Membrane glycophorins in Sta blood group erythrocytes

Structural and immunochemical studies of glycophorins isolated from erythrocytes of an individual homozygous for the M Sta blood group phenotype are described. Reactivities with specific monoclonal antibodies indicated that two major M and N glycophorins were present. The M and N Sta glycophorins were resolved by Lens culinaris lectin affinity chromatography. The N species was not held on the lectin but the M species, like control alpha glycophorins, was retained and could be eluted with alpha-methylmannoside. The two proteins were present in almost equimolar amounts. Studies of the CNBr fragments provided evidence that the structure of M Sta glycophorin is the same as that of the usual M alpha glycophorin but that the N Sta glycophorin is a variant. The amino-terminal octapeptides of the M and N species were similar in amino acid and carbohydrate composition to those isolated, respectively, from M and N alpha glycophorins. The studies focused on CNBr glycopeptide B that, in control alpha glycophorins, extends from amino acid residues 9 to 81. The fragment from the M species exhibited properties identical to those of the corresponding fragment of control alpha glycophorins in terms of size, chromatographic behavior, amino acid and carbohydrate contents and compositions, the presence of O-glycosidically linked saccharides and a single Asn-linked carbohydrate unit. The structures of the O-linked units were inferred experimentally to be NeuAc(alpha 2,3)Gal-(beta 1,3)GalNAc and NeuAc(alpha 2,3)Gal(beta 1,3) [NeuAc(alpha 2,6)]GalNAc, present in a ratio similar to that found in controls; and the Asn-linked unit also appeared to be as in the control. The tryptic glycopeptide pattern of the M Sta glycophorin CNBr fragment B was identical to the pattern of the corresponding control fragment, and the composition of the tryptic peptides suggested sequence identity with the control fragment. In contrast, the N Sta glycophorin yielded two CNBr glycopeptides B; both contained fewer amino acid residues and virtually lacked Man and GlcNAc, indicating the absence of the Asn-linked carbohydrate. The much decreased levels of these carbohydrates in the intact N protein, corroborated the latter finding. The O-glycosidic saccharides appeared similar to those found in control alpha glycophorins. However, the tryptic glycopeptide pattern of the variant differed from control M or N alpha glycophorins, suggesting a deletion of a large segment of the molecule near residues 40-61 and/or a substitution of methionine for a residue upstream from residue 40.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

A glycopeptide resistant to exoglycosidases

The carboxyl group of the terminal N-acetylneuraminic acid residue of the glycopeptide, prepared from α₁-acid glycoprotein by protease digestion, was esterified with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, and then reduced with sodium borohydride. The reduced glycopeptide, thus prepared, containing the reduced N-acetylneuraminic acid, was resistant to hydrolysis by neuraminidase, and consequently to other exoglycosidases. The penultimate β-d-galactosyl residue of the oligosaccharide chain of the reduced glycopeptide was hydrolyzed by β-d-galactosidase only after the removal of the terminal, reduced, sialic acid by mild hydrolysis with acid. The reduced glycopeptide should be a useful substrate for the assay of endoglycosidases in the presence of exoenzymes. It should also find use as a carbon source in the growth of endoglycosidase-elaborating bacteria.     

Precipitation of concanavalin A by a high mannose type glycopeptide

The interactions of a high mannose type glycopeptide with Concanavalin A has been investigated by quantitative precipitation analysis. The equivalence points of the precipitin curves indicate that the glycopeptide is bivalent for lectin binding. These results and others demonstrate that there are two lectin binding sites per molecule of the glycopeptide: one site on the alpha (1-6) arm of the core beta-mannose residue involving a trimannosyl moiety, and another site on the alpha (1-3) arm of the core beta-mannose residue involving an alpha (1-2) mannobiosyl group. The two sites are unequal in their affinities, and bind by different mechanisms. These results are related to the possible structure-function properties of high mannose type of glycopeptides on the surface of cells.     

Isolation and characterization of a blood group active glycopeptide from porcine aorta

A fucose-rich glycopeptide was prepared from the pronase digest of porcine thoracic aorta by gel-filtration through Sephadex G-100, DEAE-Sephadex A-25 column chromatography and alpha-amylase digestion. This glycopeptide was electrophoretically homogeneous. The large molecular size and chemical composition suggested that this glycopeptide was derived from mucin-type glycoprotein. The results of the beta-elimination reaction indicated that this glycopeptide contained the O-glycosidic linkages between galactosamine and serine/threonine. This glycopeptide exhibited blood group A and H activities. The present study revealed that the porcine thoracic aorta contains a blood group antigen of mucin-type glycoprotein nature.     

Presence of two endo-beta-N-acetylglucosaminidases in human kidney

We have isolated for the first time two kinds of endo-beta-N-acetylglucosaminidases (E-beta-GNases) simultaneously from human kidney. E-beta-GNase 1 was purified by water extraction, ammonium sulfate fractionation, and chromatography on Sephadex-G-200, DEAE-Sephadex, concanavalin A-Sepharose and Hypatite C columns. After the DEAE-Sephadex step, 107 units of E-beta-GNase 1 with a specific activity of 0.53 units/mg was obtained and after hydroxyapatite column, the enzyme recovery was 26 units with a specific activity of 10.4 units/mg. This enzyme hydrolyzed the high mannose-type asparaginylglycopeptide efficiently and had little activity toward the complex-type glycopeptide. This enzyme had an pH optimum at about 4.5 and was not inhibited by acetate ion. The Asn residue in a glycopeptide appeared not to be an important recognition site for E-beta-GNase 1 to express its activity because the acetylation or the dansylation of Asn residues as well as the elimination of Asn residue from the glycopeptide did not change the susceptibility of the oligosaccharide to E-beta-GNase 1. E-beta-GNase 2 was purified by water extraction, ammonium sulfate fractionation, and chromatography on Sephadex G-200, DEAE-Sephadex, concanavalin A-Sepharose, and Mono S columns. This enzyme was purified about 110-fold with 6.6% recovery. E-beta-GNase 2 was found to be a novel type of E-beta-GNase that hydrolyzed both the high mannose-type and the complex-type oligosaccharide with chitobiosyl group at the reducing end and without the Asn. E-beta-GNase 2 activity was found to be dependent on a L-aspartamido-beta-D-N-acetylglucosamine amidohydrolase (Asn-GNase) for the hydrolysis of asparaginylglycopeptide. Asn-GNase cleaved off the Asn residue from the glycopeptide, and the resulting oligosaccharide was hydrolyzed by E-beta-GNase 2. Because the acetylation or the dansylation of Asn residue in a glycopeptide rendered the glycopeptide resistant to Asn-GNase, the use of the modified asparaginylglycopeptide could not reveal the existence of E-beta-GNase 2 activity. The pH optimum of E-beta-GNase was found to be about 3.5. Like beta-hexosaminidases, this enzyme was inhibited by acetate ion, suggesting the recognition of GlcNAc moiety by this enzyme.     

Expanding the Scope of Native Chemical Ligation in Glycopeptide Synthesis

Glycoprotein is one of the important biopolymer in a biological system. In order to understand the complex correlation between the exact oligosaccharide structure of the glycoprotein and its function, preparation of homogeneous glycoprotein is to be essential. For such a purpose, chemical synthesis is one of the most promising methods to obtain homogeneous glycoproteins. Glycopolypeptide, which is a backbone of glycoprotein and an essential intermediate for glycoprotein synthesis, can be obtained through coupling of peptide and glycopeptide segments because straightforward synthesis of such a long glycopolypeptide is still a challenging task. Native chemical ligation (NCL) is one of the powerful methods for the coupling reaction of peptides, however, despite extensive investigation, NCL has site limitation for the coupling. In this context, we discovered NCL at serine site, where is a highly conserved amino acid residue in glycoproteins. This reaction strategy is owed to conversion reaction of cysteine residue to serine residue after conventional NCL. This conversion reaction is consisted of three steps; S-methylation of cysteine, CNBr reaction to afford O-ester linked peptide, and O to N acyl shift to get native peptide linkage with serine residue. During extensive investigation of the strategy, we found new reaction media for CNBr reaction, which is the key reaction in the strategy. This enabled us to synthesize not only N-linked glycopeptides but also O-linked sialyl glycopeptides. Thus we could demonstrate the usefulness of this new glycopeptide ligation strategy. In this short review, we will introduce our newly developed cysteine to serine conversion reaction which will expand the application of NCL in peptide as well as glycopeptide synthesis.     

Interterminal distance and flexibility of a triantennary glycopeptide as measured by resonance energy transfer

Three geometric isomers of a single triantennary glycopeptide, each containing two fluorophores attached to terminal positions in the molecule, were used to probe distance and flexibility of the oligosaccharide in solution. A dansyl group (energy acceptor) was attached to the C6 of Gal at either position 6', 6, or 8, and a naphthyl-2-acetyl group (energy donor) was coupled to the N terminus of the Ala-Asn peptide. (formula; see text) Resonance energy-transfer measurements revealed an average distance of approximately 22, 18, and 17 A between the donor and the acceptor attached to either the 6, 8, or 6' Gal residue, respectively. The lifetime of the donor's emission was nearly a single-exponential decay of 27 ns (96%), whereas the decay of the donor with proximally attached acceptor was fit by nonlinear least-squares analysis to a multiexponential for each glycopeptide probe. Fitting with a Lorentzian function revealed spatially distinct donor/acceptor distances presumably arising from glycopeptide branch flexibility. The results suggest that the acceptor located at Gal 8 is the most rigid relative to the donor with a single population of distances centered at 18.4 A. In contrast, the acceptor attached to either Gal 6' or 6 displayed two populations of different distances from the donor. The Gal 6 isomer contained a major population with average donor/acceptor separation distance of 21.7 A and a minor population with average separation distance of 9.7 A. Similarly, the Gal 6' isomer showed a major population with donor/acceptor separation distance of 18.3 A and a minor population with separation distance of 11.7 A. These data support the earlier conclusions that the Man alpha(1----6)Man linkage found in the core pentasaccharide of all branched N-linked oligosaccharides is flexible. In addition, the data suggest that the branch containing Gal 6 is also flexible in the triantennary glycopeptide.     

THE AFFINITY OF THE FUCOSE-BINDING LECTIN FROM LOTUS TETRAGONOLOBUS FOR GLYCOPEPTIDES AND OLIGOSACCHARIDES ACCUMULATING IN FUCOSIDOSIS

Abstract— The affinity of the fucose-binding lectin from Lotus tetragonolobus for fuco-oligosaccharides accumulating in the brain and other tissues of a patient with fucosidosis was studied by two methods: by inhibition of the co-precipitation of the lectin with porcine stomach mucin and by one-step affinity chromatography on a column of the lectin bound to Sepharose-4B. Both methods indicated that the lectin had greater affinity for the disaccharide Fuc(α, 1-6)GlcNAc than for either the main fucosidosis storage material in brain, a fuco-dekasaccharide, or the heterogeneous fuco-glycopeptide fractions obtained from normal human and rat brain glycoproteins. Our results suggest that the fucose residue linked α(1-6) to the N -acetylglucosamine residue involved in the N -glycosidic linkage to asparagine is not available to the lectin in the intact N -glycosidic chains of normal brain glycopeptide fractions and that the lectin has poor affinity for the Fuc(α, 1-3)Glc N Ac linkage in rat brain glycoproteins.     

Modification of amino groups of human-erythrocyte glycoproteins and the new concept on the structural basis of M and M blood-group specificity.

1. Various kinds of modification of amino groups of M and N blood group glycoproteins abolished their capacity to inhibit rabbit and human anti-M and anit-N sera. 2. The reversible modification of amino groups revealed that M and N blood group activity was restored after the removal of amino-group-blocking residues. 3. Modification of amino groups had an entirely different effect on the reactivity of red cell glycoproteins with Vicia graminea agglutinin. The serological activity of N glycoprotein towards Vicia graminea anti-N agglutinin was unchanged, whereas the weak activity of M glycoprotein towards this anti-N agglutinin was increased to the level of the of N glycoprotein. 4. These results indicate that there is a structural difference between M and N glycoproteins, which resides beyond the oligosaccharide chains. It suggests in turn that M and N blood group specificity is determined by amino acid sequence in the peptide chains of red cell glycoproteins.     

Two monoclonal antibodies highly specific for the blood group N determinant

Two monoclonal IgM antibodies, 179K and 35/5F, obtained following immunization of mice with A₂,MN or O,MN human erythrocytes, agglutinate NN and MN red cells strongly, and MM erythrocytes weakly. As shown by hemagglutination inhibition and solid phase ELISA, both antibodies are highly specific for the blood group N determinant. They react with N glycoprotein, its amino-terminal glycopeptides and with Ss glycoprotein (glycophorin B), which carries the blood group N determinant. They fail to react with M glycoprotein, M glycoprotein-derived glycopeptides, or with internal glycopeptides derived from N glycoprotein. Reaction of the antibodies with N glycoprotein is abolished by desialylation, periodate oxidation/borohydride reduction, orN-acetylation of the glycoprotein. Thus, the antibodies are specific for an epitope which includes sialylated oligosaccharide chain(s) and is located in the region of the amino-terminal leucine residue of N glycoprotein. MMU^– erythrocytes, lacking both blood group N and Ss glycophorin are non-reactive. Agglutination of MMU⁺ erythrocytes by the anti-N antibodies occursvia interaction with glycophorin B and correlates with the Ss phenotype of red cells MM,S erythrocytes are usually more strongly, agglutinated than MM,ss cells. The agglutination of MM erythrocytes decreases markedly as the pH is increased from 6 to 8, while agglutination of NN red cells is much less affected by shifts in pH over this range. As a result, both monoclonal antibodies are highly anti-N specific typing reagents when the agglutination assay is carried out at pH 8.     

Human blood-group MN and precursor specificities: structural and biological aspects

The human blood-group MM and NN antigens carry 2 to 4 immunodominant groupings per repeating subunit and differ only by one sialic acid residue per immunodominant group. This residue covers in the MM antigen the β-D-galactopyranosyl group that is terminal in the N immunodominant structure and that, together with a terminal α-linked N-acetylneuraminic acid residue, is responsible for N specificity. M specificity was readily converted into N specificity by mild acid treatment. N structure is the immediate biochemical precursor of M structure, and M and N antigenic specificities are not determined by two allelic genes as believed hitherto. The NN antigen was inactivated by β-D-galactosidase as well as by removal of N-acetylneuraminic acid. Some of the reactivities of the NN antigen, lost upon β-D-galactosidase treatment, reappeared on subsequent partial N-acetylneuraminic acid removal. The structure uncovered by complete sialic acid depletion of MN antigens is the Thomsen—Friedenreich T antigen, the specificity of which is determined by β-D-galactopyranosyl groups. β-D-Galactosidase treatment transformed the T antigen into one possessing Tn activity. The significance of blood-group MN active substances extends to human breast cancer, where MN antigens were found in benign and malignant glands, but some of their precursors in cancerous tissue only.     

Novel trivalent anti-influenza reagent

We designed and synthesized novel trivalent anti-influenza reagents. Sialyllactose was located at the terminal of each valence which aimed to block each receptor-binding site of the hemagglutinin (HA) trimer on the surface of the virus. Structural analyses were carried out with a model which was constructed with a computer simulation. A previously reported cyclic glycopeptide blocker [Ohta, T.; Miura, N.; Fujitani, N.; Nakajima, F.; Niikura, K.; Sadamoto, R.; Guo, C.-T.; Suzuki, T.; Suzuki, Y.; Monde, K.; Nishimura, S.-I. Angew. Chem. Int. Ed., 2003, 42, 5186] bound to the HA in the model. The analyses suggest that the glutamine residue in the cyclic peptide bearing Neu5Acα2,3Galβ1,4Glc trisaccharide via a linker interacts with the Gln189 in HA through hydrogen bonding. The present anti-influenza reagents likely interact with a glutamine residue included in the vicinity of Gln189. A plague reduction assay of the influenza virus, A/PR/8/1934 (H1N1), was performed in MDCK cells to evaluate for the synthesized compounds to inhibit viral replication. One of the compounds showed approximately 85% inhibition at the concentration of 400 μM at 4 °C.     

The sugar part of kappa-caseins from cow milk and colostrum and its microheterogeneity.

Cow kappa-casein contains only three different sugars (Gal, GalNAc, NeuNAc). However detailed analyses achieved mainly by gas liquid chromatography suggested a microheterogeneity at the sugar level. After alkaline borohydride treatment, filtration on Bio-Gel P4 and paper chromatography, different carbohydrate parts were obtained. The two main compounds had the following molar compositions: GalNAc (1), Gal(1) and NeuNAc (1) and GalNAc (1), Gal(1) and NeuNAc (2). From these data and our previous sequence studies, some formulae of the polysaccharide part were proposed. One of them was closely related to the sugar sequence of a glycopeptide with MN activity which was in agreement with our observation concerning a cross antigenic reactivity between the N blood group substances and the caseinoglycopeptides. All the polysaccharide parts isolated from colostrum caseinoglycopeptide were much more complex than those obtained from the normal glycopeptide, confirming an evolution of the sugar part as a function of time after parturition.     

Glycopeptides from rat brain glycoproteins

The lipid-free protein residue of rat brain tissue was treated with papain to solubilize the heteropolysaccharide chains of the tissue glycoproteins. The glycopeptides were separated into non-dialyzable and dialyzable glycopeptide preparations. Each preparation was then sorted out into groups of glycopeptides by means of electrophoresis and gel filtration. The quantitatively predominant glycopeptides were the alkali-stable glycopeptides (Group A) which accounted for 64% of the glycopeptide carbohydrate recovered from rat brain. Most of the group A glycopeptides appeared in the non-dialyzable preparation. The molecular weight of the glycopeptides of Group A ranged from approximately 5200–3700. The largest glycopeptide molecule in this mixture possessed the highest electrophoretic mobility and contained one fucose, four N-acetylneuraminic acid (NANA), six N-acetylglucosamine, four galactose, and three mannose residues per molecule. The spectrum of glycopeptides isolated in this group showed a progressive decrease in NANA rsidues, NANA and galactose residues, and NANA, galactose, and N-acetylglucosamine residues which could be correlated with a progressive decline in molecular weight and electrophoretic mobility. Some of the glycopeptides in each fraction recovered from this group of glycopeptides contained sulfate ester groups.A second group of glycopeptides (Group C glycopeptides) accounted for 25% of the total glycoprotein carbohydrate recovered from rat brain. These were recoverd from the dialyzable glycopeptide preparation, and resolved into three fractions by column electrophoresis. These glycopeptides do not contain sulfate, are composed predominately of mannose and N-acetylglucosamine, and possess a molecular weight of approximately 3000.Several minor groups of glycopeptides were detected. Alkali-labile glycopeptides (Group B) appeared in the non-dialyzable glycopeptide preparation. The dialyzable glycopeptide preparation contained glycopeptides (Group E) which contained N-acetylgalactosamine and glucose. These had a molecular weight of approximately 2000. Group D glycopeptides recovered from the dialyzable glycopeptide preparation contained variable amounts of NANA, mannose, galactose, N-acetylglucosamine, and sulfate. These possessed a molecular weight of approximately 2900.     

Efficient synthesis of O-linked glycopeptide by a transglycosylation using endo alpha-N-acetylgalactosaminidase from Streptomyces sp.

Gal beta-(1-->3)-GalNAc-linked hexapeptide was synthesized by a transglycosylation using Gal beta-(1-->3)-GalNAc beta-pNP as a donor and a serine-containing hexapeptide as an acceptor using endo GalNAc-ase from Streptomyces sp.. The Gal beta-(1-->3)-GalNAc residue was transferred to the hydroxyl group of the serine residue of the peptide. The total yield of the glycopeptide via this process was better than that of the chemoenzymatic method. This process was confirmed to be a versatile method for the synthesis of O-linked glycopeptides.     

Structure of the UDP-glucosyltransferase GtfB that modifies the heptapeptide aglycone in the biosynthesis of vancomycin group antibiotics

BACKGROUND: Members of the vancomycin group of glycopeptide antibiotics have an oxidatively crosslinked heptapeptide scaffold decorated at the hydroxyl groups of 4-OH-Phegly4 or beta-OH-Tyr6 with mono- (residue 6) or disaccharides (residue 4). The disaccharide in vancomycin itself is L-vancosamine-1,2-glucose, and in chloroeremomycin it is L-4-epi-vancosamine-1,2-glucose. The sugars and their substituents play an important role in efficacy, particularly against vancomycin-resistant pathogenic enterococci. RESULTS: The glucosyltransferase, GtfB, that transfers the glucose residue from UDP-glucose to the 4-OH-Phegly4 residue of the vancomycin aglycone, initiating the glycosylation pathway in chloroeremomycin maturation, has been crystallized, and its structure has been determined by X-ray analysis at 1.8 A resolution. The enzyme has a two-domain structure, with a deep interdomain cleft identified as the likely site of UDP-glucose binding. A hydrophobic patch on the surface of the N-terminal domain is proposed to be the binding site of the aglycone substrate. Mutagenesis has revealed Asp332 as the best candidate for the general base in the glucosyltransfer reaction. CONCLUSIONS: The structure of GtfB places it in a growing group of glycosyltransferases, including Escherichia coli MurG and a beta-glucosyltransferase from T4 phage, which together form a subclass of the glycosyltransferase superfamily and give insights into the recognition of the NDP-sugar and aglycone cosubstrates. A single major interdomain linker between the N- and C- terminal domains suggests that reprogramming of sugar transfer or aglycone recognition in the antibiotic glycosyltransferases, including the glycopeptide and also the macrolide antibiotics, will be facilitated by this structural information.     

Synthesis of tumor associated sialyl-T-glycopeptides and their immunogenicity.

Sialyl-T-glycopeptides were synthesized by solid-phase techniques, using a PEGA resin as the solid support. An appropriately protected building block containing alpha-Neu5Ac-(2 --> 3)-beta-Gal-(1 --> 3)-alpha-GalN3-(1-->) attached to Fmoc-Thr/Ser-OPfp was employed in a solid phase glycopeptide assembly of a 10-mer glycopeptide, using a general Fmoc/OPfp-ester strategy. Reduction of the azido group of the GalN3 residue was effected on solid-phase, using DTT and DBU. After acidolytic cleavage from the resin, the methyl ester of the sialic acid residue and acetyl groups were removed with 30% NaOMe/MeOH in MeOH and water pH 14, at -30 degrees C for 2 h. At this low temperature, the highly basic conditions did not result in any detectable beta-elimination. However, one O-acetyl group, located at the 2-position of the Gal was resistant to hydrolysis. To remove this remaining acetyl group, reaction with hydrazine hydrate in CHCl3 and MeOH at room temperature for 2.5 h was successful. The two target sequences of sialyl-T-glycopeptides were obtained in good yield. In contrast to the the analogs carrying the T-antigen, the Sial-T-glycopeptides were nonimmunogenic, supporting the idea that the sialylation is a method of circumventing the recognition by the immune system.     

The role of carbohydrate in the blood group N-related epitopes recognised by three new monoclonal antibodies

The specificity of three new monoclonal anti-glycophorin antibodies, reacting preferentially with blood group N antigen, was characterized by means of untreated, enzymatically and chemically modified M and N glycoproteins. All antibodies recognized the NH₂-terminal Leu residue and its amino group, but differed in some other features, including the role of carbohydrate in the epitopes. One of the antibodies (631/3B4, IgM) showed an unusual two-directional dependence of activity on the degree of antigen desialylation. The progressive desialylation of N glycoprotein first caused a strongly increased binding to the epitope, followed by a complete loss of activity. The epitopes for the two remaining antibodies (648/4B5 and 650/4B5, both IgG₁) showed reactivity independent of sialylation, but dependent on the presence of Gal-GalNAc-units. Release of the disaccharide byO-glycanase treatment of N glycoprotein abolished its reactivity with both antibodies.     

Defined geometry of binding between triantennary glycopeptide and the asialoglycoprotein receptor of rat heptocytes

Three derivatives of a triantennary glycopeptide, each containing a single uniquely located 6-amino-galactose residue at either position 6', 6, or 8, were modified at the 6-amino group by attachment of a photolyzable reagent and radiolabeled by iodination of tyrosine. These were allowed to bind to the asialoglycoprotein receptor of isolated rat hepatocytes and photolyzed for affinity labeling. (formula; see text) Each probe specifically labeled either the major (RHL1) or minor (RHL2/3) subunits which comprise the receptor. A photolyzable group attached to galactose residue 6 6' specifically radiolabeled RHL1, whereas a photolyzable group attached to galactose 8 specifically labeled RHL2/3. Photoaffinity labeling of a soluble rat hepatic lectin preparation demonstrated that the minor subunits (RHL2/3) were no longer labeled by the triantennary probe with a photolyzable group at galactose 8. The inhibitory potency of a variety of complex glycopeptides against radiolabeled ligand binding to both rat hepatocytes and soluble lectin are in agreement with photoaffinity results that galactose 8 of triantennary glycopeptide is of unique importance by binding solely to the minor subunits (RHL2/3) of the asialoglycoprotein receptor on hepatocytes. Conversely, galactose residues 6 and 6' bind specifically to the major subunit (RHL1), indicating a precise binding geometry between the trivalent ligand and lectin.