734 (1988) Wecksler, M., Levy, A., Jaffé, W.G.: Mitogenic effects of extracts of Canavalia ensiformis and Concanavalina A. Acta Cient. Venezo. 19 (4), 154–156 (1968) Powell, A.E., Leon, M.A.: Reversible interaction of human lymphocytes with the mitogen concanavalin A. Exp. Cell. Res. 62 , 315–325 (1970) Yamashita, U., Shirakawa, F., Nakamura, H.: Production of interleukin 1 by adult T cell leukemia (ATL) cell lines. J. Immunol. 138 (10), 3284–3289 (1987) Ree, H.J.: Lectin histochemistry of malignant tumors. II. Concanavalin A: a new histochemical marker for macrophage-histiocytes in follicular lymphoma. Cancer. 51 , 1639–1646 (1983) Anti-inflammatory: anti-necrotic effects of lectins from Canavalia ensiformis and Canavalia brasiliensis in experimental acute pancreatitis. Glycoconj. J. 39 , 599–608 (2022) Edelman, G.M., Cunningham, B.A., Reeke, G.N., Jr, Becker, J.W., Waxdal, M.J., Wang, J.L.: The covalent and three-dimensional structure of concanavalin A. Proc. Natl. Acad. Sci. U S A. 69 , 2580–2584 (1972) Becker, J.W., Reeke, G.N. Jr., Wang, J.L.: The covalent and three-dimensional structure of concanavalin A. III. Structure of the monomer and its interactions with metals and saccharides. J. Biol. Chem. 250 , 1513–1524 (1975) Barber, B.H., Carver, J.P.: Magnetic resonance studies of concanavalin A: CONFORMATIONAL CHANGES INDUCED BY Ca 2+ and alpha-methyl-D-mannopyranoside. Can. J. 53 , 371–379 (1975) Smith, E.E., Gunja Smith, Z.H., Goldstein, I.J.: Protein-carbohydrate interaction: A turbidimeteric study of the interaction of Concanavalin A with amylopectin and glycogen and some of their enzymic and chemical degradation products. Biochem. J. 107 , 715–724 (1968) Allen, A.K., Desai, N.N., Neuberger, A.: The purification of the glycoprotein lectin from the broad bean (Vicia faba) and a comparison of this properties with lectins of similar specificity. Biochem. J. 155 , 127–135 (1976) Goldstein, I.J., Hollerman, C.E., Smith, E.K.: Protein-carbohydrate interaction II. Inhibition studies on the interaction of concanavalin A with polysaccharides. Biochemistry. 4 , 876–883 (1965) Baenziger, J.U., Fiete, D.: Structural determinants of concanavalin A specificity for oligosaccharides. J. Biol. Chem. 254 , 2400–2407 (1979) Hardman, K.D., Goldstein, I.J.: The Structure and Activity of Concanavalin A. In: Atassi, M.Z. (ed.) Immunochemistry of proteins, vol. 2, pp. 373–416. Plenum Press, New York (1977) Debray, H., Montreuil, J.: Structural basis for the affinity of four insolubilized lectins, with a specifivity for α-d-mannose, towards various glycopeptides with the N-glycosylamine linkage and related oligosaccharides. J. Biosci. 5 , 93–100 (1983) Chervenak, M.C., Toone, E.J.: Calorimetric analysis of the binding of lectins with overlapping carbohydrate-binding ligand specificities. Biochemistry. 34 , 5685–5695 (1995) Mann, D.A., Kanai, M., Maly, D.J., Kiessling, L.L.: Probing low affinity and multivalent interactions with surface plasmon resonance: ligands for Concanavalin A. J. Am. Chem. Soc. 120 , 10575–10582 (1998) Dimick, S.M., Powell, S.C., McMahon, S.A., Moothoo, D.N., Naismith, J.H., Toone, E.J.: On the meaning of affinity: Cluster glycoside effect and Concanavalin A. J. Am. Chem. Soc. 121 , 10286–10296 (1999) Barre, A., Bourne, Y., Van Damme, E.J.M., Peumans, W.J., Rougé, P.: Mannose-binding plant lectins: Different structural scaffolds for a common sugar-recognition process. Biochimie. 83 , 645–651 (2001) Srinivas, O., Mitra, N., Surolia, A., Jayaraman, N.: Photoswitchable cluster glycosides as tools to probe carbohydrate-protein interaction: synthesis and lectin-binding studies of azobenzene containing multivalent sugar ligands. Glycobiology. 15 , 861–873 (2005) Wu, A.M.: Polyvalency of Glycotopes and their conformational features in glycans as the most powerful recognition factors for the glycan-lectin interactions. In: Banerjee, D. K. (ed.) Glycome: the hidden code in biology , Chap. 14, pp. 287–305. Nova Science, New York (2021) Duk, M., Lisowska, E., Wu, J.H., Wu, A.M.: The biotin/avidin-mediated microtiter plate lectin assay with the use of chemically modified glycoprotein ligand. Anal. Biochem. 221 , 266–272 (1994) Lisowska, E., Duk, M., Wu, A.M.: Preparation of biotinylated lectins and application in microtiter plate assays and western blotting. In: Meier, T., Fahrenholz, F. (eds.) A Laboratory Guide to Biotin-Labeling in Biomolecule Analysis, vol. 7, pp. 115–129. Biomethods, Birkhauser Verlag Basel (1996) Wu, A.M., Lisowska, E., Duk, M., Yang, Z.: Lectins as tools in glycoconjugate research. Glycoconj. J. 26 , 899–913 (2009) Wu, A.M., Liu, J.H.: Lectins and ELLSA as powerful tools for Glycoconjugate Recognition Analyses. Glycoconj. J. 36 , 175–183 (2019) Fournier, T., Medjoubi-N, N., Porquet, D.: Alpha-1-acid glycoprotein. Biochim. Biophys. Acta. 1482 , 157–171 (2000) Clemeston, K.J.: α1-Acid glycoprotein (pp.174), Fibrinogen (pp.185) of Chap. 9; Mintreuil, J., Spik, G., Nazurier, J.: Lactotransferrins (lactoferrins) (pp.206) of Chap. 10;, Hughes, R.C.: Lamiinin (pp. 535–540) of Chap. 14. In: Montreuil, J., Vliegenthart, J.F.G., and Schachter, H. (eds) Glycoptoeins II , Vol 29b, Elsevier Science B. V., Amsterdam (1997) Orczyk-Pawilowicz, M., Hirnle, L., Katnik-Prastowska, I.: Alterations of N-glycan branching and expression of sialic acid on amniotic fluid alpha-1-acid glycoprotein derived from second and third trimesters of normal and prolonged pregnancies. Clinica Chimica Acta 367 , 96 – 92 (2006) Fournet, B., Montreuil, J., Strecker, G., Dorland, L., Haverkamp, J., Vliegenthart, J.F.G., Binette, J.P., Schmid, K.: Determination of the primary structures of 16 asialo-carbohydrate units derived from human plasma alpha 1-acid glycoprotein by 360-MHZ 1H NMR spectroscopy and permethylation analysis. Biochemistry. 17 , 5206–5214 (1978) Shiyan, S.D., Bovin, N.V.: Carbohydrate composition and immunomodulatory activity of different glycoforms of alpha1-acid glycoprotein. Glycoconj. J. 14 , 631–638 (1997) Brock, J.H.: The physiology of lactoferrin. Biochem. Cell. Biol. 80 , 1–6 (2002) Hughes, G.J., Reason, A.J., Savoy, L.-A., Jaton, J.-C., Frutiger-Hughes, S.: Carbohydrate moieties in human secretory component. Biochim. Biophys. Acta. 1434 , 86–93 (1999) Farnaud, S., Evans, R.W.: Lactoferrin–a multifunctional protein with antimicrobial properties. Mol. Immunol. 40 , 395–405 (2003) Engel, J.: Laminins and other strange proteins. Biochemistry. 31 , 10643–10651 (1992) Tamm, I., Horsfall, F.L.: Characterization and Separation of an Inhibitor of Viral Hemagglutination Present in Urine. Proc. Soc. Exp. Biol. Med. 74 , 108–114 (1950) Hard, K., Van, Z.G., Moonen, P., Kamerling, J.P., Vliegenthart, F.G.: The Ash-linked carbohydrate chains of human Tamm-Horsfall glycoprotein of one male: Novel sulfated and novel N-acetylgalactosamine-containing N-linked carbohydrate chains. Eur. J. Biochem. 209 , 895–915 (1992) Lehle, L., Tanner, W.: Yeast mannoproteins and cell wall architecture. In: Montreuil, J., Vliegenthart, J.F.G., Schachter, H. (eds.) Glycoproteins, vol. 29a, pp. 476–477. Elsevier Science B. V., Amsterdam (1997). Chap. 7 Yoshida, Y., Naito, E., Mizukoshi, H., Watanabe, Y., Kimura, K., Yokoi, W., Sato, T., Okumura, T., Ito, M., Sawada, H.: Side-Chain Structure of Cell Surface Polysaccharide, Mannan, Affects Hypocholesterolemic Activity of Yeast. J. Agric. Food Chem. 57 , 8003–8009 (2009) Gemmill, T.R., Trimble, R.B.: Overview of N - and O-linked oligosaccharide structures found in various yeast species. Biochim. Biophys. Acta. 1426 , 227–237 (1999) Carbohydrates in Chap. 16, Campbell, M.K., Farrell, S.O. (eds) Biochemistry, 7 th edition. pp. 470–471. Brooks / Cole, Cengage Learning: (2012) Troy, I.I.: F.A.: Polysialylation: from bacteria to brains. Glycobiology. 2 , 5–23, Review (1992) a Jennings, H.J.: Capsular Polysaccharides as Vaccine Candidates. Curr. Top. Microbiol. Immunol. 150 , 97–127 (1990) Tettamanti, G., Pigman, W.: Purification and characterization of bovine and ovine submaxillary mucins. Arch. Biochem. Biophys. 124 , 41–45 (1968) Herp, A., Borelli, C., Wu, A.M.: Biochemistry and lectin binding properties of mammalian salivary mucous glycoproteins, In: Wu, A.M. (ed.) The Molecular Immunology of Complex Carbohydrates. Adv. Exp. Med. Biol. 228 , pp. 395–435. Kluwer Academic Publishers, Plenum Press, New York and London (1988) Wu, A.M., Csako, G., Herp, A.: Structure, biosynthesis, and function of salivary mucins. Mol Cell Biochem. 137 (1), 39–55, Review (1994) Wu, A.M., Herp, A., Khoo, K.H., Yu, S.Y., Yang, Z.: Glycomic mapping of O - and N -linked glycans from major rat sublingual mucin. Glycoconj. J. 25 , 199–212 (2008) Kabat, E.A.: Purification of Blood Group Substances. In: Kabat, E.A. (ed.) Blood Group Substances: Their Chemistry and Immunochemistry, pp. 135–139. Academic Press, New York (1956). Chap. 4 Wu, A.M.: Structural concepts of the blood group A, B, H, Le a , Le b , I and i active glycoproteins purified from human ovarian cyst fluid. In: Wu, A.M. (ed.) The Molecular Immunology of Complex Carbohydrates. Adv. Exp. Med. Biol. 228 , pp. 351–394. Kluwer Academic Publishers, Plenum Press, New York and London (1988) Wu, A.M.: Human Blood Group ABH / Ii , Le a , b , x , y , and Sialyl Le a , x Glycotopes Internal Structures; and Immunochemical Roles of Human Ovarian Cyst Glycoproteins. In: Chap. 3, The Molecular Immunology of Complex Carbohydrates-3 (Wu, A.M. ed.). Adv. Exp. Med. Biol. 705 : 33–52 (2011) Wu, A.M.: Glycan structures and their recognition roles in the human blood group ABH/Ii, Le a, b, x, y and Sialyl Le a,x active cyst glycoproteins. Glycoconj. J. 36 , 495–507 (2019) Wu, A.M.: Loci and motifs of the GalNAcα1→3/ O related glycotopes in the mammalian glycoconjugates and their lectin recognition roles. Glycoconj. J. 39 , 565–710 (2022) Wieruszeski, J.-M., Michalski, J.-C., Montreuil, J., Strecker, G., Peter-Katalinic, J., Egge, H., van Halbeek, H., Mutsaers, J.H.G.M., Vliegenthart, J.F.G.: Structure of the monosialyl oligosaccharides derived from salivary gland mucin glycoproteins of the Chinese swiftlet (genus Collocalia ). J. Biol. Chem. 262 , 6650–6657 (1987) Kamerling, J.P., Vliegenthart, J.F.G.: Hemocyanins, schemes 2 (pp.123–142) of Chap. 6. In: Montreuil, J., Vliegenthart, J.F.G., Schachter, H. (eds.) Glycoptoeins II, vol. 29b. Elsevier Science B. V., Amsterdam (1997) Yamamoto, K., Tsuji, T., Irimura, T., Osawa, T.: The Structure of Carbohydrate Unit B of Porcine Thyroglobulin. Biochem. J. 195 , 701–713 (1981) Tables Tables 1 to 6 are available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files ConATables.doc Cite Share Download PDF Status: Published Journal Publication published 11 Sep, 2023 Read the published version in Glycoconjugate Journal → Version 1 posted Editorial decision: Major revision 05 Mar, 2023 Reviews received at journal 13 Feb, 2023 Reviewers agreed at journal 08 Feb, 2023 Reviewers invited by journal 08 Feb, 2023 Editor assigned by journal 06 Feb, 2023 Submission checks completed at journal 02 Feb, 2023 First submitted to journal 31 Jan, 2023 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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-2533417","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":172791408,"identity":"9fb6c01f-4c6f-458f-ad79-37c8d0032fb6","order_by":0,"name":"Albert M. Wu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA2klEQVRIiWNgGAWjYLCCDwwMPHxQNmMDMToYZwC1sCG0MBPWwswDJIjXwj/tjJm0bY6dDBsD8+PXPAw2shsO8B+TwKdF4naOmXTutmSgw9jMrHkY0ow3HGBmw6uF4XbuNqAWZqAWHjZjHobDiSAtN/DpkAdpsdxWD9Pyn7AWA5AWxm2HQVqYH/MwHCCsxfB2/mfL3m3HediY2cwY5xgkG888zGz+A58WudtpiTd+bqu252dvfvzhTYWdbN/xxscG+LQgADMDMKAMwAziAfMHEhSPglEwCkbBCAIAMHE+DNcauR4AAAAASUVORK5CYII=","orcid":"","institution":"Chang Gung University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Albert","middleName":"M.","lastName":"Wu","suffix":""}],"badges":[],"createdAt":"2023-01-31 10:59:29","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2533417/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2533417/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s10719-023-10129-4","type":"published","date":"2023-09-11T15:01:19+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":32554101,"identity":"eeb4e884-67ec-420b-8206-b5198b0e6a04","added_by":"auto","created_at":"2023-02-06 23:24:58","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":92571,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eProposal Con A glycotopes (RF-4) and their derivatives (RF-5) in four examples of mammalian \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eN\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e-Glycans. \u003c/strong\u003e*The shaded and dotted area are suggested for the glycotopes of the Con A, in which No. of Man and are expressed by script of 3 or 5, \u003cstrong\u003eM\u003c/strong\u003e\u003csub\u003e3\u003c/sub\u003e\u003cstrong\u003e \u003c/strong\u003e(\u003cstrong\u003eM\u003c/strong\u003e) / \u003cstrong\u003eM\u003c/strong\u003e\u003csub\u003e5\u003c/sub\u003e and dashed line closed are Man with No. of GlcNAcβ1→2, 4 and 6 Manα1→ branches (i to iv), in which Two \u003cstrong\u003eM\u003c/strong\u003e\u003csub\u003eMono \u003c/sub\u003ein #1; one \u003cstrong\u003eM\u003c/strong\u003e\u003csub\u003eTetra\u003c/sub\u003e in #2; one \u003cstrong\u003eM\u003c/strong\u003e\u003csub\u003eTri\u003c/sub\u003e in #3 and one \u003cstrong\u003eM\u003c/strong\u003e\u003csub\u003eDi\u003c/sub\u003e in #4. \u003cstrong\u003eC\u003c/strong\u003e, GlcNAcβ1→4GlcNAcβ; \u003cstrong\u003eC\u003c/strong\u003e\u003csub\u003ef\u003c/sub\u003e, GlcNAcβ1→4(LFucα1→6)GlcNAcβ.\u003c/p\u003e","description":"","filename":"ConAFigs1.png","url":"https://assets-eu.researchsquare.com/files/rs-2533417/v1/41a9d89a4cb75214c6eea2ab.png"},{"id":32554099,"identity":"898096f2-96c5-4190-8abf-01b8749534a3","added_by":"auto","created_at":"2023-02-06 23:24:58","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":71198,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eChemical structures of three kinds of polysaccharides- yeast Mannan, glycogen and colominic acid.\u003c/strong\u003e Their recognition intensities are illustrated in Table 1.\u003c/p\u003e","description":"","filename":"ConAFigs2.png","url":"https://assets-eu.researchsquare.com/files/rs-2533417/v1/5724b50ab6088d3c9a784842.png"},{"id":32554534,"identity":"be41d2f6-5c77-4d86-9442-a468b365c989","added_by":"auto","created_at":"2023-02-06 23:32:58","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":181402,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ea The generalized internal structures of carbohydrate chains of blood group active and sialyl glycoproteins, isolated from human ovarian cyst fluid.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(i) The four-branched structures (#1 to #4) shown above represents the internal portion of the carbohydrate moiety of blood group substances, to which the residues responsible for A, B, H, Le\u003csup\u003ea\u003c/sup\u003e, Le\u003csup\u003eb\u003c/sup\u003e, and unknown glycotopes (Le\u003csup\u003ex\u003c/sup\u003e and Le\u003csup\u003ey\u003c/sup\u003e) by the years of 1988 are attached, [58-60].\u0026nbsp; The numbers in parentheses #[1] to #[12] indicate the site of attachment for the human blood group A, B, H, Le\u003csup\u003ea\u003c/sup\u003e, Le\u003csup\u003eb\u003c/sup\u003e, Le\u003csup\u003ex\u003c/sup\u003e and Le\u003csup\u003ey\u003c/sup\u003e determinants (ii).\u0026nbsp; Most of the carbohydrate chains isolated are parts of this structure including short chains of T\u003csub\u003eα\u003c/sub\u003e and \u003cstrong\u003eTn\u003c/strong\u003e determinants.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e(ii) The core structure of a Le\u003csup\u003ea\u003c/sup\u003e, Le\u003csup\u003ex\u003c/sup\u003e, sLe\u003csup\u003ea\u003c/sup\u003e and sLe\u003csup\u003ex\u003c/sup\u003e active glycoprotein isolated from human ovarian cyst fluid (HOC 350) is proposed to be a simple (short) type chain, which is composed of a linear tetra-saccharides of Core 1 extension ,\u0026nbsp;\u0026nbsp; (I\u003csub\u003eβ\u003c/sub\u003e or II\u003csub\u003eβ\u003c/sub\u003e1→3T\u003csub\u003eα\u003c/sub\u003e, i.e. combination of one unit of I\u003csub\u003eβ\u003c/sub\u003e or II\u003csub\u003eβ\u003c/sub\u003e and the T\u003csub\u003eα\u003c/sub\u003e sequence) and a longer Branch 3 with an additional I\u003csub\u003eβ\u003c/sub\u003e/II\u003csub\u003eβ\u003c/sub\u003e unit and Branch 4, with NeuAcα2→3/6 linked to Gal at the nonreducing end and LFucα1→3/4 linked to GlcNAc [58-60].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eb Attachment site #\u003c/strong\u003e[ ] \u003cstrong\u003eof the key sugars to the internal structure of glycan chains of human ovarian cyst \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eO\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e-glycoprotein in Fig. 3a\u003c/strong\u003e [58-60].\u003c/p\u003e","description":"","filename":"ConAFigs3.png","url":"https://assets-eu.researchsquare.com/files/rs-2533417/v1/ebc9b13ba78bbe222556bb73.png"},{"id":32554533,"identity":"8c82ef04-81da-404c-8d1f-d0da8a51c567","added_by":"auto","created_at":"2023-02-06 23:32:58","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":63875,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eChemical structures of four \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eO\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e-glycans in the salivary glycoproteins of mammalian \u003c/strong\u003e[52-55]\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"ConAFigs4.png","url":"https://assets-eu.researchsquare.com/files/rs-2533417/v1/b1ae9194e62b5c54bc7893e2.png"},{"id":43301358,"identity":"e7038710-5c9a-4b4b-84a8-928b668a3324","added_by":"auto","created_at":"2023-09-18 15:10:12","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1606859,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2533417/v1/1ac7c8fe-35a2-4199-ac63-f099aa248634.pdf"},{"id":32554102,"identity":"31f07dcc-1ffb-4fa6-895f-9a3141a82e0f","added_by":"auto","created_at":"2023-02-06 23:24:58","extension":"doc","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":608256,"visible":true,"origin":"","legend":"","description":"","filename":"ConATables.doc","url":"https://assets-eu.researchsquare.com/files/rs-2533417/v1/bacd1a09d6dc6f6a6f4bf2fe.doc"}],"financialInterests":"No competing interests reported.","formattedTitle":"Roles of the structural units, glycotopes / mammalian N-glycans for Con A - Glycan interactions, their codes, and their recognition Factors","fulltext":[{"header":"Introduction","content":"\u003cp\u003eLectins are an important class of proteins or glycoproteins of non-immune origin that bind non-covalently to characteristic carbohydrate structures with specificity or selectivity [\u003cspan additionalcitationids=\"CR2 CR3\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. They have been widely used as tools to probe the structural and functional roles of cell surface carbohydrates and to fractionate soluble or membrane glycoproteins of diverse origins and defined as applied lectins [\u003cspan additionalcitationids=\"CR2 CR3 CR4 CR5 CR6\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Based on lectin-reactive monosaccharides with the highest affinity, the applied lectins have been divided in five specificity groups [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. These are: (i) the mannose/glucose-binding lectins, (ii) the \u003cem\u003eN\u003c/em\u003e-acetylgalactosamine/galactose-binding lectins, (iii) the acetylglucosamine-lectins, (iv) the l-fucose-binding lectins and (v) sialic acid-binding lectins [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. The mannose/glucose-binding lectins comprise a large group of agglutinins present in the family Leguminosae, primarily in seeds. Prominent among these lectins are those from the jack bean (Con A, \u003cem\u003eCanavalia ensiformis\u003c/em\u003e; concanavalin A), the lentil (\u003cem\u003eLens culinaris\u003c/em\u003e), the pea (PSA, \u003cem\u003ePisum sativum\u003c/em\u003e), the fava bean (\u003cem\u003eVicia faba\u003c/em\u003e) and the common vetch (\u003cem\u003eVicia cracca\u003c/em\u003e). All these agglutinins are metalloproteins requiring metal ions e.g. Ca\u003csup\u003e2+\u003c/sup\u003e and/or Mn\u003csup\u003e2+\u003c/sup\u003e for their carbohydrate-binding activity [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. After late 1980s, many other related lectins were continuously reported [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan additionalcitationids=\"CR9 CR10\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], these are including a novel lectin (Morniga M) from Mulberry (\u003cem\u003eMorus nigra\u003c/em\u003e) Bark [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], Garlic (\u003cem\u003eAllium sativum\u003c/em\u003e), ramsons (\u003cem\u003eAllium ursinum\u003c/em\u003e) blubs [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], \u003cem\u003eClivia miniata\u003c/em\u003e [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], and \u003cem\u003eGalanthus nivalis\u003c/em\u003e agglutinins (GNA, Snowdrop bulb) [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eConcanavalin A (Con A) has been recognized as one of the most well established and useful lectins from plant seeds [\u003cspan additionalcitationids=\"CR2 CR3\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Its physical chemical properties and carbohydrate-bnding properties have been intensively documented. It was first isolated crystallized and found to require metal ions for its activity; it was also shown to precipitate glycogen; to agglutinate various erythrocytes; to precipitate many glycoproteins, such as blood group substances / immunoglobulins, and to react with a variety of bacterial and animal cells [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. During the past five decades, it has been applied to initiate cell differentiation and division [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]; to stimulate T-cells to produce IL 1-like factors [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]; and to demonstrate macrophage histiocytes in pathological specimens [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Recently, Con A has been further demonstrated to have a potential to prevent cell death in experimental acute pancreatitis [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. The native Con A is composed of four identical subunits of Mr 2.65\u0026sdot;10\u003csup\u003e4\u003c/sup\u003e daltons each. Each of the four subunit of lectin is a compactly folded, dome-shaped structure [\u003cspan additionalcitationids=\"CR2 CR3\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Each subunit binds Ca\u003csup\u003e+\u0026thinsp;2\u003c/sup\u003e and/or Mn\u003csup\u003e+\u0026thinsp;2\u003c/sup\u003e metal atoms and one saccharide. [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Under pH 5.6, Con A is in a dimer form, the succinylated Con A is one of these dimer forms and has the same glycan specificity. This succinylated Con A is more soluble and stable than these of natural one and can stimulate DNA synthesis in mouse splenocytes [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e], but it loses its ability to precipitate polysaccharides [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Their differences among recognition capacities and ranges are being investingated by our established approaches- ELLSA and inhibition assay.\u003c/p\u003e \u003cp\u003eAlthough the recognition mechanism and factors of many Manα1\u0026rarr; / Glcα1\u0026rarr; specific lectins have been investigated by a great number of methods. These include calorimetric titration, quantitative precipitin assays, surface plasmon resonance, crystal structure etc. [\u003cspan additionalcitationids=\"CR2 CR3\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan additionalcitationids=\"CR21 CR22 CR23 CR24 CR25 CR26 CR27 CR28 CR29\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. But, the concept of the Man related \u003cb\u003ecode\u003c/b\u003e was still ignored by the most investigators. Furthermore, their roles among the structural units, glycotopes and their polyvalency of glycotopes have not clearly described [\u003cspan additionalcitationids=\"CR2 CR3\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. In this study, the recognition roles of Con A were analyzed by our well established method- the enzyme linked lectinosorbent (ELLSA) and inhibition assays [\u003cspan additionalcitationids=\"CR33 CR34\" citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e], using a panel of natural polyvalent glycotopes (glycotope containing glycan masses) as well as an array of mono-, di-, structural units and glycotopes. Based on all the data provided, a Manα1\u0026rarr; related \u003cb\u003ecode\u003c/b\u003e for Con A- mammalian \u003cem\u003eN\u003c/em\u003e-glycoprotein interactions was constructed and documented. Furthermore, a table of three biomasses of six Recognition Factors (RFs) was proposed. These two themes should provide one of most constructive advances in this field since 1980s.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cp\u003e\u003cspan class=\"BoldItalic\"\u003eMammalian N-glycoproteins\u003c/span\u003e\u0026ndash;\u003c/p\u003e\n\u003cp\u003eMost mammalian \u003cem\u003eN\u003c/em\u003e-glycoproteins, shown in the Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e [\u003cspan class=\"CitationRef\"\u003e36\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e44\u003c/span\u003e], \u0026alpha;- acid gp, bovine lactoferrin, porcine thyrogobulin, and laminin etc. were purchased from Sigma.\u003c/p\u003e\n\u003cp\u003eTHGP, Tamm-Horsfall glycoproteins provided by the late Dr. W.M. Watkins (University of London, Royal Postgraduate Medical School, Hammersmith Hospital, London, UK), were isolated with 0.58 \u003cem\u003eM\u003c/em\u003e NaCl from the urine of donors with the Sd (a+) or Sd (a-) blood group by the method of Tamm and Horsfall [\u003cspan class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e46\u003c/span\u003e]. The precipitated material was lyophilized, and its lipid content was removed with 9:1, 2:1, and 1:2 chloroform-methanol treatment and further purified as described.\u003c/p\u003e\n\u003cp\u003e\u003cspan class=\"BoldItalic\"\u003ePolysaccharides\u003c/span\u003e\u0026ndash;\u003c/p\u003e\n\u003cp\u003eYeast mannan [\u003cspan class=\"CitationRef\"\u003e47\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e49\u003c/span\u003e], Glycogen [\u003cspan class=\"CitationRef\"\u003e50\u003c/span\u003e], and colominic acid (poly-2,8-\u003cem\u003eN\u003c/em\u003e-acetylneuraminic acid capsular polysaccharide, [Neu5Ac\u0026alpha;2-8Neu5Ac]\u003csub\u003en\u003c/sub\u003e) from \u003cem\u003eE. coli\u003c/em\u003e [\u003cspan class=\"CitationRef\"\u003e51\u003c/span\u003e] were also ordered from Sigma (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cspan class=\"BoldItalic\"\u003eMammalian N-/O- glycoprotein\u003c/span\u003e\u0026ndash;\u003c/p\u003e\n\u003cp\u003eRSL, Rat sublingual glycoprotein- Rat salivary glands of the adult Sprague\u0026ndash;Dawleys obtained from Pel Freeze Biologicals, Inc., Rogers, AR, was a mixture of sublingual and submandibular glands. The RSL-major was isolated according to a modified method used for the preparation of bovine and ovine submandibular mucin [\u003cspan class=\"CitationRef\"\u003e52\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e54\u003c/span\u003e]. Its \u003cem\u003eN\u003c/em\u003e-glycans of RSL-major were determined to be non-bisected hybrid types similarly carrying a sialylated type II chain (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e) [\u003cspan class=\"CitationRef\"\u003e55\u003c/span\u003e], while the predominant \u003cem\u003eO\u003c/em\u003e-glycans carried on the rat sublingual mucin were defined as sialylated core 3 and 4 types (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cspan class=\"BoldItalic\"\u003eHuman Cyst O-glycoproteins (HOC)\u003c/span\u003e \u0026ndash;\u003c/p\u003e\n\u003cp\u003eHuman blood group ABH active cyst gps (HOC), such as Cyst MSS, Mcdon, Beach, and Tighe etc, were prepared from human ovarian cyst fluid and their structures and recognition properties have been investigated for over 70 years [\u003cspan class=\"CitationRef\"\u003e56\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e60\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003eThe carbohydrate chains of HOC consist of multiple saccharide branches attached by \u003cem\u003eO\u003c/em\u003e-glycosidic linkages at their internal reducing ends to serine or threonine residues of the polypeptide backbone [\u003cspan class=\"CitationRef\"\u003e58\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e60\u003c/span\u003e]. The over all structures were mapped and constructed shown in Figs.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ea, b, it has a linear structure of tetra monosaccharides with four branches of one Gal\u0026beta;1\u0026rarr;3GlcNAc\u0026beta;1\u0026rarr;, \u003cstrong\u003eI\u003c/strong\u003e\u003csub\u003e\u0026beta;\u003c/sub\u003e, and three Gal\u0026beta;1\u0026rarr;4GlcNAc\u0026beta;1\u0026rarr;, \u003cstrong\u003eII\u003c/strong\u003e\u003csub\u003e\u0026beta;\u003c/sub\u003e, to which GalNAc\u0026alpha;1\u0026rarr;3, LFuc\u0026alpha;1\u0026rarr;2 and/or, LFuc\u0026alpha;1\u0026rarr;3 or 4 key sugars are attached at the appropriate locations to form glycotope \u003cstrong\u003eA\u003c/strong\u003e\u003csub\u003eh\u003c/sub\u003e and/or \u003cstrong\u003eA\u003c/strong\u003e\u003csub\u003eh\u003c/sub\u003e-\u003cstrong\u003eLe\u003c/strong\u003e\u003csup\u003eb.y\u003c/sup\u003e. This linear tetrasaccharide is composed of one unit of Type \u003cstrong\u003eI\u003c/strong\u003e precursor unit at the nonreducing end and Type \u003cstrong\u003eIII\u003c/strong\u003e precursor (Gal\u0026beta;1\u0026rarr;3GalNAc\u0026alpha;1\u0026rarr;\u003cem\u003eO\u003c/em\u003e, \u003cstrong\u003eT\u003c/strong\u003e\u003csub\u003e\u0026alpha;\u003c/sub\u003e, Core 1 determinant) at the other end, linked to Ser or Thr of the protein moiety. The Branch \u003cstrong\u003e1\u003c/strong\u003e, Type \u003cstrong\u003eI\u003c/strong\u003e precursor and Branch \u003cstrong\u003e2\u003c/strong\u003e, Type \u003cstrong\u003eII\u003c/strong\u003e precursor are linked to Gal at the nonreducing end of this linear tetra-structure. In Branch \u003cstrong\u003e3\u003c/strong\u003e, two kinds of linkages between GlcNAc of Branch \u003cstrong\u003e3\u003c/strong\u003e and crypto \u003cstrong\u003eTn\u003c/strong\u003e glycotope were identified. These are GlcNAc\u0026beta;\u003csub\u003e1.6\u003c/sub\u003e and GlcNAc\u0026beta;\u003csub\u003e1.3\u003c/sub\u003e to the carbon-6 of the GalNAc of \u003cstrong\u003eTn\u003c/strong\u003e glycotope at the reducing end. The length of Branch \u003cstrong\u003e3\u003c/strong\u003e is often increased by adding more saccharides to a combination of Type \u003cstrong\u003eI\u003c/strong\u003e and/or Type \u003cstrong\u003eII\u003c/strong\u003e chains. The Branch \u003cstrong\u003e4\u003c/strong\u003e is made up of Type \u003cstrong\u003eII\u003c/strong\u003e precursor unit, which in turn is \u0026beta;\u003csub\u003e1.6\u003c/sub\u003e linked to the Gal end of the \u003cstrong\u003eT\u003c/strong\u003e\u003csub\u003e\u0026alpha;\u003c/sub\u003e glycotopes (Type \u003cstrong\u003eIII\u003c/strong\u003e precursor sequence) determinant.\u003c/p\u003e\n\u003cp\u003e\u003cspan class=\"BoldItalic\"\u003eMammalian Salivary O-glycans\u003c/span\u003e\u0026ndash;\u003c/p\u003e\n\u003cp\u003eAll salivary- \u003cem\u003eO\u003c/em\u003e-glycans- ovine salivary mucin (OSM), porcine salivary mucin (PSM), bovine sub-mandibular gps (BSM) and armadillo submandibular gp etc. were purified according to the method of Tettamanti and Pigman (1968) and its modifications [\u003cspan class=\"CitationRef\"\u003e52\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e55\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003e\u003cspan class=\"BoldItalic\"\u003eBird nest O-glycoprotein\u003c/span\u003e\u0026ndash;\u003c/p\u003e\n\u003cp\u003eThe crude bird nest-cementing substance from the salivary gland of Chinese swiftlets (genus \u003cem\u003eCollocalia\u003c/em\u003e) was extracted with distilled H\u003csub\u003e2\u003c/sub\u003eO at 60\u0026deg;C for 20 min from the commercial bird nest substance (Kim Hing Co., Singapore) [\u003cspan class=\"CitationRef\"\u003e61\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003e\u003cspan class=\"BoldItalic\"\u003eLigands used for the inhibition assay\u003c/span\u003e\u0026ndash;\u003c/p\u003e\n\u003cp\u003eMono-, di-, and oligosaccharides (structural units/glycotopes) illustrated in Tables\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e/4 were purchased from Sigma (St. Louis, Mo, USA) and Dextra (Reading, Berkshire, UK).\u003c/p\u003e\n\u003cp\u003e\u003cspan class=\"BoldItalic\"\u003eCon A and Biotinylation\u003c/span\u003e\u0026ndash;\u003c/p\u003e\n\u003cp\u003eConcanavalin A (Con A) was purchased from Vector (Burlingame, Ca, USA). Biotinylation of ConA was performed as the procedures described by Duk \u003cem\u003eet al\u003c/em\u003e. [\u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e35\u003c/span\u003e], in which the biotinamidocaproate-\u003cem\u003eN\u003c/em\u003e-hydroxy-succinimide ester (biotin ester; from Sigma Chemical, St. Louis, Mo., USA), the lectin [200 \u0026micro;g/250 \u0026micro;l phosphate-buffered saline (PBS; 0.14 \u003cem\u003eM\u003c/em\u003e NaCl, 0.027 \u003cem\u003eM\u003c/em\u003e KCl, 0.081 \u003cem\u003eM\u003c/em\u003e Na\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e, 0.0014 \u003cem\u003eM\u003c/em\u003e KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, pH 8.0)] was mixed with 400 \u0026micro;l of the biotin ester solution (100 \u0026micro;g biotin ester/200 \u0026micro;g lectin) for 30 min at room temperature. The biotinylated lectin was dialyzed for 2\u0026ndash;3 h against ddH\u003csub\u003e2\u003c/sub\u003eO and overnight against Tris-buffered saline (TBS; 0.05 \u003cem\u003eM\u003c/em\u003e Tris-HCl, 0.15 \u003cem\u003eM\u003c/em\u003e NaCl, pH 8.0). After dialysis, the sample volume was adjusted to 1 ml with TBS, and 20 \u0026micro;l of 5% sodium azide was added (yielding a 200 \u0026micro;g/ml solution of Con A in 0.1% NaN\u003csub\u003e3\u003c/sub\u003e) [\u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e\n\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n\u003ch2\u003eEnzyme-linked lectin sorbent assay (ELLSA)\u003c/h2\u003e\n\u003cp\u003eELLSA was performed according to the conditions and procedures described by Duk et al. (1994) [\u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e33\u003c/span\u003e]. The volume of each reagent applied to the to the 96-well microtiter plate was 50 \u0026micro;l/well, and all incubations, except for coating, were performed at room temperature (20\u0026ndash;25\u0026deg;C). All reagents were diluted with TBS containing 0.05% Tween 20 (TBS-T; 0.05 M Tris-HCl, 0.15 M NaCl, pH 7.35), unless otherwise indicated. Plates were washed with TBS-T between incubations.\u003c/p\u003e\n\u003cp\u003e\u003cspan class=\"BoldItalic\"\u003eInhibition assay of Con A-glycan interactions\u003c/span\u003e [ ]\u003c/p\u003e\n\u003cp\u003eAs suggested by Duk \u003cem\u003eet al.\u003c/em\u003e in 1994 [\u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e33\u003c/span\u003e], serially diluted ligands were mixed with an equal volume of a fixed amount of Con A. The control lectin sample was diluted two-fold with TBS-T. After 1h at room temperature the samples were read in the binding assay, as described above. The inhibitory activity was estimated from the inhibition curve and is expressed as the amount of inhibitor (ng or nmol per well) giving 50% inhibition of the control lectin binding.\u003c/p\u003e\n\u003cp\u003eAll experiments were performed in duplicates or triplicates, and the data represent mean values of the results. The standard deviation did not exceed 10% and in most experiments was less than 5% of the mean value. The blank wells, where coating or addition of Con A was omitted, gave low absorbance values (below 0.1) after 2h and 4h incubation with the substrate at room temperature in the dark. It showed that blocking the wells before lectin addition was not necessary when Tween 20 was present in the TBS.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eI. Con A \u0026ndash; glycan interactions to define their intensities and avidities (RF-6).\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn order to understand the roles of the mammalian \u003cem\u003eN\u003c/em\u003e-glycans and their glycotopes and structural units in the Con A \u0026ndash; glycan interactions, the avidities of Con A for selected glycans were analyzed by the ELLSA. The profiles of the results obtained with \u003cem\u003eN\u003c/em\u003e-glycans / \u003cem\u003eO\u003c/em\u003e-glycans and polysaccharides are summarized in Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e. Among the glycan biomasses tested, Con A reacted strongly with all \u003cem\u003eN\u003c/em\u003e-glycans tested (#1 to #12) with binding avidity over 8+ (A\u003csub\u003e405\u003c/sub\u003e\u0026thinsp;\u0026gt;\u0026thinsp;4.0). It also reacted strongly with yeast mannan and glycogen (#16 and #17), but not with most human blood group ABH active cyst \u003cem\u003eO\u003c/em\u003e-glycans and salivary \u003cem\u003eO\u003c/em\u003e-glycans tested (#19 to #36). These results were further confirmed by inhibition assay and shown in the next section (II).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eII. Inhibition of Con A \u0026ndash; asialo bovin \u0026alpha;\u003c/strong\u003e \u003csub\u003e \u003cstrong\u003e1\u003c/strong\u003e \u003c/sub\u003e \u003cstrong\u003e-acid gp (\u003c/strong\u003e \u003cspan class=\"BoldItalic\"\u003eN\u003c/span\u003e \u003cstrong\u003e-glycoprotein) interaction by various\u003c/strong\u003e \u003cspan class=\"BoldItalic\"\u003eN\u003c/span\u003e\u003cstrong\u003e- and\u003c/strong\u003e \u003cspan class=\"BoldItalic\"\u003eO\u003c/span\u003e\u003cstrong\u003e- glycoproteins and natural polysaccharides to demonstrate their intensities (RF-6).\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe abilities of various gps to inhibit the binding of Con A to asialo bovin \u0026alpha;\u003csub\u003e1\u003c/sub\u003e-acid gp (the poly oligomannosyl residues, \u003cstrong\u003eM\u003c/strong\u003e glycotope containing \u003cem\u003eN\u003c/em\u003e-glycans) were analyzed by inhibition of ELLSA. A summary of the results is given in Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e. Among the gps and polysaccharides tested, Yeast mannan is the most potent one, which expressed by Mass R.P., is 1.7\u0026sdot;10\u003csup\u003e3\u003c/sup\u003e more potent than monomeric Man. All other \u003cem\u003eN\u003c/em\u003e-glycoproteins and glycogen tested were also active. But, all \u003cem\u003eO\u003c/em\u003e-glycoproteins tested were poor or inactive.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIII. Inhibition of Con A \u0026ndash;\u003c/strong\u003e \u003cspan class=\"BoldItalic\"\u003eN\u003c/span\u003e\u003cstrong\u003e-glycan Interactions by the mammalian\u003c/strong\u003e \u003cspan class=\"BoldItalic\"\u003eN\u003c/span\u003e\u003cstrong\u003e-glycans, glycotopes, structural units and mono/oligosaccharides to establish their Recognition Factors of #1 to 5.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe ablilty of various glycan ligands to block the binding of Con A to \u003cem\u003eN\u003c/em\u003e-glyco protein (asialo bovine \u0026alpha;\u003csub\u003e1\u003c/sub\u003e-acid gp) was analyzed by the inhibition of ELLSA. The amount required for 50% inhibition of the binding of Con A to asialo bovin\u0026alpha;\u003csub\u003e1\u003c/sub\u003e-acid gp are listed in Tables\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e and \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e. Among over 40 ligands tested, two Man containing penta-saccharide (#1 and #2 in Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e), expressed by \u003cstrong\u003eM\u003c/strong\u003eolar \u003cstrong\u003eR\u003c/strong\u003eelative \u003cstrong\u003eP\u003c/strong\u003eotency (Molar R.P.), were the best, and 1.0\u0026sdot;10\u003csup\u003e3\u003c/sup\u003e more potent the monomeric Man. \u003cstrong\u003eM\u003c/strong\u003e (\u003cstrong\u003eM\u003c/strong\u003e\u003csub\u003e3\u003c/sub\u003e), Man \u0026alpha;1\u0026rarr;3, \u0026alpha;1\u0026rarr;6, Mannotriose) is also the active one, and about 2/3 as potent as penta-one (#1 and #2). The recognition specificity of Con A towards ligands in decreasing order as follows: \u0026alpha;1\u0026rarr;3, \u0026alpha;1\u0026rarr;6 Mannopentaose (\u003cstrong\u003eM\u003c/strong\u003e\u003csub\u003e5\u003c/sub\u003e) and Biantennary \u003cem\u003eN\u003c/em\u003e-linked core pentasaccharide (\u003cstrong\u003eM\u003c/strong\u003e\u003csub\u003eDi\u003c/sub\u003e)\u0026thinsp;\u0026ge;\u0026thinsp;\u0026alpha;1\u0026rarr;3, \u0026alpha;1\u0026rarr;6 Mannotriose (\u003cstrong\u003eM\u003c/strong\u003e\u003csub\u003eDi\u003c/sub\u003e)\u0026thinsp;\u0026gt;\u0026thinsp;Man\u0026alpha;1\u0026rarr;3\u003cstrong\u003eMan\u003c/strong\u003e (\u0026alpha;1\u0026rarr;3Mannobiose), Man\u0026alpha;1\u0026rarr;2\u003cstrong\u003eMan\u003c/strong\u003e (\u0026alpha;1\u0026rarr;2Mannobiose), Man\u0026alpha;1\u0026rarr;6\u003cstrong\u003eMan\u003c/strong\u003e (\u0026alpha;1\u0026rarr;6Mannobiose)\u0026thinsp;\u003cstrong\u003e\u0026gt;\u003c/strong\u003e\u0026thinsp;Man\u0026alpha;1\u0026rarr;4\u003cstrong\u003eMan\u003c/strong\u003e (\u0026alpha;1\u0026rarr;4Mannobiose)\u0026thinsp;\u0026gt;\u0026thinsp;GlcNAc\u0026beta;1\u0026rarr;2\u003cstrong\u003eMan\u003c/strong\u003e (\u0026beta;1\u0026rarr;2 N-Acetyl glucosamine-mannose)\u0026thinsp;\u0026gt;\u0026thinsp;Man\u0026alpha;1\u0026rarr;/Glc\u0026alpha;1\u0026rarr; \u0026gt; \u003cstrong\u003eMan\u0026thinsp;\u0026gt;\u0026thinsp;\u0026gt;\u003c/strong\u003e\u0026thinsp;Glc and Gal / GalNAc were inactive.\u003c/p\u003e\n"},{"header":"Discussion","content":"\u003cp\u003e\u003cstrong\u003e1. Enzyme-linked lectinosorbent assay (ELLSA) and inhibition as one of the most useful and powerful tools to establish the glycan code and Recognition Factors of Lectin (GBP)- glycan interactions.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBased on practical and economic considerations, the speed and accuracy of the assay, the amount of glycans, lectins, ligands required, and the range of binding intensities, ELLSA should be one of the best approaches to estimate the Recognition Factors involoved in the lectin-glycan interactions and to analyze the recognition sites of the lectins [\u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e35\u003c/span\u003e]. This approach has been applied to our Gal and GalNAc related glycan assay for over two decades and provided another angle to look at the mechanism of lectin-glycan interactions.\u003c/p\u003e\n\u003cp\u003eBecause of limitation of the technical approaches and availability of reagents, previous studies on the powers of recognition factors of Con A were restricted to plant/microbial glycans and some of \u003cem\u003eN\u003c/em\u003e-glycans. The knowledge of the code system and the contribution of the polyvalent forms of the glycotopes in the mammalian glycans- Con A interactions were still incomplete. In this report, a Man\u0026alpha;1\u0026rarr; related code system was constructed and a concept of three different glycan biomasses / six recognition factors of the interaction processes were illustrated (Tables\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ea, b). These two themes should be one of the most valuable contributions in glycoconjugates since 1980s.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2. Establishment of the glycan code for Man\u0026alpha;1\u0026rarr; / Glc\u0026alpha;1\u0026rarr; specific lectin (Con A)-glycan interactions.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe glycan codes for Gal/GalNAc specific lectin-glycan interactions has been defined since early 1990 [\u003cspan class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e31\u003c/span\u003e]. It is one of the best and easy ways to elucidate the structures and functional roles of Gal/GalNAc related glycan complexes. But, the glycan codes for Man\u0026alpha;1\u0026rarr; and Glc\u0026alpha;1\u0026rarr; specific lectins and their interactions with mammalian \u003cem\u003eN\u003c/em\u003e-glycans have not been well illustrated. Combination of the results of this study and the previous data, a Man related code has been documented. In this code system (Tables\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ea, b), \u003cstrong\u003eM\u003c/strong\u003e\u003csub\u003e3\u003c/sub\u003e \u003cstrong\u003e(M)\u003c/strong\u003e, Man\u0026alpha;1\u0026rarr;6(Man\u0026alpha;1\u0026rarr;3) Mantriose is suggested as an essential and core element for all mammalian \u003cem\u003eN\u003c/em\u003e-glycan chains. When they are expanded to various derivatives and/or the complex structures of \u003cem\u003eN\u003c/em\u003e-glycans to perform their biological functions, two different Man related codes, as shown in Fig.\u0026nbsp;5a, was suggested to express by both the number of Man and the number of GlcNAc\u0026beta;1\u0026rarr; branches, i.e:\u003c/p\u003e\n\u003col\u003e\n\u003cli\u003e\n\u003cp\u003e\u003cstrong\u003eM\u003c/strong\u003e: The number of Man present in the \u003cem\u003eN\u003c/em\u003e-glycan chain is expressed by the subscript of \u003cstrong\u003eM\u003c/strong\u003e with 3 or 5 as \u003cstrong\u003eM\u003c/strong\u003e\u003csub\u003e3\u003c/sub\u003e (\u003cstrong\u003eM\u003c/strong\u003e) or \u003cstrong\u003eM\u003c/strong\u003e\u003csub\u003e5\u003c/sub\u003e.\u003c/p\u003e\n\u003c/li\u003e\n\u003cli\u003e\n\u003cp\u003eBranches from \u003cstrong\u003eM\u003c/strong\u003e: The number of GlcNAc\u0026alpha;1\u0026rarr; or \u003cstrong\u003eI\u003c/strong\u003e\u003csub\u003e\u0026beta;\u003c/sub\u003e/\u003cstrong\u003eII\u003c/strong\u003e\u003csub\u003e\u0026beta;\u003c/sub\u003e (Gal\u0026beta;1\u0026rarr;3/4GlcNAc\u0026beta;1\u0026rarr;) branches present in the \u003cem\u003eN\u003c/em\u003e-glycan chain is illustrated by the subscript of Di to Penta of \u003cstrong\u003eM\u003c/strong\u003e, i.e \u003cstrong\u003eM\u003c/strong\u003e\u003csub\u003eDi\u003c/sub\u003e to \u003cstrong\u003eM\u003c/strong\u003e\u003csub\u003ePenta or more\u003c/sub\u003e.\u003c/p\u003e\n\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003e\u003cstrong\u003e3. Expressions of the intensities and avidities of Recognition Factors in the lectins - glycan interactions by the Molar Relative Potency [RFs- (i) to (v)] and the Mass Relative Potency [RF-(vi)].\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Factors involved in the recognition process of the lectin-glycan interactions are defined as \u003cstrong\u003eR\u003c/strong\u003eecognition \u003cstrong\u003eF\u003c/strong\u003eactors (RFs) [\u003cspan class=\"CitationRef\"\u003e31\u003c/span\u003e]. During the past 70 years, six of them have been selected and applied to demonstrate the mammalian Gal/GalNAc related glycoconjugate- glycan interactions [\u003cspan class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e60\u003c/span\u003e]. They are: (i) Sub-monosaccharide specificity: anomers and epimers in the pyranose form of monosaccharides; (ii) seven biomedical impotence of monosaccharide: Gal, GalNAc, GlcNAc, Man,Glc, lFuc, and NeuNAc; (iii) Reactivities toward mammalian structural units (di), oligo \u0026ndash;saccharides, and \u003cstrong\u003eTn\u003c/strong\u003e glycotope in a decreasing order; (iv) The most active ligand (Glycotope) among the structural units and related oligo glycans; (v) Cluster forms of glycan structural units and glycotopes and/or \u003cstrong\u003eTn\u003c/strong\u003e in glycopeptides; (vi) P\u003cstrong\u003eolyvalent\u003c/strong\u003e structural units/glycotopes and their resulting configuration / conformation present in the natural marco-complex carbohydrates. Although RFs of the most (ii)-(iv) [i.e RF-2 to RF-5] are the weak ones, while they are the essential and core elements in the recognition process, which are similar to amino acids peptide sequence in a protein. They have also been used to classify the applied lectins [\u003cspan class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e35\u003c/span\u003e]. As the intensities and avidities of the polyvalency of the Gal/GalNAc related glycotopes and their resulting conformational features [RF-(vi)] has been found to be the critical factor for the most mechanism of lectin-glycan interactions. Their intensities can be increased 1.0\u0026times;10\u003csup\u003e6\u003c/sup\u003e times more than their monomeric status [\u003cspan class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e35\u003c/span\u003e]. Therefore, the RF-(vi), in the Gal/GalNAc system was coined as \u003cstrong\u003esuper\u003c/strong\u003e recognition factor for the mechanism of lectin-glycan interactions.\u003c/p\u003e\n\u003cp\u003eIn order to demonstrate all possible intensities and powers of Recognition Factors (RFs) involved in the most Man\u0026alpha;1\u0026rarr; and Glc\u0026alpha;1\u0026rarr; specific-glycan interactions, two kind of relative potencies have been applied to express their powers and intensities. These two are: (a) \u003cstrong\u003eM\u003c/strong\u003eolar \u003cstrong\u003eR\u003c/strong\u003eecognition \u003cstrong\u003eF\u003c/strong\u003eactors (\u003cstrong\u003eM\u003c/strong\u003eolar \u003cstrong\u003eR.P.\u003c/strong\u003e) for RFs of (i) to (v); (b) \u003cstrong\u003eM\u003c/strong\u003eass \u003cstrong\u003eR\u003c/strong\u003eecognition \u003cstrong\u003eF\u003c/strong\u003eactors (\u003cstrong\u003eM\u003c/strong\u003eass \u003cstrong\u003eR.P.\u003c/strong\u003e) for RFs of (v) and (vi). At the present time, the intensity and avidity of the recognition factor- (vi) has to be expressed by \u003cstrong\u003eMass\u003c/strong\u003e R.P. only. This is due to the number of the glycotope residues and their resulting conformation features in the most of natural \u003cem\u003eN\u003c/em\u003e-glycan complexes have not been identified and estimated. It may take a great number of decades or centuries to reach to the Molar level. At the age of the data of RF-6 become available to express by the Molar R.P., it is sure that the actural data should be much higher (stronger) than these of Mass unit illustrated in this report.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4. Classification of three glycan masses and six Recognition Factors for the Con A- natural macro glycan interactions.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCon A has been found to have a wide range of recognition capacity among the natural Man\u0026alpha;1\u0026rarr;, Glc\u0026alpha;1\u0026rarr; and GlcNAc containing macro-glycans (glycan biomasses). In this report, three glycan biomasses were selected (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eb). These are: (1) Mammalian \u003cem\u003eN\u003c/em\u003e-glycans (Man\u0026alpha;1\u0026rarr;), (2) Yeast mannan (Man\u0026alpha;1\u0026rarr;) and (3) Glycogen (Glc\u0026alpha;1\u0026rarr;4 and/or Glc\u0026alpha;1\u0026rarr;6). Both yeast mannan and glycogen have their own structural advantages with the poly forms of structural units and/or glycotopes at their \u003cstrong\u003eterminal\u003c/strong\u003e ends. While most structural units and/or glycotope in the mammalian \u003cem\u003eN\u003c/em\u003e-glycans are in the crypto forms with \u0026beta;1\u0026rarr;4 linked to chitin disaccharide (GlcNAc\u0026beta;1\u0026rarr;4GlcNAc\u0026beta;1\u0026rarr;Asn in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e) at reducing end. Thus, their intensities may be affected by shielding (masking) effects in the recognition process. Furthermore, the number of \u003cem\u003eN\u003c/em\u003e-glycan chains are much less than these in \u003cem\u003eO\u003c/em\u003e-glycoporteins. However, Con A still has an ability to overcome these weakness and to keep as one of the most powerful, useful and economic reagents to detect the mammalian \u003cem\u003eN\u003c/em\u003e-glycans [\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e5. Accommodation (Recognition) sizes and sites of Con A for mammalian\u003c/strong\u003e \u003cspan class=\"BoldItalic\"\u003eN\u003c/span\u003e\u003cstrong\u003e-glycan interactions.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe loci on a lectin molecule to accommodate the structures of RF-(i) to (iv) [RF-1 to RF-4] is defined as the combining sites of a lectin and glycan, in which the most active structural unit(s), RF-(iv) has been chosen as the glycotope of a lectin. From all the data obtained from the profile of lectin-glycan interactions and inhibitory potencies of the ligands (RFs), the shape and size of recognition site of a lectin can be plotted. As shown in Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e, four potential structural units (RF-3) were chosen for Con A- i.e #4, Man\u0026alpha;1\u0026rarr;3Man (\u0026alpha;1, 3Mannobiose); #5, Man\u0026alpha;1\u0026rarr;2Man (\u0026alpha;1, 2Mannobiose); #6, Man\u0026alpha;1\u0026rarr;6Man (\u0026alpha;1, 6Mannobiose); #7, Man\u0026alpha;1\u0026rarr;4Man (\u0026alpha;1, 4Mannobiose). They are 18 to 33 more potent than monomeric Man. Three glycotopes (RF-4) were selected [#1 to #3, i.e \u003cstrong\u003eM\u003c/strong\u003e\u003csub\u003e3\u003c/sub\u003e (\u0026alpha;1\u0026rarr;3, \u0026alpha;1\u0026rarr;6 Mannotriose); \u003cstrong\u003eM\u003c/strong\u003e\u003csub\u003e5\u003c/sub\u003e (\u0026alpha;1\u0026rarr;3, \u0026alpha;1\u0026rarr;6 Mannopentaose), and \u003cstrong\u003eM\u003c/strong\u003e\u003csub\u003eDi\u003c/sub\u003e, (Biantennary GlcNAc\u0026beta;1\u0026rarr;4M or Biantennary \u003cem\u003eN\u003c/em\u003e-linked core pentasaccharide) in Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e]. They are 6.7\u0026times;10\u003csup\u003e2\u003c/sup\u003e to 1.0\u0026times;10\u003csup\u003e3\u003c/sup\u003e more potent than monomeric Man.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e6. Construction of Man\u0026alpha;1\u0026rarr; code and its Recognition Factors (iii) to (vi) from the recognition intensities and structural profiles of both Con A and Morniga M.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMorniga M is a jacalin-related and mannose-specific lectin isolated from the bark of the mulberry \u003cem\u003e(Morus nigra)\u003c/em\u003e [\u003cspan class=\"CitationRef\"\u003e8\u003c/span\u003e]. It is the first Man\u0026alpha;1\u0026rarr; specific lectin, as shown in Tables\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ea to c, analyzed by our established method- ELLSA / inhibition assay [\u003cspan class=\"CitationRef\"\u003e8\u003c/span\u003e]. It was shown that the binding affinity of Morniga M for ligands, expressed by Mass R.P., can be ranked in decreasing order as follows: mammalian glycoproteins carrying multiple \u003cem\u003eN\u003c/em\u003e-glycan chains\u0026thinsp;\u0026gt;\u0026thinsp;\u0026gt;\u0026thinsp;\u003cem\u003eN\u003c/em\u003e-glycan chain with a single trimannosyl core, [\u003cstrong\u003eM\u003c/strong\u003e or \u003cstrong\u003eM\u003c/strong\u003e\u003csub\u003e3\u003c/sub\u003e, Man\u0026alpha;1\u0026rarr;6(Man\u0026alpha;1\u0026rarr;3) Man], Penta-Man oligomer [\u003cstrong\u003eM\u003c/strong\u003e\u003csub\u003e5\u003c/sub\u003e, Man\u0026alpha;1\u0026rarr;6(Man\u0026alpha;1\u0026rarr;3)Man\u0026alpha;1\u0026rarr;6(Man\u0026alpha;1\u0026rarr;3)Man]\u0026thinsp;\u0026ge;\u0026thinsp;Man\u0026alpha;1\u0026rarr;2, 3 or 6 Man\u0026thinsp;\u0026gt;\u0026thinsp;Man\u0026thinsp;\u0026gt;\u0026thinsp;GlcNAc/Glc, while Gal, GalNAc and LFuc were inactive. Mapping the interaction profiles of both lectins, it is concluded that (1) both Man\u0026alpha;1\u0026rarr; specific lectins can recognize many the cryptic forms of Man related structural units and glycotopes in the \u003cem\u003eN\u003c/em\u003e-glycans (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e and Tables\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ea, \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ec); (2) the presence of multiple \u003cem\u003eN\u003c/em\u003e-glycan chains in the most glycoproteins enhances their activity (Tables\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ea,b); (3) the other mammalian structural units, such as Gal/GalNAc related codes (structural units)and glycotopes were poor or inactive; (4) as the Con A also reacted well with most mammalian \u003cem\u003eN\u003c/em\u003e-glycans, it is provided more constructive evidences to establish the concept of the Man\u0026alpha;1\u0026rarr; related codes, the structural units, and glycotopes (Tables\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ea/b); (5) The data of Con A reacted well yeast mannan and glycogens (Tables\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e and \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ea) vs Morniga M was inactive [\u003cspan class=\"CitationRef\"\u003e8\u003c/span\u003e] provide an additional evidence to support the concept of most lectins have their own binding characters [\u003cspan class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n\u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n\u003c/div\u003e"},{"header":"Conclusion And Highlights","content":"\u003cp\u003eThe recognition mechanism of Con A has been studied intensively and applied to glycoconjugates and glycobiology for over 80 years. However, the roles and the relationships among mammalian structural units, glycotopes, \u003cem\u003eN\u003c/em\u003e-glycan chains, and their polyvalent forms in \u003cem\u003eN\u003c/em\u003e-glycoproteins involved in the Con A- glycan interactions have not been well defined and organzied. In this study, they were azalyzed by our well developed method- the enzyme linked lectinosorbent (ELLSA) and inhibition assay. Based on all data obtained, Con A has a relatively broad and wide recognition range for both Man\u0026alpha;1\u0026rarr; and Glc\u0026alpha;1\u0026rarr; related glycans. It reacted strongly with yeast mannan, and glycogen, and a wide range of mammalian \u003cem\u003eN\u003c/em\u003e-glycans, including the rat sublingual gp (RSL), human Tamm-Horsfall glycoprotein (THGP), thyroglobin and lactosferrin. Furthermore, the Man code system, expressed by both numbers of Man and GlcNAc\u0026beta;1\u0026rarr; branches (\u003cstrong\u003eM\u003c/strong\u003e\u003csub\u003e3\u003c/sub\u003e to \u003cstrong\u003eM\u003c/strong\u003e\u003csub\u003e9\u003c/sub\u003e / \u003cstrong\u003eM\u003c/strong\u003e\u003csub\u003eMono to Penta etc\u003c/sub\u003e.), and \u003cstrong\u003ea table of three Man\u0026alpha;1\u0026rarr; and Glc\u0026alpha;1\u0026rarr; related biomasses of six recognition factors involved in the Con A-glycan interactions\u003c/strong\u003e should be two of the valuable advances in the field of glycosciences since 1980s. Recognition Factors of several more Man\u0026alpha;1\u0026rarr; specific lectins are being studied in our lab.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003col\u003e\n\u003cli\u003eLectin: C(G)BP, Carbohydrate (Glycan) Binding Protein; Con A, Concanavalin A lectin; Morniga-M, \u003cem\u003eMorus nigra\u003c/em\u003e lectin; Lentil, \u003cem\u003eLens culinaris\u003c/em\u003e lectin; PSA, Pea (\u003cem\u003ePisum sativum\u003c/em\u003e) lectin.\u003c/li\u003e\n\u003cli\u003eGlycoproteins: THGP, Tamm-Horsfall glycoprotein; HOC, cyst gps: glycoproteins isolated from human ovarian cyst fluid; RSL, rat sublingual gp-major; OSM, ovine submandibular gp-major; PSM, porcine salivary mucin-major; BSM, bovine submandibular gp-major.\u003c/li\u003e\n\u003cli\u003eMammalian monosaccharide: Man, D-manno pyranose; Glc, D-glucopyranose; GlcNAc,\u003cem\u003e N\u003c/em\u003e-acetylglucosamine; Gal, D-galactopyranose; GalNAc, \u003cem\u003eN\u003c/em\u003e-acetylgalactosamine; GalN, Galactosamine; LFuc, 6-deoxy-L-galactopyranose; Neu5Ac, SA, NeuAc, sialic acid; LacNAc, \u003cem\u003eN\u003c/em\u003e-acetyllactosamine.\u003c/li\u003e\n\u003cli\u003eFor Man codes, two kinds of abbreviations (\u003cstrong\u003eM\u003c/strong\u003e\u003csub\u003e3 to 9\u003c/sub\u003e and \u003csub\u003eMono - Penta\u003c/sub\u003e) are used to expresses the structural units and the glycotopes in the \u003cem\u003eN\u003c/em\u003e-Glycan chains.\u0026nbsp;\u003cstrong\u003eM\u003c/strong\u003e\u003csub\u003e3 \u003c/sub\u003e/\u003cstrong\u003e M\u003c/strong\u003e\u003csub\u003e5\u003c/sub\u003e: The No. of subscript in \u003cstrong\u003eM\u003c/strong\u003ean indicates the No. of Man in the structures of the mammalian \u003cem\u003eN\u003c/em\u003e-glycans;\u0026nbsp;\u003cstrong\u003eM\u003c/strong\u003e or\u003cstrong\u003e M\u003c/strong\u003e\u003csub\u003e3\u003c/sub\u003e, Man\u0026alpha;1\u0026rarr;6(Man\u0026alpha;1\u0026rarr;3)Man, or \u0026alpha;1\u0026rarr;3, \u0026alpha;1\u0026rarr;6 Mannotriose;\u0026nbsp;\u003cstrong\u003eM\u003c/strong\u003e\u003csub\u003e5\u003c/sub\u003e, Man\u0026alpha;1\u0026rarr;6(Man\u0026alpha;1\u0026rarr;3)Man\u0026alpha;1\u0026rarr;6(Man\u0026alpha;1\u0026rarr;3)Man, or \u0026alpha;1\u0026rarr;3, \u0026alpha;1\u0026rarr;6 Mannopentaose; ii.\u003cstrong\u003e M\u003c/strong\u003e\u003csub\u003eMono to Tetra\u003c/sub\u003e: The Mono to Tetra or more of subscript in \u003cstrong\u003eM\u003c/strong\u003ean indicates the No. of GlcNAc\u0026beta;1\u0026rarr; or \u003cstrong\u003eII\u003c/strong\u003e\u003csub\u003eb\u003c/sub\u003e branches in the structures of the mammalian \u003cem\u003eN\u003c/em\u003e-glycan chain;\u003c/li\u003e\n\u003c/ol\u003e\n\u003col start=\"5\"\u003e\n\u003cli\u003eThe structural units, glycotopes and codes and the structures of human blood group antigens in the mammalian \u003cem\u003eO\u003c/em\u003e-glycans.\u0026nbsp;\u003cstrong\u003eTn\u003c/strong\u003e, GalNAca1\u0026rarr;Ser/Thr;\u0026nbsp;\u003cstrong\u003eT\u003c/strong\u003e, Thomsen-Friedenreich disaccharide; \u003cstrong\u003eT\u003c/strong\u003e\u003csub\u003ea\u003c/sub\u003e, Galb1\u0026rarr;3GalNAca1\u0026rarr;;\u0026nbsp;\u003cstrong\u003eA\u003c/strong\u003e, GalNAc\u0026alpha;1\u0026rarr;3Gal; \u003cstrong\u003eA\u003c/strong\u003e\u003csub\u003eh\u003c/sub\u003e, GalNAc\u0026alpha;1\u0026rarr;3(LFuc\u0026alpha;1\u0026rarr;2)Gal (Human blood group A glycotope);\u0026nbsp;\u003cstrong\u003eB\u003c/strong\u003e, Gal\u0026alpha;1\u0026rarr;3Gal;\u003cstrong\u003e B\u003c/strong\u003e\u003csub\u003eh\u003c/sub\u003e, Gal\u0026alpha;1\u0026rarr;3(LFuc\u0026alpha;1\u0026rarr;2)Gal (Human blood group B glycotope);\u0026nbsp;\u003cstrong\u003eH\u003c/strong\u003e, LFuc\u0026alpha;1\u0026rarr;2Gal (Human blood group O (H) glycotope); \u003cstrong\u003eh\u003c/strong\u003e, crypto LFuc\u0026alpha;1\u0026rarr;2Gal;\u0026nbsp;\u003cstrong\u003eI\u003c/strong\u003e\u003csub\u003e\u0026beta;\u003c/sub\u003e, Gal\u0026beta;1\u0026rarr;3GlcNAc\u0026beta;1\u0026rarr;, human blood group type \u003cstrong\u003eI\u003c/strong\u003e precursor sequence;\u0026nbsp;\u003cstrong\u003eII\u003c/strong\u003e\u003csub\u003e\u0026beta;\u003c/sub\u003e, Gal\u0026beta;1\u0026rarr;4GlcNAc\u0026beta;, human blood group type \u003cstrong\u003eII\u003c/strong\u003e precursor sequence;\u0026nbsp;\u003cstrong\u003eLe\u003csup\u003ea\u003c/sup\u003e\u003c/strong\u003e, Lewis\u003csup\u003ea\u003c/sup\u003e, Gal\u0026beta;1\u0026rarr;3(Fuc\u0026alpha;1\u0026rarr;4)GlcNAc;\u0026nbsp;\u003cstrong\u003eLe\u003csup\u003eb\u003c/sup\u003e\u003c/strong\u003e, Lewis\u003csup\u003eb\u003c/sup\u003e, Fuc\u0026alpha;1\u0026rarr;2Gal\u0026beta;1\u0026rarr;3(Fuc\u0026alpha;1\u0026rarr;4)GlcNAc;\u0026nbsp;\u003cstrong\u003eLe\u003csup\u003ex\u003c/sup\u003e\u003c/strong\u003e, Lewis\u003csup\u003ex\u003c/sup\u003e, Gal\u0026beta;1\u0026rarr;4(Fuc\u0026alpha;1\u0026rarr;3)GlcNAc;\u0026nbsp;\u003cstrong\u003eLe\u003csup\u003ey\u003c/sup\u003e\u003c/strong\u003e, Lewis\u003csup\u003ey\u003c/sup\u003e, Fuc\u0026alpha;1\u0026rarr;2Gal\u0026beta;1\u0026rarr;4(Fuc\u0026alpha;1\u0026rarr;3)GlcNAc;\u0026nbsp;\u003cstrong\u003esLe\u003csup\u003ea\u003c/sup\u003e\u003c/strong\u003e, sialyl Lewis\u003csup\u003ea\u003c/sup\u003e, NeuAc\u0026alpha;2\u0026rarr;3Gal\u0026beta;1\u0026rarr;3(Fuc\u0026alpha;1\u0026rarr;4)GlcNAc;\u0026nbsp;\u003cstrong\u003esLe\u003csup\u003ex\u003c/sup\u003e\u003c/strong\u003e, sialyl Lewis\u003csup\u003ex\u003c/sup\u003e, NeuAc\u0026alpha;2\u0026rarr;3Gal\u0026beta;1\u0026rarr;4(Fuc\u0026alpha;1\u0026rarr;3)GlcNAc;\u0026nbsp;\u003c/li\u003e\n\u003cli\u003e\u003cstrong\u003eC\u003c/strong\u003e, GlcNAc\u0026beta;1\u0026rarr;4GlcNAc, chitin disaccharide;\u0026nbsp;\u003cstrong\u003eC\u003c/strong\u003e\u003csub\u003ef\u003c/sub\u003e, GlcNAc\u0026beta;1\u0026rarr;4(LFuc\u0026alpha;1\u0026rarr;6)GlcNAc.\u003c/li\u003e\n\u003cli\u003eRFs: Six Recognition Factors have been selected and defined- RF-1 to RF-6 or RF-(i) to (vi). Molar R.P.: Molar Relative Potency for the avidities and/or intensities of RF-1 to RF-5 or RF-(i) to RF-(v);\u003cbr /\u003e Mass R.P.: Mass Relative Potency for the avidity and/or intensity of RF-6 or RF-(vi).\u003c/li\u003e\n\u003cli\u003eELLSA, Enzyme linked lectinosorbent assay; ELLISA, inhibition assay.\u003c/li\u003e\n\u003cli\u003eOthers: TBS, Tris-HCl buffered saline; TBS-T, TBS with Tween 20; PBS, phosphate-buffered saline; MMCO, molecular mass cut off.\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by CGU research grant, BMRP 008, BMRP 530, and MICCs Forever Fund (in process), Kwei-San, Tao-yuan, Taiwan.\u003c/p\u003e\n\u003cp\u003eThe author would like to thanks Dr. Ming-Sung Tsai and Ms. Chien-Ching Wu for their immunochemical analyses, and Ms. \u003cstrong\u003eKo\u003c/strong\u003e\u0026rsquo;s typing assistance.\\\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contribution\u003c/strong\u003e\u003cstrong\u003es\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIt is only one author.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIt has been indicated at the end of our manuscript as this work was supported by CGU research grant, BMRP 008, BMRP 530, and MICCs Forever Fund (in process), Kwei-San, Tao-yuan, Taiwan.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIt has been described in the tables of our manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis article does not contain any studies with human participants or animals performed by the author.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe author has no competing interests to declare that are relevant to the content of this article.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eGoldstein, I.J., Poretz, R.D.: Isolation, physicochemical characterization, and carbohydrate-binding specificity of lectins. In: Liener, I.E., Sharon, N., Goldstein, I.J. (eds.) The lectins: properties, functions, and applications in biology and medicine. Chapter 2, pp. 35\u0026ndash;244. Orlando Academic Press (1986)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGoldstein, I.J., Winter, H.C., Poretz, R.D.: Plant lectins: tools for the study of complex carbohydrates. In: Montreuil, J., Vliegenthart, J.F.G. and Schachter, H. (eds.) \u003cem\u003eGlycoproteins II\u003c/em\u003e. New comprehensive biochemistry, Vol. 29b, Chap. 12, pp.\u0026nbsp;403\u0026ndash;470. Elsevier Science B. V., Amsterdam (1997)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSharon, N., Lis, H.: Specificity and affinity. In: Sharon, N., Lis, H. (eds.) Lectins, pp. 63\u0026ndash;103. Kluwer Academic Publishers, Dordrecht, The Netherlands (2003). Chap. 4\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSharon, N., Lis, H.: Lectins as Cell Recognition Molecules. Science. \u003cb\u003e246\u003c/b\u003e, 227\u0026ndash;234 (1989)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWu, A.M., Sugii, S.J., Herp, A.: A Guide for Carbohydrate specificities of Lectins. In: Wu, A.M. (ed.) \u003cem\u003eThe Molecular Immunology of Complex Carbohydrates.\u003c/em\u003e Adv. Exp. Med. Biol. \u003cb\u003e228\u003c/b\u003e, the appendix, pp.\u0026nbsp;817\u0026ndash;853. Plenum Press, New York and London (1988)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWu, A.M., Song, S.C., Tsai, M.S., Herp, A.: A guide to the carbohydrate specificities of applied lectins-2 (updated in 2000). In: Wu, A.M. (ed.) \u003cem\u003eThe Molecular Immunology of Complex Carbohydrates-2.\u003c/em\u003e Adv. Exp. Med. Biol. \u003cb\u003e491\u003c/b\u003e, pp.\u0026nbsp;551\u0026ndash;585. Kluwer Acadmeic/Plenum Publishers (2001)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWu, A.M., Liu, J.H., Singh, T., Yang, Z.: Recognition Roles of mammalian structural units and polyvalency in lectin-glycan interactions. In: Wu, A.M. (ed.) \u003cem\u003eThe Molecular Immunology of Complex Carbohydrates-3\u003c/em\u003e, Chap. 6, Adv. Exp. Med. Biol., \u003cb\u003e705\u003c/b\u003e: pp.\u0026nbsp;99\u0026ndash;116. Springer New York Dordrecht Heidelberg London (2011)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWu, A.M., Wu, J.H., Singh, T., Chu, K.C., Peumans, W.J., Roug\u0026eacute;, P., Van Damme, E.J.M.: A Novel Lectin (Morniga M) from Mulberry (\u003cem\u003eMorus nigra\u003c/em\u003e) Bark Recognizes Oligomannosyl Residues in N-Glycans. J. Biomed. Sci. \u003cb\u003e11\u003c/b\u003e, 874\u0026ndash;885 (2004)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKaku, H., Goldstein, I.J., Van Damme, E.J.M., Peumans, W.J.: New mannose-specific lectins from garlic (\u003cem\u003eAllium sativum\u003c/em\u003e) and ramsons (\u003cem\u003eAllium ursinum\u003c/em\u003e) blubs. Carbohydr. Res. \u003cb\u003e229\u003c/b\u003e, 347\u0026ndash;353 (1992)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVan Damme, E.J.M., Smeets, K., Van Leuven, F., Peumans, W.J.: Molecular cloning of mannose-binding lectins from \u003cem\u003eClivia miniata\u003c/em\u003e. Plant. Mol. Biol. \u003cb\u003e24\u003c/b\u003e, 825\u0026ndash;830 (1994)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSybuya, N., Goldstein, I.J., Van Damme, E.J.M., Peumans, W.J.: Binding properties of a mannose-specific lectin from the snowdrop (Galanthus nivalis) bulb. J. Biol. Chem. \u003cb\u003e263\u003c/b\u003e, 728\u0026ndash;734 (1988)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWecksler, M., Levy, A., Jaff\u0026eacute;, W.G.: Mitogenic effects of extracts of Canavalia ensiformis and Concanavalina A. Acta Cient. Venezo. \u003cb\u003e19\u003c/b\u003e(4), 154\u0026ndash;156 (1968)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePowell, A.E., Leon, M.A.: Reversible interaction of human lymphocytes with the mitogen concanavalin A. Exp. Cell. Res. \u003cb\u003e62\u003c/b\u003e, 315\u0026ndash;325 (1970)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYamashita, U., Shirakawa, F., Nakamura, H.: Production of interleukin 1 by adult T cell leukemia (ATL) cell lines. J. Immunol. \u003cb\u003e138\u003c/b\u003e(10), 3284\u0026ndash;3289 (1987)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRee, H.J.: Lectin histochemistry of malignant tumors. II. Concanavalin A: a new histochemical marker for macrophage-histiocytes in follicular lymphoma. Cancer. \u003cb\u003e51\u003c/b\u003e, 1639\u0026ndash;1646 (1983)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAnti-inflammatory: anti-necrotic effects of lectins from \u003cem\u003eCanavalia ensiformis\u003c/em\u003e and \u003cem\u003eCanavalia brasiliensis\u003c/em\u003e in experimental acute pancreatitis. Glycoconj. J. \u003cb\u003e39\u003c/b\u003e, 599\u0026ndash;608 (2022)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEdelman, G.M., Cunningham, B.A., Reeke, G.N., Jr, Becker, J.W., Waxdal, M.J., Wang, J.L.: The covalent and three-dimensional structure of concanavalin A. Proc. Natl. Acad. Sci. U S A. \u003cb\u003e69\u003c/b\u003e, 2580\u0026ndash;2584 (1972)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBecker, J.W., Reeke, G.N. Jr., Wang, J.L.: The covalent and three-dimensional structure of concanavalin A. III. Structure of the monomer and its interactions with metals and saccharides. J. Biol. Chem. \u003cb\u003e250\u003c/b\u003e, 1513\u0026ndash;1524 (1975)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBarber, B.H., Carver, J.P.: Magnetic resonance studies of concanavalin A: CONFORMATIONAL CHANGES INDUCED BY Ca\u003csup\u003e2+\u003c/sup\u003e and alpha-methyl-D-mannopyranoside. Can. J. \u003cb\u003e53\u003c/b\u003e, 371\u0026ndash;379 (1975)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSmith, E.E., Gunja Smith, Z.H., Goldstein, I.J.: Protein-carbohydrate interaction: A turbidimeteric study of the interaction of Concanavalin A with amylopectin and glycogen and some of their enzymic and chemical degradation products. Biochem. J. \u003cb\u003e107\u003c/b\u003e, 715\u0026ndash;724 (1968)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAllen, A.K., Desai, N.N., Neuberger, A.: The purification of the glycoprotein lectin from the broad bean (Vicia faba) and a comparison of this properties with lectins of similar specificity. Biochem. J. \u003cb\u003e155\u003c/b\u003e, 127\u0026ndash;135 (1976)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGoldstein, I.J., Hollerman, C.E., Smith, E.K.: Protein-carbohydrate interaction II. Inhibition studies on the interaction of concanavalin A with polysaccharides. Biochemistry. \u003cb\u003e4\u003c/b\u003e, 876\u0026ndash;883 (1965)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBaenziger, J.U., Fiete, D.: Structural determinants of concanavalin A specificity for oligosaccharides. J. Biol. Chem. \u003cb\u003e254\u003c/b\u003e, 2400\u0026ndash;2407 (1979)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHardman, K.D., Goldstein, I.J.: The Structure and Activity of Concanavalin A. In: Atassi, M.Z. (ed.) Immunochemistry of proteins, vol. 2, pp. 373\u0026ndash;416. Plenum Press, New York (1977)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDebray, H., Montreuil, J.: Structural basis for the affinity of four insolubilized lectins, with a specifivity for α-d-mannose, towards various glycopeptides with the N-glycosylamine linkage and related oligosaccharides. J. Biosci. \u003cb\u003e5\u003c/b\u003e, 93\u0026ndash;100 (1983)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChervenak, M.C., Toone, E.J.: Calorimetric analysis of the binding of lectins with overlapping carbohydrate-binding ligand specificities. Biochemistry. \u003cb\u003e34\u003c/b\u003e, 5685\u0026ndash;5695 (1995)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMann, D.A., Kanai, M., Maly, D.J., Kiessling, L.L.: Probing low affinity and multivalent interactions with surface plasmon resonance: ligands for Concanavalin A. J. Am. Chem. Soc. \u003cb\u003e120\u003c/b\u003e, 10575\u0026ndash;10582 (1998)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDimick, S.M., Powell, S.C., McMahon, S.A., Moothoo, D.N., Naismith, J.H., Toone, E.J.: On the meaning of affinity: Cluster glycoside effect and Concanavalin A. J. Am. Chem. Soc. \u003cb\u003e121\u003c/b\u003e, 10286\u0026ndash;10296 (1999)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBarre, A., Bourne, Y., Van Damme, E.J.M., Peumans, W.J., Roug\u0026eacute;, P.: Mannose-binding plant lectins: Different structural scaffolds for a common sugar-recognition process. Biochimie. \u003cb\u003e83\u003c/b\u003e, 645\u0026ndash;651 (2001)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSrinivas, O., Mitra, N., Surolia, A., Jayaraman, N.: Photoswitchable cluster glycosides as tools to probe carbohydrate-protein interaction: synthesis and lectin-binding studies of azobenzene containing multivalent sugar ligands. Glycobiology. \u003cb\u003e15\u003c/b\u003e, 861\u0026ndash;873 (2005)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWu, A.M.: Polyvalency of Glycotopes and their conformational features in glycans as the most powerful recognition factors for the glycan-lectin interactions. In: Banerjee, D. K. (ed.) \u003cem\u003eGlycome: the hidden code in biology\u003c/em\u003e, Chap. 14, pp.\u0026nbsp;287\u0026ndash;305. Nova Science, New York (2021)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDuk, M., Lisowska, E., Wu, J.H., Wu, A.M.: The biotin/avidin-mediated microtiter plate lectin assay with the use of chemically modified glycoprotein ligand. Anal. Biochem. \u003cb\u003e221\u003c/b\u003e, 266\u0026ndash;272 (1994)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLisowska, E., Duk, M., Wu, A.M.: Preparation of biotinylated lectins and application in microtiter plate assays and western blotting. In: Meier, T., Fahrenholz, F. (eds.) A Laboratory Guide to Biotin-Labeling in Biomolecule Analysis, vol. 7, pp. 115\u0026ndash;129. Biomethods, Birkhauser Verlag Basel (1996)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWu, A.M., Lisowska, E., Duk, M., Yang, Z.: Lectins as tools in glycoconjugate research. Glycoconj. J. \u003cb\u003e26\u003c/b\u003e, 899\u0026ndash;913 (2009)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWu, A.M., Liu, J.H.: Lectins and ELLSA as powerful tools for Glycoconjugate Recognition Analyses. Glycoconj. J. \u003cb\u003e36\u003c/b\u003e, 175\u0026ndash;183 (2019)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFournier, T., Medjoubi-N, N., Porquet, D.: Alpha-1-acid glycoprotein. Biochim. Biophys. Acta. \u003cb\u003e1482\u003c/b\u003e, 157\u0026ndash;171 (2000)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eClemeston, K.J.: α1-Acid glycoprotein (pp.174), Fibrinogen (pp.185) of Chap. 9; Mintreuil, J., Spik, G., Nazurier, J.: Lactotransferrins (lactoferrins) (pp.206) of Chap. 10;, Hughes, R.C.: Lamiinin (pp.\u0026nbsp;535\u0026ndash;540) of Chap. 14. In: Montreuil, J., Vliegenthart, J.F.G., and Schachter, H. (eds) \u003cem\u003eGlycoptoeins II\u003c/em\u003e, Vol 29b, Elsevier Science B. V., Amsterdam (1997)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOrczyk-Pawilowicz, M., Hirnle, L., Katnik-Prastowska, I.: Alterations of N-glycan branching and expression of sialic acid on amniotic fluid alpha-1-acid glycoprotein derived from second and third trimesters of normal and prolonged pregnancies. \u003cem\u003eClinica Chimica Acta\u003c/em\u003e \u003cb\u003e367\u003c/b\u003e, 96 \u0026ndash; 92 (2006)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFournet, B., Montreuil, J., Strecker, G., Dorland, L., Haverkamp, J., Vliegenthart, J.F.G., Binette, J.P., Schmid, K.: Determination of the primary structures of 16 asialo-carbohydrate units derived from human plasma alpha 1-acid glycoprotein by 360-MHZ 1H NMR spectroscopy and permethylation analysis. Biochemistry. \u003cb\u003e17\u003c/b\u003e, 5206\u0026ndash;5214 (1978)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShiyan, S.D., Bovin, N.V.: Carbohydrate composition and immunomodulatory activity of different glycoforms of alpha1-acid glycoprotein. Glycoconj. J. \u003cb\u003e14\u003c/b\u003e, 631\u0026ndash;638 (1997)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBrock, J.H.: The physiology of lactoferrin. Biochem. Cell. Biol. \u003cb\u003e80\u003c/b\u003e, 1\u0026ndash;6 (2002)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHughes, G.J., Reason, A.J., Savoy, L.-A., Jaton, J.-C., Frutiger-Hughes, S.: Carbohydrate moieties in human secretory component. Biochim. Biophys. Acta. \u003cb\u003e1434\u003c/b\u003e, 86\u0026ndash;93 (1999)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFarnaud, S., Evans, R.W.: Lactoferrin\u0026ndash;a multifunctional protein with antimicrobial properties. Mol. Immunol. \u003cb\u003e40\u003c/b\u003e, 395\u0026ndash;405 (2003)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEngel, J.: Laminins and other strange proteins. Biochemistry. \u003cb\u003e31\u003c/b\u003e, 10643\u0026ndash;10651 (1992)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTamm, I., Horsfall, F.L.: Characterization and Separation of an Inhibitor of Viral Hemagglutination Present in Urine. Proc. Soc. Exp. Biol. Med. \u003cb\u003e74\u003c/b\u003e, 108\u0026ndash;114 (1950)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHard, K., Van, Z.G., Moonen, P., Kamerling, J.P., Vliegenthart, F.G.: The Ash-linked carbohydrate chains of human Tamm-Horsfall glycoprotein of one male: Novel sulfated and novel N-acetylgalactosamine-containing N-linked carbohydrate chains. Eur. J. Biochem. \u003cb\u003e209\u003c/b\u003e, 895\u0026ndash;915 (1992)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLehle, L., Tanner, W.: Yeast mannoproteins and cell wall architecture. In: Montreuil, J., Vliegenthart, J.F.G., Schachter, H. (eds.) Glycoproteins, vol. 29a, pp. 476\u0026ndash;477. Elsevier Science B. V., Amsterdam (1997). Chap. 7\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYoshida, Y., Naito, E., Mizukoshi, H., Watanabe, Y., Kimura, K., Yokoi, W., Sato, T., Okumura, T., Ito, M., Sawada, H.: Side-Chain Structure of Cell Surface Polysaccharide, Mannan, Affects Hypocholesterolemic Activity of Yeast. J. Agric. Food Chem. \u003cb\u003e57\u003c/b\u003e, 8003\u0026ndash;8009 (2009)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGemmill, T.R., Trimble, R.B.: Overview of \u003cb\u003eN\u003c/b\u003e- and O-linked oligosaccharide structures found in various yeast species. Biochim. Biophys. Acta. \u003cb\u003e1426\u003c/b\u003e, 227\u0026ndash;237 (1999)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCarbohydrates in Chap. 16, Campbell, M.K., Farrell, S.O. (eds) Biochemistry, 7 th edition. pp.\u0026nbsp;470\u0026ndash;471. Brooks / Cole, Cengage Learning: (2012)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTroy, I.I.: F.A.: Polysialylation: from bacteria to brains. Glycobiology. \u003cb\u003e2\u003c/b\u003e, 5\u0026ndash;23, Review (1992)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ea Jennings, H.J.: Capsular Polysaccharides as Vaccine Candidates. Curr. Top. Microbiol. Immunol. \u003cb\u003e150\u003c/b\u003e, 97\u0026ndash;127 (1990)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTettamanti, G., Pigman, W.: Purification and characterization of bovine and ovine submaxillary mucins. Arch. Biochem. Biophys. \u003cb\u003e124\u003c/b\u003e, 41\u0026ndash;45 (1968)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHerp, A., Borelli, C., Wu, A.M.: Biochemistry and lectin binding properties of mammalian salivary mucous glycoproteins, In: Wu, A.M. (ed.) \u003cem\u003eThe Molecular Immunology of Complex Carbohydrates.\u003c/em\u003e Adv. Exp. Med. Biol. \u003cb\u003e228\u003c/b\u003e, pp.\u0026nbsp;395\u0026ndash;435. Kluwer Academic Publishers, Plenum Press, New York and London (1988)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWu, A.M., Csako, G., Herp, A.: Structure, biosynthesis, and function of salivary mucins. Mol Cell Biochem. \u003cb\u003e137\u003c/b\u003e(1), 39\u0026ndash;55, Review (1994)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWu, A.M., Herp, A., Khoo, K.H., Yu, S.Y., Yang, Z.: Glycomic mapping of \u003cem\u003eO\u003c/em\u003e- and \u003cem\u003eN\u003c/em\u003e-linked glycans from major rat sublingual mucin. Glycoconj. J. \u003cb\u003e25\u003c/b\u003e, 199\u0026ndash;212 (2008)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKabat, E.A.: Purification of Blood Group Substances. In: Kabat, E.A. (ed.) Blood Group Substances: Their Chemistry and Immunochemistry, pp. 135\u0026ndash;139. Academic Press, New York (1956). Chap. 4\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWu, A.M.: Structural concepts of the blood group A, B, H, Le\u003csup\u003ea\u003c/sup\u003e, Le\u003csup\u003eb\u003c/sup\u003e, I and i active glycoproteins purified from human ovarian cyst fluid. In: Wu, A.M. (ed.) \u003cem\u003eThe Molecular Immunology of Complex Carbohydrates.\u003c/em\u003e Adv. Exp. Med. Biol. \u003cb\u003e228\u003c/b\u003e, pp.\u0026nbsp;351\u0026ndash;394. Kluwer Academic Publishers, Plenum Press, New York and London (1988)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWu, A.M.: Human Blood Group \u003cb\u003eABH\u003c/b\u003e/\u003cb\u003eIi\u003c/b\u003e, \u003cb\u003eLe\u003c/b\u003e\u003csup\u003e\u003cb\u003ea\u003c/b\u003e,\u003cb\u003eb\u003c/b\u003e,\u003cb\u003ex\u003c/b\u003e,\u003cb\u003ey\u003c/b\u003e\u003c/sup\u003e, and \u003cb\u003eSialyl Le\u003c/b\u003e\u003csup\u003e\u003cb\u003ea\u003c/b\u003e,\u003cb\u003ex\u003c/b\u003e\u003c/sup\u003e Glycotopes Internal Structures; and Immunochemical Roles of Human Ovarian Cyst Glycoproteins. In: Chap. 3, \u003cem\u003eThe Molecular Immunology of Complex Carbohydrates-3\u003c/em\u003e (Wu, A.M. ed.). \u003cem\u003eAdv. Exp. Med. Biol.\u003c/em\u003e \u003cb\u003e705\u003c/b\u003e: 33\u0026ndash;52 (2011)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWu, A.M.: Glycan structures and their recognition roles in the human blood group \u003cb\u003eABH/Ii, Le\u003c/b\u003e\u003csup\u003e\u003cb\u003ea, b, x, y\u003c/b\u003e\u003c/sup\u003e and \u003cb\u003eSialyl Le\u003c/b\u003e\u003csup\u003e\u003cb\u003ea,x\u003c/b\u003e\u003c/sup\u003e active cyst glycoproteins. Glycoconj. J. \u003cb\u003e36\u003c/b\u003e, 495\u0026ndash;507 (2019)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWu, A.M.: Loci and motifs of the GalNAcα1\u0026rarr;3/\u003cem\u003eO\u003c/em\u003e related glycotopes in the mammalian glycoconjugates and their lectin recognition roles. Glycoconj. J. \u003cb\u003e39\u003c/b\u003e, 565\u0026ndash;710 (2022)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWieruszeski, J.-M., Michalski, J.-C., Montreuil, J., Strecker, G., Peter-Katalinic, J., Egge, H., van Halbeek, H., Mutsaers, J.H.G.M., Vliegenthart, J.F.G.: Structure of the monosialyl oligosaccharides derived from salivary gland mucin glycoproteins of the Chinese swiftlet (genus \u003cem\u003eCollocalia\u003c/em\u003e). J. Biol. Chem. \u003cb\u003e262\u003c/b\u003e, 6650\u0026ndash;6657 (1987)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKamerling, J.P., Vliegenthart, J.F.G.: Hemocyanins, schemes 2 (pp.123\u0026ndash;142) of Chap. 6. In: Montreuil, J., Vliegenthart, J.F.G., Schachter, H. (eds.) Glycoptoeins II, vol. 29b. Elsevier Science B. V., Amsterdam (1997)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYamamoto, K., Tsuji, T., Irimura, T., Osawa, T.: The Structure of Carbohydrate Unit B of Porcine Thyroglobulin. Biochem. J. \u003cb\u003e195\u003c/b\u003e, 701\u0026ndash;713 (1981)\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables 1 to 6 are available in the Supplementary Files section.\u003c/p\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":"glycoconjugate-journal","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"glyc","sideBox":"Learn more about [Glycoconjugate Journal](http://link.springer.com/journal/10719)","snPcode":"10719","submissionUrl":"https://submission.nature.com/new-submission/10719/3","title":"Glycoconjugate Journal","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Con A, Plant lectins, Glycoprotein binding proteins, Carbohydrate specificities, Recognition Factors, Man structural units, glycotopes, N-Glycan, and Man / N-Glycan Codes.","lastPublishedDoi":"10.21203/rs.3.rs-2533417/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2533417/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe binding property of Con A has been studied intensively and applied widely to glycoconjugates / glycobiology for over 80 years. \u0026nbsp;However, its role and functional relationship of Con A with these mammalian structural units, glycotopes, \u003cem\u003eN\u003c/em\u003e-glycan chains, as well as their polyvalent forms in \u003cem\u003eN\u003c/em\u003e-glycoproteins involved in the Con A- glycan interactions have not been all defined and organzied.\u0026nbsp; In this study, the recognition factors involved in these inteactions were azalyzed by our well developed method- the enzyme linked lectinosorbent (ELLSA) and inhibition assay. \u0026nbsp;Based on all data obtained, it is concluded that Con A, as previous report, has a relatively broad and wide recognition ability with the Manα1→ and Glcα1→ related glycans. \u0026nbsp;In addition to it reacted strongly with yeast mannan and glycogens, it also bound well with a large number of mammalian \u003cem\u003eN\u003c/em\u003e-glycans, including the \u003cem\u003eN\u003c/em\u003e-glycans of rat sublingual gp (RSL), human Tamm-Horsfall glycoprotein (THGP), thyroglobin and lactosferrin.\u0026nbsp; The recognition specificity of Con A towards ligands, expressed by \u003cstrong\u003eM\u003c/strong\u003eolar \u003cstrong\u003eR\u003c/strong\u003eelative \u003cstrong\u003eP\u003c/strong\u003eotency (Molar R.P.), in a decreasing order is as follows: a1→3, a1→6 Mannopentaose (\u003cstrong\u003eM\u003c/strong\u003e\u003csub\u003e5\u003c/sub\u003e)\u003cstrong\u003e \u003c/strong\u003eand\u003cstrong\u003e \u003c/strong\u003eBiantennary \u003cem\u003eN\u003c/em\u003e-linked core pentasaccharide (\u003cstrong\u003eM\u003c/strong\u003e\u003csub\u003eDi\u003c/sub\u003e) ≥ a1→3, a1→6 Mannotriose (\u003cstrong\u003eM\u003c/strong\u003e\u003csub\u003e3\u003c/sub\u003e)\u003cstrong\u003e \u003c/strong\u003e\u0026gt; Mana1→3Man (α1→3Mannobiose), Mana1→2Man (α1→2Mannobiose), Mana1→6Man (α1→6Mannobiose), Mana1→4Man (α1→4Mannobiose) \u0026gt; GlcNAcb1→2\u003cstrong\u003eMan\u003c/strong\u003e (b1→2 N-Acetyl glucosamine-mannose) \u0026gt; Mana1→/Glcα1→ \u0026gt; \u003cstrong\u003eMan\u003c/strong\u003e \u003cstrong\u003e\u0026gt;\u003c/strong\u003e Glc,\u003cstrong\u003e \u003c/strong\u003ewhile Gal / GalNAc were inactive.\u0026nbsp; Furthermore, the Man related code system, in this study, is proposed to express by both numbers of Man and GlcNAcb1→ branches (\u003cstrong\u003eM\u003c/strong\u003e\u003csub\u003e3\u003c/sub\u003e to \u003cstrong\u003eM\u003c/strong\u003e\u003csub\u003e9\u003c/sub\u003e / \u003cstrong\u003eM\u003c/strong\u003e\u003csub\u003eMono to Penta etc.\u003c/sub\u003e) and \u003cstrong\u003ea table of three Mana1→ and Glca1→ related biomasses of six recognition factors involved in the Con A-glycan interactions\u003c/strong\u003e has also been demonstrated.\u0026nbsp; These themes should be one of the most valuable advances since 1980s.\u003c/p\u003e","manuscriptTitle":"Roles of the structural units, glycotopes / mammalian N-glycans for Con A - Glycan interactions, their codes, and their recognition Factors","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-02-06 23:24:52","doi":"10.21203/rs.3.rs-2533417/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2023-03-05T10:38:37+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2023-02-13T10:38:39+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"ca4bc962-0f50-40c3-aa1c-ab87a5472c26","date":"2023-02-08T20:55:37+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2023-02-08T20:50:07+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2023-02-06T14:26:12+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2023-02-02T12:54:34+00:00","index":"","fulltext":""},{"type":"submitted","content":"Glycoconjugate Journal","date":"2023-01-31T10:58:20+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"glycoconjugate-journal","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"glyc","sideBox":"Learn more about [Glycoconjugate Journal](http://link.springer.com/journal/10719)","snPcode":"10719","submissionUrl":"https://submission.nature.com/new-submission/10719/3","title":"Glycoconjugate Journal","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"7e0db10f-719e-4b3d-a889-80d9586d44ae","owner":[],"postedDate":"February 6th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2023-09-18T15:07:40+00:00","versionOfRecord":{"articleIdentity":"rs-2533417","link":"https://doi.org/10.1007/s10719-023-10129-4","journal":{"identity":"glycoconjugate-journal","isVorOnly":false,"title":"Glycoconjugate Journal"},"publishedOn":"2023-09-11 15:01:19","publishedOnDateReadable":"September 11th, 2023"},"versionCreatedAt":"2023-02-06 23:24:52","video":"","vorDoi":"10.1007/s10719-023-10129-4","vorDoiUrl":"https://doi.org/10.1007/s10719-023-10129-4","workflowStages":[]},"version":"v1","identity":"rs-2533417","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2533417","identity":"rs-2533417","version":["v1"]},"buildId":"rHA-KDH7Qsr4HCuvH75dn","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}