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

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 well defined and organized. In this study, the recognition factors involved in these interactions were analyzed by our well developed method- the enzyme linked lectinosorbent (ELLSA) and inhibition assay. Based on all the data obtained, it is concluded that Con A, as previously reported, has a relatively broad and wide recognition ability of the Manα1 → and Glcα1 → related glycans. It reacted not only strongly with yeast mannan and glycogens, but 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), thyroglobulin and lactoferrin. The recognition specificity of Con A towards ligands, expressed by Molar Relative Potency (Molar R.P.), in a decreasing order is as follows: α1 → 3, α1 → 6 Mannopentaose (M5) and Biantennary N-linked core pentasaccharide (MDi) ≥ α1 → 3, α1 → 6 Mannotriose (M3) > Manα1 → 3Man (α1 → 3Mannobiose), Manα1 → 2Man (α1 → 2Mannobiose), Manα1 → 6Man (α1 → 6Mannobiose), Manα1 → 4Man (α1 → 4Mannobiose) > GlcNAcβ1 → 2Man (β1 → 2 N-Acetyl glucosamine-mannose) > Manα1 → /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 GlcNAcβ1 → branches (M3 to M9 / MMono to Penta etc.) and a table of three Manα1 → and Glcα1 → 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.


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][2][3][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][2][3][4][5][6][7].Based on lectinreactive 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/glucosebinding 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][2][3]5].After late 1980s, many other related lectins were continuously reported [6,[8][9][10][11], these are including a novel lectin (Morniga M) from Mulberry (Morus nigra) Bark [8], Garlic (Allium sativum), ramsons (Allium ursinum) bulbs [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][2][3][4].Its physicochemical and carbohydratebinding properties have been intensively documented.It was first isolated and 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][2][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, domeshaped structure [1][2][3][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 investigated by our established approaches-ELLSA and inhibition assay.
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][2][3][4][20][21][22][23][24][25][26][27][28][29][30].However, the concept of the Man related code has not been addressed by the most investigators.Furthermore, their roles among the structural units, glycotopes and their polyvalency of glycotopes have not been clearly described [1-4, 24, 31].In this study, the recognition roles of mechanism and factors of Con A were analyzed by our well established methodthe enzyme linked lectinosorbent (ELLSA) and inhibition assays [32][33][34][35], using a panel of natural polyvalent glycotopes (glycotope containing glycan masses) as well as an array of mono-, di-, structural units, glycotopes, and oligoglycotopes.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.

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][53][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).

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].

Enzyme-linked lectin sorbent assay (ELLSA)
ELLSA was performed according to the conditions and procedures described by Duk et al. [32,33].The volume of each reagent applied 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 1 h 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 2 h and 4 h 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.

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 "Inhibition of Con A -asialo bovin α 1 -acid gp (N-glycoprotein) interaction by various Nand O-glycoproteins and natural polysaccharides to demonstrate their intensities (RF-6)".

Inhibition of Con A -asialo bovin α 1 -acid gp (N-glycoprotein) interaction by various N-and Oglycoproteins 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 are expressed by Mass R.P., is 1.7 × 10 3 more potent than monomeric Man.All Yeast Mannan [47][48][49] Mannan isolated from yeast Saccharomyces cerevisiae, consists mostly of Man with its backbone of α1,6 glycan, side chains of α1,2 and α1,3 glycan, and exhibits presence of Man-phosphate in the side chain of this specific yeast species.The outer chain structure of mannan is demonstrated as below: PO4 -: phosphate ii.Glycogen [50]   Glcα1→4Glc, (Glcα1→4)1 to 9 at terminal end A highly branched unit (Glcα1→6Glc), about one every 10 residues.When the size of n is small, the intensities of recognition Factor of (RF-6) are poor or inactive in interactions.
[ Neu5Ac  (ii) In the years of early 2000s, sialyl Le x , sialyl Le a , Le x and Le y glycotopes of sialyl cyst glycoproteins were defined and characterized [5].other N-glycoproteins and glycogen tested were also active.But, all O-glycoproteins tested were poor or inactive.
a The inhibitory activity was estimated from the inhibition curves and is expressed as the nanogram amount of inhibitor required to produce 50% inhibition of the lectin binding to asialo bovine α 1 -acid coated on microtiter plates; Total volume, 50 μl b To express by Mass Relative Potency of Con A, in which monomeric Man (#14 in Table 2) was taken as 1.0 c More suggested structural units are illustrated in Fig. 2 and Table 6 a Amount of various glycan ligands giving 50% inhibition of Con A (5 ng/50 μl) − asialo bovine α 1 -acid (50 ng/50 μl).The inhibitory activity was estimated from the inhibition curves and is expressed as the nanomole amount of inhibitor required for 50% inhibition; Total volume 50 μl; b For molar relative potency of Con A (Molar R.P.) of Man (#14) was taken as 1.0 b #1 to #3 are selected as glycotopes, and #4 to #7 as structural units (Man codes) and #4 / #6 in bold are important ones for Con A toward mammalian glycoconjugates

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][33][34][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 and 6).These two themes should be one of the most valuable contributions in glycoconjugates since 1980s.

Expressions of the intensities and avidities of recognition factors in the lectin-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 lectinglycan interactions are defined as Recognition Factors (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 monosaccharides of biomedical importance: 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) Polyvalent structural units/glycotopes and their resulting configuration / conformation present in the natural marco-complex carbohydrates.Although RFs of the most (ii)-(v) [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) Molar Recognition Factors (Molar R.P.) for RFs of (i) to (v); (b) Mass Recognition Factors (Mass 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 be expressed by the Molar M, M 5 , and

M
Mono to Tetra are the glycan codes for Mammalian N-glycans and yeast Mannan.Some of their structures are shown in Table 5 Table 6 (continued)

proteins)
Combination of various Man glycotope derivatires (RF-5) to form yeast mannan as Fig. 2(i) [47].Polyvalent forms of the combination of RF-5 i and RF-5 ii ([] n ) [50].R.P., it is sure that the actual data should be much higher (stronger) than these of Mass unit illustrated in this report.

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 6).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][2][3].

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 7, 8 and 9, 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 and 9); (2) the presence of multiple N-glycan chains in the most glycoproteins enhances their activity (Tables 7 and 8); (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 provided more constructive evidences to establish the concept of the Manα1 → related codes, the structural units, and glycotopes (Tables 5 and 6); (5) The data of Con A reacted well with yeast mannan and glycogens (Tables 1 and 7) vs Morniga M was inactive [8].This data provide an additional evidence to support the concept that most lectins have their own binding characters [5,6].

Conclusion, highlights and future plans
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 organized.In this study, they were azalyzed by our well developed method-the enzyme linked lectinosorbent (ELLSA) and inhibition assay.Based on all the data obtained [1][2][3][4][20][21][22][23][24][25][26][27][28][29][30], it is concluded that Con A  [1][2][3][4][20][21][22][23][24][25][26][27][28][29][30] will arranged as another independent topic as a continuation of the "Lectins as tools in glycoconjugate research".and the "Lectins and ELLSA as powerful tools for Glycoconjugate Recognition Analyses".[34,35].As some data and several points have to be further evaluated and confirmed, especially the batches of Con A used, the amount of biomasses added and the other traditional methods used for analyses such as the inhibitions of hemagglutination and quantitative assays, which should be much closer to nature and biology to fit the policy of glycoconjugates.

For 6 (
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 Man indicates the No. of Man in the structures of the mammalian N-glycans M or M 3 Manα1 → 6(Manα1 → 3)Man, or α1 → 3Manα1 → 3)Man, or α1 → 3, α1 → 6 Mannopentaose M Mono to Tetra The Mono to Tetra or more of subscript in Man indicates the No. of GlcNAcβ1 → or II β 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 GalNAcα1 → Ser/Thr T Thomsen-Friedenreich disaccharide

1 .
Major glycan structures of rat sublingual glycoprotein.The major N-glycans are non-bisected hybrid type structures, with (b) and without (a) core fucosylation.(B) Chemical structures of RSL O-glycans, this part of structures are shown in Fig. 4-iv.The major O-glycans are based on cores 3 (c) and 4 (d-f), dominated by a single most abundant disialylated core 4 structure (e).

Fig. 1
Fig. 1 Proposed Con A glycotopes (RF-4) and their derivatives (RF-5) in four examples of mammalian N-Glycans.*The shaded and dotted area are suggested for the glycotopes of the Con A, in which No. of Man are expressed by script of 3 or 5, M 3 (M) / M 5 and dashed line closed are Man with No. of GlcNAcβ1→2, 4 and 6 Manα1→ branches (i to iv), in which Two M Mono in #1; one M Tetra in #2; one M Tri in #3 and one M Di in #4.C, GlcNAcβ1→4GlcNAcβ; C f , GlcNAcβ1→4(lFucα1→6)GlcNAcβ ◂ (i) By 1980s, the structural concept of cyst glycoproteins (HOC): in which Le x and Le y were still considered as new genes with unknown functions.

Fig. 3 aFig. 3 Fig. 4
Fig. 3 a The generalized internal structures of carbohydrate chains of blood group active and sialyl glycoproteins, isolated from human ovarian cyst fluid.(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 a , Le b , and unknown glycotopes (Le x and Le y ) by the years of 1988 are attached,[58][59][60].The numbers in parentheses #[1] to #[12] indicate the site of attachment for the human blood group A, B, H, Le a , Le b , Le x and Le y determinants (ii).Most of the carbohydrate chains isolated are parts of this structure including short

I 2 .
. Outer Man complex (i to iv, Terminal end) II.Part of core of the internal Max complex 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 .

Table 1
Chemical structures of three kinds of polysaccharides-yeast Mannan, glycogen and colominic acid.Their recognition intensities are illustrated in

Table 3
Molar Relative Potencies of Con A toward ligands and haptens for RF-1 to 4

Di to oligosaccharide structural units and glycotopes
The data of the ligands or happens have not reached 50% inhibition are illustrated, are listed in Table3.

Table 4
Maximal quantities of various glycan ligands, structural units giving negligible or weak inhibition of the Con A (5 ng/well) -asialo bovine α 1 -acid gp (10 ng/well) a

Table 6
Profiles of three glycan biomasses and Six Recognition Factors of Con A-Glycan interactions

Table 7
Recognition Intensities and avidities of Morniga-M and Con A toward mammalian N-and O-glycoproteins, yeast mannan and glycogen for RF-6 analyzed by ELLSA a

Table 8
Intensities and avidities of Con A from jack beans and [8]ata obtained from Table2and ref.[8].The inhibitory activity was estimated from the inhibition curves and is expressed as the nanogram amount of inhibitor required to produce 50% inhibition of the lectin binding to asialo bovine α 1 -acid coated microtiter plates; Total volume, 50 μl; For Mass Relative potency (Mass R.P.) for RF-6, Nano gram of Man was taken as 1.0 b The symbols in parentheses are codes of glycans c N.D. not determined, N.A. not available d Tested > 2.4 × 10 4 ng and no 50% inhibition data obtained (vs. 3 ng for Yeast Mannan in Table2)

Table 9
[8]ar Relative Potencies of Morniga-M and Con A toward monosaccharides, structural units and Glycotopes (RFs-#2 to #4) analyzed by ELLSA and inhibition assay a Data obtained from Table3and ref.[8].The inhibitory activity was expressed as the amount of inhibitor giving 50% inhibition.Total volume was 50 μl b Molar Relative potency = Molar Quantity of Man required for 50% inhibition was taken as 1.0 /Molar Quantity of sample required for 50% inhibition c N.D. not determined d N.A., not available due to N.D. or don't reach 50% inhibitions has a relatively broad and wide recognition range for both Manα1 → and Glcα1 → related glycans.It reacted not only strongly with yeast mannan and glycogen, but also with a wide range of mammalian N-glycans, including the rat sublingual gp (RSL), human Tamm-Horsfall glycoprotein (THGP), thyroglobulin and lactoferrin, in which the Man code system, expressed by both numbers of Man and GlcNAcβ1 → branches (M 3 to M 9 / M Mono to Penta etc. ).Furthermore, a a

table with three Manα1 → and Glcα1 → related biomasses of six recognition factors involved in the Con A-glycan interactions
is introduced.These should be several valuable advances in the fields of glycoscience since 1980s.Recognition Factors of more Manα1 → specific lectins are being studied.Comparison of the affinity / dissociation constants from frontal affinity chromatography, equilibrium dialysis, and isothermal calorimetry provided by other reports and approaches