Concanavalin A interactions with asparagine-linked glycopeptides. Bivalency of high mannose and bisected hybrid type glycopeptides

We have previously reported that concanavalin A (ConA) is precipitated by a high mannose type glycopeptide (Brewer, C. F. (1979) Biochem. Biophys. Res. Commun. 90, 117-122; Bhattacharyya, L., and Brewer, C. F. (1986) Biochem. Biophys. Res. Commun. 137, 670-674). In the present study, we have investigated the ability of a series of high mannose and bisected hybrid type glycopeptides to bind and precipitate the lectin. The modes of binding of the glycopeptides were studied by nuclear magnetic relaxation dispersion (NMRD) techniques, and their affinities were determined by hemagglutination inhibition measurements. The stoichiometries of the precipitation reactions were investigated by quantitative precipitation analysis. The equivalence zones (regions of maximum precipitation) of the precipitin curves indicate that certain high mannose and bisected hybrid type glycopeptides are bivalent for lectin binding. From the NMRD and precipitation data, we have identified two protein binding sites on each glycopeptide: one site on the alpha(1-6) arm of the core beta-mannose residue involving a trimannosyl moiety which binds with high affinity (primary site); and the other site on the alpha(1-3) arm of the core beta-mannose residue involving an alpha-mannose residue(s), which binds with lower affinity (secondary site). These two types of sites bind to ConA by different mechanisms. Certain bisected hybrid type glycopeptides were found to possess only the primary ConA binding sites, but not the secondary sites, and hence were able to bind but not precipitate the lectin. Other related glycopeptides have only the secondary type sites and thus exhibit low affinity and are unable to precipitate the protein. The results are related to the possible structure-function properties of cell-surface glycopeptides.     

Structural basis for the recognition of complex-type biantennary oligosaccharides by Pterocarpus angolensis lectin

The crystal structure of Pterocarpus angolensis lectin is determined in its ligand-free state, in complex with the fucosylated biantennary complex type decasaccharide NA2F, and in complex with a series of smaller oligosaccharide constituents of NA2F. These results together with thermodynamic binding data indicate that the complete oligosaccharide binding site of the lectin consists of five subsites allowing the specific recognition of the pentasaccharide GlcNAc beta(1-2)Man alpha(1-3)[GlcNAc beta(1-2)Man alpha(1-6)]Man. The mannose on the 1-6 arm occupies the monosaccharide binding site while the GlcNAc residue on this arm occupies a subsite that is almost identical to that of concanavalin A (con A). The core mannose and the GlcNAc beta(1-2)Man moiety on the 1-3 arm on the other hand occupy a series of subsites distinct from those of con A.     

Concanavalin A interactions with asparagine-linked glycopeptides. Bivalency of bisected complex type oligosaccharides

In the preceding paper (Bhattacharyya, L., Ceccarini, C., Lorenzoni, P., and Brewer, C.F. (1987) J. Biol. Chem. 262, 1288-1293), we have demonstrated that certain high mannose and bisected hybrid type glycopeptides are bivalent for concanavalin A (ConA) binding. In the present study, we have investigated the interactions of ConA with a series of synthetic nonbisected and bisected complex type oligosaccharides and related glycopeptides. The modes of binding of the carbohydrates were studied by nuclear magnetic relaxation dispersion techniques, and their affinities were determined by hemagglutination inhibition measurements. We find that certain bisected complex type oligosaccharides are capable of binding and precipitating the lectin. The corresponding nonbisected analogs, however, bind but do not precipitate the protein. The stoichiometries of the precipitin reactions were investigated by quantitative precipitation analyses. The equivalence zones (regions of maximum precipitation) of the precipitin curves indicate that the bisected complex type oligosaccharides are bivalent for lectin binding. Data for the nonbisected analogs are consistent with their being univalent. The nuclear magnetic relaxation dispersion and precipitation data indicate that nonbisected and bisected complex type carbohydrates bind with different mechanisms and conformations. The former class binds by extended site interactions with the protein involving the 2 alpha-mannose residues on the alpha(1-6) and alpha(1-3) arms of the core beta-mannose residue. The latter class binds by only 1 of these 2 mannose residues, which leaves the other mannose residue free to bind to a second ConA molecule. The role of the bisecting GlcNAc residue in affecting the binding properties of complex type carbohydrates to ConA is discussed, and the results are related to the possible structure-function properties of complex type glycopeptides on the surface of cells.     

Precipitation of the D-galactose specific lectin from Erythrina indica by a triantennary complex type oligosaccharide

We have previously demonstrated that a high mannose type glycopeptide is bivalent for binding Concanavalin A (Con A) and can precipitate the lectin (Bhattacharyya L. and Brewer, C.F. (1986) Biochem. Biophys. Res. Commun. 137, 670-674). The present results show that a triantennary complex type oligosaccharide containing nonreducing terminal galactose residues can precipitate the D-galactose/N-acetyl-D-galactosamine specific lectin from Erythrina indica (EIL). The interactions of the oligosaccharide with EIL was investigated by quantitative precipitin analysis. The equivalence point of the precipitin curve indicated that the glycopeptide is trivalent for EIL binding. These results indicate that each arm of the oligosaccharide can independently bind separate lectin molecules leading to precipitation of the complex. These findings are discussed in terms of the possible biological structure-function properties of complex type oligosaccharides.     

Primary structure of an N-glycosidic carbohydrate unit derived from Sophora japonica lectin

The lectin isolated from Sophora japonica seeds is a glycoprotein which binds oligosaccharides with non-reducing terminal Gal beta(1----3/4)GlcNac beta 1----units. The carbohydrate moiety of the lectin is composed of fucose, xylose, mannose and N-acetylglucosamine. The major glycopeptide of the lectin, prepared by pronase digestion, was derivatized with fluorescein isothiocyanate, purified by PAGE and examined by exoglycosidase digestion as well as purified by gel filtration through Bio-Gel P6-DG and investigated by methylation analysis and 400-MHz 1H-NMR spectroscopy. The primary structure of the glycopeptide was established to be as follows. (Formula: see text). Structures similar to this containing a (beta 1-2)xylosyl substituent on the core beta-mannosyl residue and an inner core (alpha 1-3)fucosyl substituent seem to occur frequently in plant glycoproteins.     

Oligosaccharide structure and amino acid sequence of the major glycopeptides of mature human beta-hexosaminidase

Human beta-hexosaminidase (EC 3.2.1.52) is a lysosomal enzyme that hydrolyzes terminal N-acetylhexosamines from GM2 ganglioside, oligosaccharides, and other carbohydrate-containing macromolecules. There are two major forms of hexosaminidase: hexosaminidase A, with the structure alpha(beta a beta b), and hexosaminidase B, 2(beta a beta b). Like other lysosomal proteins, hexosaminidase is targeted to its destination via glycosylation and processing in the rough endoplasmic reticulum and Golgi apparatus. Phosphorylation of specific mannose residues allows binding of the protein to the phosphomannosyl receptor and transfer to the lysosome. In order to define the structure and placement of the oligosaccharides in mature hexosaminidase and thus identify candidate mannose 6-phosphate recipient sites, the major tryptic/chymotryptic glycopeptides from each isozyme were purified by reverse-phase high-performance liquid chromatography. Two major concanavalin A binding glycopeptides, localized to the beta b chain, and one non concanavalin A binding glycopeptide, localized to the beta a chain, were found associated with the beta-subunit in both hexosaminidase A and hexosaminidase B. A single major concanavalin A binding glycopeptide was found to be associated with the alpha subunit of hexosaminidase A. The oligosaccharide structures were determined by nuclear magnetic resonance spectrometry. Two of them, the alpha and one of the beta b glycans, contained a Man3-GlcNAc2 structure, while the remaining one on the beta b chain was composed of a mixture of Man5-7-GlcNAc2 glycans. The unique glycopeptide associated with the beta a chain contained a single GlcNAc residue. Thus, all three mature polypeptides comprising the alpha and beta subunits of hexosaminidase contain carbohydrate, the structures of which have the appearance of being partially degraded in the lysosome. In the alpha chain we found only one possible site for in vivo phosphorylation. In the beta it is unclear if only one or all three of the sites could have contained phosphate. However, mature placental hexosaminidase A and B can be rephosphorylated in vitro. This requires the presence of an oligosaccharide containing an alpha 1,2-linked mannose residue. Only the single Man6-7 (of the Man5-7-GlcNAc2 glycans) containing site on the beta b chain retains this type of residue. Therefore, this site may act as the sole in vitro substrate in both of the mature isozymes for the phosphotransferase.     

Primary structure and carbohydrate binding specificity of a potent anti-HIV lectin isolated from the filamentous cyanobacterium Oscillatoria agardhii

The primary structure of a lectin, designated Oscillatoria agardhii agglutinin (OAA), isolated from the freshwater cyanobacterium O. agardhii NIES-204 was determined by the combination of Edman degradation and electron spray ionization-mass spectrometry. OAA is a polypeptide (Mr 13,925) consisting of two tandem repeats. Interestingly, each repeat sequence of OAA showed a high degree of similarity to those of a myxobacterium, Myxococcus xanthus hemagglutinin, and a marine red alga Eucheuma serra lectin. A systematic binding assay with pyridylaminated oligosaccharides revealed that OAA exclusively binds to high mannose (HM)-type N-glycans but not to other N-glycans, including complex types, hybrid types, and the pentasaccharide core or oligosaccharides from glycolipids. OAA did not interact with any of free mono- and oligomannoses that are constituents of the branched oligomannosides. These results suggest that the core disaccharide, GlcNAc-GlcNAc, is also essential for binding to OAA. The binding activity of OAA to HM type N-glycans was dramatically decreased when alpha1-2 Man was attached to alpha1-3 Man branched from the alpha1-6 Man of the pentasaccharide core. This specificity of OAA for HM-type oligosaccharides is distinct from other HM-binding lectins. Kinetic analysis with an HM heptasaccharide revealed that OAA possesses two carbohydrate binding sites per molecule, with an association constant of 2.41x10(8) m-1. Furthermore, OAA potently inhibits human immunodeficiency virus replication in MT-4 cells (EC50=44.5 nm). Thus, we have found a novel lectin family sharing similar structure and carbohydrate binding specificity among bacteria, cyanobacteria, and marine algae.     

The catalytic and lectin domains of UDP-GalNAc:polypeptide alpha-N-Acetylgalactosaminyltransferase function in concert to direct glycosylation site selection

UDP-GalNAc:polypeptide alpha-N-Acetylgalactosaminyltransferases (ppGalNAcTs), a family (EC 2.4.1.41) of enzymes that initiate mucin-type O-glycosylation, are structurally composed of a catalytic domain and a lectin domain. Previous studies have suggested that the lectin domain modulates the glycosylation of glycopeptide substrates and may underlie the strict glycopeptide specificity of some isoforms (ppGalNAcT-7 and -10). Using a set of synthetic peptides and glycopeptides based upon the sequence of the mucin, MUC5AC, we have examined the activity and glycosylation site preference of lectin domain deletion and exchange constructs of the peptide/glycopeptide transferase ppGalNAcT-2 (hT2) and the glycopeptide transferase ppGalNAcT-10 (hT10). We demonstrate that the lectin domain of hT2 directs glycosylation site selection for glycopeptide substrates. Pre-steady-state kinetic measurements show that this effect is attributable to two mechanisms, either lectin domain-aided substrate binding or lectin domain-aided product release following glycosylation. We find that glycosylation of peptide substrates by hT10 requires binding of existing GalNAcs on the substrate to either its catalytic or lectin domain, thereby resulting in its apparent strict glycopeptide specificity. These results highlight the existence of two modes of site selection used by these ppGalNAcTs: local sequence recognition by the catalytic domain and the concerted recognition of distal sites of prior glycosylation together with local sequence binding mediated, respectively, by the lectin and catalytic domains. The latter mode may facilitate the glycosylation of serine or threonine residues, which occur in sequence contexts that would not be efficiently glycosylated by the catalytic domain alone. Local sequence recognition by the catalytic domain differs between hT2 and hT10 in that hT10 requires a pre-existing GalNAc residue while hT2 does not.     

The B4 lectin from Vicia villosa seeds interacts with N-acetylgalactosamine residues alpha-linked to serine or threonine residues in cell surface glycoproteins

We have examined the carbohydrate binding specificity of the B4 lectin from Vicia villosa seeds. The B4 lectin agglutinates Tn-exposed erythrocytes specifically and binds to these erythrocytes (1.4 X 10(6) sites/cell) with an association constant of 4.2 X 10(7) M-1. The concentrations of saccharides and glycopeptides of defined structure which cause 50% inhibition of B4 lectin binding to Tn-exposed erythrocytes were determined. N-Acetylgalactosamine is the best monosaccharide inhibitor, causing 50% inhibition of binding at a concentration of 0.04 mM. Other monosaccharides inhibit lectin binding in the following order of decreasing potency: N-acetylgalactosamine greater than methyl-alpha-galactopyranoside greater than p-nitrophenyl-alpha- or beta-galactopyranoside greater than methyl-beta-galactopyranoside, galactose greater than galactosamine greater than mannose, N-acetylglucosamine. The disaccharide Gal beta 1,3GalNAc causes 50% inhibition of binding at a concentration of 2.8 mM, a concentration similar to that of the p-nitrophenyl-alpha- or beta-galactopyranosides. Glycopeptides containing O-glycosidically linked oligosaccharide units are significantly more potent inhibitors of lectin binding than the oligosaccharide units alone. The most potent glycopeptide inhibitor is a fetuin glycopeptide containing two alpha-linked N-acetylgalactosamine units. This glycopeptide causes 50% inhibition of lectin binding at a concentration of 0.00034 mM and probably closely resembles the B4 lectin binding site on Tn-exposed erythrocytes.     

A structural basis for four distinct elution profiles on concanavalin A--Sepharose affinity chromatography of glycopeptides.

Twelve 14C-acetylated glycopeptides have been subjected to affinity chromatography on concanvalin A (Con A)--Sepharose at pH 7.5. The elution profiles could be classified into four distinct patterns. The first pattern showed no retardation of glycopeptide on the column and was elicited with a glycopeptide having three peripheral oligosaccharide chains: (abstract:see text). Such glycopeptides have only a single mannose residue capable of interacting with Con A--Sepharose; an interacting mannose residue is either an alpha-linked nonreducing terminal residue or an alpha-linked 2-O-substituted residue. The second type of profile showed a retarded elution of glycopeptide with buffer lacking methyl alpha-D-glucopyranoside (indicative of weak interaction with the column) and was given by glycopeptides with the structures: (abstract: see text) where R1 is either H or a sialyl residue. The third profile type showed tight binding of glycopeptide to Con A--Sepharose and elution as a sharp peak with 0.1 M methyl alpha-D-glucopyranoside; glycopeptides giving this pattern had the structures: (abstract: see text) where R2 is either H, glcNAc, Gal-beta 1,4-GlcNAc, or sialyl-Gal-beta 1,4-GlcNAc. These glycopeptides all have two interacting mannose residues, the mimimum required for binding to the column; one of these mannose residues must, however, be a terminal residue to obtain tight binding and sharp elution. The fourth profile type showed tight binding of glycopeptide to the column but elution with 0.1 M methyl alpha-D-glucopyranoside resulted in a broad peak indicating very tight binding; glycopeptides showing this behaviour had the structures: (abstract: see text) where R3 is either GlcNAc,Gal-beta 1,4-GlcNAc, or sialyl-Gal-beta 1,4-GlcNAc.Therefore it can be concluded that although a minimum of two interacting mannose residues is required for binding to Con A--Sepharose, the residues linked to these mannoses can either strengthen or weaken binding to the column.     

High affinity interaction between a bivalve C-type lectin and a biantennary complex-type N-glycan revealed by crystallography and microcalorimetry

Codakine is an abundant 14-kDa mannose-binding C-type lectin isolated from the gills of the sea bivalve Codakia orbicularis. Binding studies using inhibition of hemagglutination indicated specificity for mannose and fucose monosaccharides. Further experiments using a glycan array demonstrated, however, a very fine specificity for N-linked biantennary complex-type glycans. An unusually high affinity was measured by titration microcalorimetry performed with a biantennary Asn-linked nonasaccharide. The crystal structure of the native lectin at 1.3A resolution revealed a new type of disulfide-bridged homodimer. Each monomer displays three intramolecular disulfide bridges and contains only one calcium ion located in the canonical binding site that is occupied by a glycerol molecule. The structure of the complex between Asn-linked nonasaccharide and codakine has been solved at 1.7A resolution. All residues could be located in the electron density map, except for the capping beta1-4-linked galactosides. The alpha1-6-linked mannose binds to calcium by coordinating the O3 and O4 hydroxyl groups. The GlcNAc moiety of the alpha1,6 arm engages in several hydrogen bonds with the protein, whereas the GlcNAc on the other antenna is stacked against Trp(108), forming an extended binding site. This is the first structural report for a bivalve lectin.     

IL-2, a lectin with specificity for high mannose glycopeptides

Utilizing a solid phase binding assay, we have demonstrated that rIL-2 binds with high affinity to the human urinary glycoprotein uromodulin. This binding is specifically inhibited by the saccharides diacetylchitobiose and Man(alpha 1-3)(Man(alpha 1-6]Man-O-methyl and by the high mannose glycopeptides Man5GlcNAc2-R and Man6GlcNAc2-R, but not by Man9GlcNAc2-R. rIL-2 also binds OVA, a glycoprotein which contains approximately 50% high mannose chains at a single glycosylation site, and to yeast mannan. This binding is inhibited by the same battery of saccharides which inhibit the binding to uromodulin. The conclusion that rIL-2 is a lectin is further supported by the observation that the sequence of IL-2 shares 27% homology with a 33-residue sequence of the carbohydrate-binding domain of human mannose-binding protein. The potential physiologic relevance of the carbohydrate binding activity is further elucidated by studies which show that 1) binding of soluble rIL-2 to immobilized uromodulin is enhanced at a pH of 4 to5 in the presence of divalent cations, and 2) neither uromodulin nor the high mannose glycopeptide Man5GlcNAc2Asn blocks the binding of rIL-2 to the IL-2R. Thus the carbohydrate-binding site of rIL-2 is distinct from the cell surface receptor-binding site, and might function preferentially in acidic microenvironments.     

Structural basis of oligomannose recognition by the Pterocarpus angolensis seed lectin

The crystal structure of a Man/Glc-specific lectin from the seeds of the bloodwood tree (Pterocarpus angolensis), a leguminous plant from central Africa, has been determined in complex with mannose and five manno-oligosaccharides. The lectin contains a classical mannose-specificity loop, but its metal-binding loop resembles that of lectins of unrelated specificity from Ulex europaeus and Maackia amurensis. As a consequence, the interactions with mannose in the primary binding site are conserved, but details of carbohydrate-binding outside the primary binding site differ from those seen in the equivalent carbohydrate complexes of concanavalin A. These observations explain the differences in their respective fine specificity profiles for oligomannoses. While Man(alpha1-3)Man and Man(alpha1-3)[Man(alpha1-6)]Man bind to PAL in low-energy conformations identical with that of ConA, Man(alpha1-6)Man is required to adopt a different conformation. Man(alpha1-2)Man can bind only in a single binding mode, in sharp contrast to ConA, which creates a higher affinity for this disaccharide by allowing two binding modes.     

On the stringent requirement of mannosyl substitution in mannooligosaccharides for the recognition by garlic (Allium sativum) lectin. A Surface Plasmon Resonance Study

The kinetics of the binding of mannooligosaccharides to the heterodimeric lectin from garlic bulbs was studied using surface plasmon resonance. The interaction of the bound lectin immobilized on the sensor chip with a selected group of high mannose oligosaccharides was monitored in real time with the change in response units. This investigation corroborates our earlier study about the special preference of garlic lectin for terminal alpha-1,2-linked mannose residues. An increase in binding propensity can be directly correlated to the addition of alpha-1,2-linked mannose to the mannooligosaccharide at its nonreducing end. Mannononase glycopeptide (Man9GlcNAc2Asn), the highest oligomer studied, exhibited the greatest binding affinity (Ka = 1.2 x 10(6) m(-1) at 25 degrees C). An analysis of these data reveals that the alpha-1,2-linked terminal mannose on the alpha-1,6 arm is the critical determinant in the recognition of mannooligosaccharides by the lectin. The association (k1) and dissociation rate constants (k(-1)) for the binding of Man9GlcNAc2Asn to Allium sativum agglutinin I are 6.1 x 10(4) m(-1) s(-1) and 4.9 x 10(-2) s(-1), respectively, at 25 degrees C. Whereas k1 increases progressively from Man3 to Man7 derivatives, and more dramatically so for Man8 and Man9 derivatives, k(-1) decreases relatively much less gradually from Man3 to Man9 structures. An unprecedented increase in the association rate constant for interaction with Allium sativum agglutinin I with the structure of the oligosaccharide ligand constitutes a significant finding in protein-sugar recognition.     

Mannose processing is an important determinant in the assembly of phosphorylated high mannose-type oligosaccharides

Phosphorylation of the high mannose-type oligosaccharides attached to newly synthesized acid hydrolases occurs in two sequential steps within the endoplasmic reticulum and the Golgi apparatus, and the products generated at the two sites differ with respect to the location of the phosphorylated mannose residue. To investigate the mechanism of this two-step phosphorylation, biosynthesis of the Man-6-P recognition marker was studied in class E Thy-1- and J774 cells metabolically labeled with [2-3H]mannose. Class E Thy-1- cells produce truncated high mannose oligosaccharides that lack 4 mannose residues from the alpha 1,6-branch of the core beta-linked mannose residue; three of the missing residues are potential phosphorylation sites. Acid hydrolases produced by these mutant cells were phosphorylated on the alpha 1,3-branch of the truncated oligosaccharide even when transport to the Golgi apparatus was inhibited. J774 cells produce normal high mannose oligosaccharides, but they secrete a large percentage of their newly synthesized acid hydrolases. The secreted enzymes contained primarily diphosphorylated units in which a phosphate was positioned to both the alpha 1,3- and alpha 1,6-branches of the core beta-linked mannose. J774 cells treated with deoxymannojirimycin continued to phosphorylate and to secrete acid hydrolases. The secreted hydrolases, however, contained only monophosphorylated oligosaccharides in which the phosphate was restricted to the alpha 1,6-branch. These results indicate that mannose residues within high mannose oligosaccharides impose constraints on the phosphorylation of their composite structures. We conclude that the two-step phosphorylation occurs as a result of a common phosphotransferase at both the pre-Golgi and Golgi locations and a change in the conformation of the oligosaccharides attached to the acid hydrolases through the action of Golgi-associated alpha-mannosidase I.     

Multiple modes of binding enhance the affinity of DC-SIGN for high mannose N-linked glycans found on viral glycoproteins

The dendritic cell surface receptor DC-SIGN and the closely related endothelial cell receptor DC-SIGNR specifically recognize high mannose N-linked carbohydrates on viral pathogens. Previous studies have shown that these receptors bind the outer trimannose branch Manalpha1-3[Manalpha1-6]Manalpha present in high mannose structures. Although the trimannoside binds to DC-SIGN or DC-SIGNR more strongly than mannose, additional affinity enhancements are observed in the presence of one or more Manalpha1-2Manalpha moieties on the nonreducing termini of oligomannose structures. The molecular basis of this enhancement has been investigated by determining crystal structures of DC-SIGN bound to a synthetic six-mannose fragment of a high mannose N-linked oligosaccharide, Manalpha1-2Manalpha1-3[Manalpha1-2Manalpha1-6]Manalpha1-6Man and to the disaccharide Manalpha1-2Man. The structures reveal mixtures of two binding modes in each case. Each mode features typical C-type lectin binding at the principal Ca2+-binding site by one mannose residue. In addition, other sugar residues form contacts unique to each binding mode. These results suggest that the affinity enhancement displayed toward oligosaccharides decorated with the Manalpha1-2Manalpha structure is due in part to multiple binding modes at the primary Ca2+ site, which provide both additional contacts and a statistical (entropic) enhancement of binding.     

Major carbohydrate structures at five glycosylation sites on murine IgM determined by high resolution 1H-NMR spectroscopy

Mouse myeloma immunoglobulin IgM heavy chains were cleaved with cyanogen bromide into nine peptide fragments, four of which contain asparagine-linked glycosylation. Three glycopeptides contain a single site, including Asn 171, 402, and 563 in the intact heavy chain. Another glycopeptide contains two sites at Asn 332 and 364. The carbohydrate containing fragments were treated with Pronase and fractionated by elution through Bio-Gel P-6. The major glycopeptides from each site were analyzed by 500 MHz 1H-NMR and the carbohydrate compositions determined by gas-liquid chromatography. The oligosaccharide located at Asn 171 is a biantennary complex and is highly sialylated. The amount of sialic acid varies, and some oligosaccharides contain alpha 1,3-galactose linked to the terminal beta 1,4-galactose. The oligosaccharides at Asn 332, Asn 364, an Asn 402 are all triantennary and are nearly completely sialylated on two branches and partially sialylated on the triantennary branch linked beta 1,4 to the core mannose. The latter is sialylated about 40% of the time for all three glycosylation sites. The major oligosaccharide located at Asn 563 is of the high mannose type. The 1H-NMR determination of structures at Asn 563 suggests that the high mannose oligosaccharide contains only three mannose residues.     

The binding characteristics and utilization of Aleuria aurantia,Lens culinaris and few other lectins in the elucidation of fucosyltransferase activities resembling cloned FT VI and apparently unique to colon cancer cells

Human colon carcinoma cell fucosyltransferase (FT) in contrast to the FTs of several human cancer cell lines, utilized GlcNAcbeta1,4GlcNAcbeta-O-Bn as an acceptor, the product being resistant to alpha1,6-L-Fucosidase and its formation being completely inhibited by LacNAc Type 2 acceptors. Further, this enzyme was twofold active towards the asialo agalacto glycopeptide as compared to the parent asialoglycopeptide. Only 60% of the GlcNAc moieties were released from [14C]fucosylated asialo agalacto triantennary glycopeptide by jack bean beta-N-acetylhexosaminidase. These alpha1,3-L-fucosylating activities on multiterminal GlcNAc residues and chitobiose were further examined by characterizing the products arising from fetuin triantennary and bovine IgG diantennary glycopeptides and their exoglycosidase-modified derivatives using lectin affinity chromatography. Utilization of [14C]fucosylated glycopeptides with cloned FTs indicated that Lens culinaris lectin and Aleuria aurantia lectin (AAL) required, respectively, the diantennary backbone and the chitobiose core alpha1,6-fucosyl residue for binding. The outer core alpha1,3- but not the alpha-1,2-fucosyl residues decreased the binding affinity of AAL. The AAL-binding fraction from [14C]fucosylated asialo fetuin, using colon carcinoma cell extract, contained 60% Endo F/PNGaseF resistant chains. Similarly AAL-binding species from [14C]fucosylated TFA-treated bovine IgG using colon carcinoma cell extract showed significant resistance to endo F/PNGaseF. However, no such resistance was found with the corresponding AAL non- and weak-binding species. Thus colon carcinoma cells have the capacity to fucosylate the chitobiose core in glycoproteins, and this alpha1,3-L-fucosylation is apparently responsible for the AAL binding of glycoproteins. A cloned FT VI was found to be very similar to this enzyme in acceptor substrate specificities. The colon cancer cell FT thus exhibits four catalytic roles, i.e., alpha1,3-L-fucosylation of: (a) Galbeta1,4GlcNAcbeta-; (b) multiterminal GlcNAc units in complex type chain; (c) the inner core chitobiose of glycopeptides and glycoproteins; and (d) the nonreducing terminal chiotobiose unit.     

Modes of binding of alpha (1-2) linked manno-oligosaccharides to concanavalin A.

Three-dimensional structures of the complexes of concanavalin A (ConA) with alpha(1-2) linked mannobiose, triose and tetraose have been generated with the X-ray crystal structure data on native ConA using the CCEM (contact criteria and energy minimization) method. All the constituting mannose residues of the oligosaccharide can reach the primary binding site of ConA (where methyl-alpha-D-mannopyranose binds). However, in all the energetically favoured complexes, either the non-reducing end or middle mannose residues of the oligosaccharide occupy the primary binding site. The middle mannose residues have marginally higher preference over the non-reducing end residue. The sugar binding site of ConA is extended and accommodates at least three alpha(1-2) linked mannose residues. Based on the present calculations two mechanisms have been proposed for the binding of alpha(1-2) linked mannotriose and tetraose to ConA.     

Fine specificities of two lectins from Cymbosema roseum seeds: a lectin specific for high-mannose oligosaccharides and a lectin specific for blood group H type II trisaccharide

The legume species of Cymbosema roseum of Diocleinae subtribe produce at least two different seed lectins. The present study demonstrates that C. roseum lectin I (CRL I) binds with high affinity to the "core" trimannoside of N-linked oligosaccharides. Cymbosema roseum lectin II (CRL II), on the other hand, binds with high affinity to the blood group H trisaccharide (Fucα1,2Galα1-4GlcNAc-). Thermodynamic and hemagglutination inhibition studies reveal the fine binding specificities of the two lectins. Data obtained with a complete set of monodeoxy analogs of the core trimannoside indicate that CRL I recognizes the 3-, 4- and 6-hydroxyl groups of the α(1,6) Man residue, the 3- and 4-hydroxyl group of the α(1,3) Man residue and the 2- and 4-hydroxyl groups of the central Man residue of the trimannoside. CRL I possesses enhanced affinities for the Man5 oligomannose glycan and a biantennary complex glycan as well as glycoproteins containing high-mannose glycans. On the other hand, CRL II distinguishes the blood group H type II epitope from the Lewis(x), Lewis(y), Lewis(a) and Lewis(b) epitopes. CRL II also distinguishes between blood group H type II and type I trisaccharides. CRL I and CRL II, respectively, possess differences in fine specificities when compared with other reported mannose and fucose recognizing lectins. This is the first report of a mannose-specific lectin (CRL I) and a blood group H type II-specific lectin (CRL II) from seeds of a member of the Diocleinae subtribe.