Microheterogeneity and oligosaccharide chains on the beta chains of HLA-DR, human major histocompatibility complex class II antigen, analyzed by the lectin-nitrocellulose sheet method

The beta chain of human histocompatibility complex class II antigen, HLA-DR, showed 4 to 5 microheterogeneous spots on a gel obtained by two-dimensional polyacrylamide gel electrophoresis. The types of oligosaccharide chains on the beta chains were analyzed by the lectin-nitrocellulose sheet method for each microheterogeneous spot with 3 cell lines of two haplotypes (HLA-DR 4,4, and 3,3). Two kinds of oligosaccharide chains were observed and were essentially the same in the microheterogeneous spots from all three cell lines. One, the oligosaccharide chain on the most basic spot (beta 1), was stained with peroxidase-coupled concanavalin A (Con A-P.O.) but not with peroxidase-coupled wheat germ agglutinin and was sensitive to endo-beta-N-acetylglucosaminidase H (endo H), indicating that it was a high-mannose type. The oligosaccharide chains on other spots that were not stained with Con A-P.O. but were stained with peroxidase-coupled Ricinus communis agglutinin were resistant to endo H. beta 2 and beta 3 were stained with E-PHA. Thus, they probably had bisected biantennary and others probably had multiantennary complex-type oligosaccharides. Sialidase experiments showed that the charge heterogeneity was due to post-translational sialylation of the oligosaccharide chains. In pulse-chase experiments, the most basic spot of beta chain (beta 1) was labeled first, beta 2 and beta 3 were labeled next, and beta 4 was labeled last. These labeling characters accorded well with the results on the oligosaccharide types mentioned above.     

Age-Dependent Changes in the Oligosaccharide Structure of the Major Myelin Glycoprotein, P0

The single oligosaccharide moiety of the major myelin glycoprotein, P0, resides in an immunoglobulin-like domain that appears to participate in homophilic binding. The studies presented here indicate that the structure of the P0 oligosaccharide from rat nerve changes as a function of Schwann cell age. Examination of 5-day-old nerve revealed that P0 contained predominantly endo-beta-N-acetylglucosaminidase H (endo H)-resistant, complex-type oligosaccharide. In contrast, P0 from adult rats had mostly endo H-sensitive carbohydrate, indicating the presence of appreciable high-mannose and/or hybrid-type oligosaccharide on the glycoprotein. The endo H-sensitive and -resistant P0 of adult nerve could be readily phosphorylated by protein kinase C, as could the complex-type P0 from 5-day-old nerve. This suggests that the glycoprotein progresses to the plasma membrane and myelin regardless of the type of oligosaccharide chain. Analysis of 35SO4(2-)-labeled P0 showed that the sulfate group was found on both endo H-sensitive and -resistant oligosaccharide. The endo H-sensitive P0 carbohydrate from adult nerve appears to be primarily of the hybrid type, as evidenced by (a) the elution profile of [3H]mannose-labeled P0 glycopeptides from adult nerve during concanavalin A chromatography and (b) the inability of P0 from adult nerve to interact with Galanthus nivalis agglutinin. The observed age-dependent changes of P0 oligosaccharide may modify the binding properties of this myelin glycoprotein.     

Incompletely processed LH molecules synthesized by rat gonadotrophs treated with inhibitors of oligosaccharide processing

Inhibitors of N-linked oligosaccharide processing are useful tools for studies on the biological function of the oligosaccharide structures in glycoprotein hormones. We have synthesized molecules of lutropin (LH) containing high-mannose- and hybrid-type oligosaccharides using rat gonadotroph-enriched primary cultures in the presence of castanospermine (a glucosidase I inhibitor) or swainsonine (a mannosidase II inhibitor), in order to compare the actions of these molecules with that of the hormone containing complex-type oligosaccharides in the activation of the receptor-adenylate cyclase system. Treatment of gonadotrophs with the above inhibitors caused an increase in the synthesis of highly basic LH molecules (pI 9.6-10.0), because addition of charged carbohydrate moieties to these molecules was prevented. Characterization of the oligosaccharide structure performed by enzymatic treatment (endoglycosidase H and neuraminidase) and the use of immobilized lectins (wheat germ agglutinin and Ricinus communis agglutinin-120) showed that these inhibitor-synthesized LH molecules contained high-mannose- and hybrid-type (asialo and sialylated) oligosaccharides. Their immunological properties were similar to that of complex-type oligosaccharide LH, but they had significantly higher receptor-binding ability in comparison with a sialylated complex-type oligosaccharide LH (about 12-fold) and an asialo complex-type oligosaccharide LH (about 3-fold). It was noted that the incompletely processed molecules were less potent than complex-type oligosaccharide LH in the activation of adenylate cyclase of Leydig cells, showing about 40-60% of the activity induced by the sialylated complex-type oligosaccharide molecule. The present data indicate that the inhibition of terminal processing of N-linked oligosaccharides by castanospermine and swainsonine impairs the full hormonal function of rat LH.     

Site-specific characterization of the N-linked oligosaccharides of a murine immunoglobulin M by high-performance liquid chromatography/electrospray mass spectrometry

Immunoglobulin M is an especially important product of the immune system because it plays a critical role in early protection against infections. In this report, the glycosylation pattern of the protective murine monoclonal IgM 12A1 to Cryptococcus neoformans polysaccharide was analyzed by high-performance liquid chromatography coupled with electrospray ionization mass spectrometry. Peptide mapping studies covering 88% of the deduced amino acid sequence indicated that of the six potential N-glycosylation sites in this antibody only five were utilized, as the tryptic peptide derived from monoclonal IgM 12A1 containing Asn-260 was recovered without carbohydrates. The oligosaccharide side chains of monoclonal IgM 12A1 were characterized at each of the N-glycosylation sites. Asn-166 possessed 20 monosialylated and nonsialylated, and fucosylated and nonfucosylated complex- and hybrid-type oligosaccharides and one high-mannose-type oligosaccharide. Thirteen oligosaccharides were attached to the site at Asn-401, including six complex-type, four hybrid-type, and three high-mannose-type oligosaccharides. Twelve hybrid-type oligosaccharides were attached to Asn-378, three of which had terminal sialic acids. Eleven hybrid-type oligosaccharides were attached to Asn-331, seven of which had terminal sialic acids. Only two high-mannose type oligosaccharides were attached to Asn-363. These results indicated great complexity in the structure and composition of oligosaccharides attached to individual IgM glycosylation sites.     

Deglycosylation of alpha 1-proteinase inhibitor by endo-beta-N-acetylglucosaminidase F

The glycosidase endo-beta-N-acetylglucosaminidase F (endo F) from Flavobacterium meningosepticum was used for the deglycosylation of rat alpha 1-proteinase inhibitor (alpha 1 PI). alpha 1 PI containing three oligosaccharide side chains of the complex type was isolated from rat serum or from the medium of rat hepatocyte primary cultures. High-mannose-type alpha 1 PI or hybrid-type alpha 1 PI was isolated from the media of hepatocytes treated with 1-deoxymannojirimycin or swainsonine, respectively. The susceptibility of complex-type alpha 1 PI to endo F was studied in the presence of various detergents. 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate and octyl glucopyranoside turned out to be most effective. In the absence of detergents, digestion of alpha 1 PI with high concentrations of endo F and/or long times of incubation led to the formation of alpha 1 PI with one and two oligosaccharide side chains. In the presence of 0.5% octyl glucopyranoside, the major cleavage products were unglycosylated alpha 1 PI and alpha 1 PI carrying one carbohydrate side chain. In contrast to the complex-type alpha 1 PI, the high-mannose type can be totally deglycosylated by endo F even in the absence of detergents. The susceptibility of the hybrid-type alpha 1 PI to endo F is between that of the complex and the high-mannose types.     

The structures of the asparagine-linked sugar chains of bovine interphotoreceptor retinol-binding protein. Occurrence of fucosylated hybrid-type oligosaccharides

The sugar chains of interphotoreceptor retinol-binding protein purified from the interphotoreceptor matrix of bovine eyes were liberated from the polypeptide portion by hydrazinolysis followed by N-acetylation and NaB[3H]4 reduction. The oligosaccharide fraction thus obtained was separated into four acidic fractions by paper electrophoresis. The four acidic fractions were confirmed to be mixtures of mono-, di-, tri-, and tetrasialyloligosaccharides. Both N-acetyl- and N-glycolylneuraminic acids were found as sialic acids of interphotoreceptor retinol-binding protein. The monosialylated oligosaccharide fraction, which accounted for 40 molar per cent of the total oligosaccharides liberated, was a mixture of the following hybrid-type oligosaccharides: (Formula: see text) This is the first time that fucosylated hybrid-type oligosaccharides have been found in any glycoprotein. The di-, tri-, and tetrasialyloligosaccharide fractions were composed of biantennary complex-type oligosaccharides, the outer chains of which are either Sia alpha 2----(3- or 6-linked)Gal beta 1----3(Sia alpha 2----6)GlcNac or Sia alpha 2----(3- or 6-linked)Gal beta 1----4GlcNAc.     

Binding site of the rat liver specific monoclonal antibody

A rat liver-specific antigen (RLSA) lost its binding ability to the corresponding monoclonal antibody after treatment with N-glycanase or sialidase, which suggested that the specific binding site might be in a portion of the sugar chain containing sialic acid. The specific antigen reacted with wheat germ agglutinin, lentil lectin, erythroagglutinating phytohemagglutinin and Ricinus communis agglutinin, but not with concanavalin A or peanut agglutinin. These results suggest that the specific antigen has asparagine-linked complex-type sugar chains which might be the binding sites of the monoclonal antibody.     

Analysis of N-linked oligosaccharide chains of glycoproteins on nitrocellulose sheets using lectin-peroxidase reagents

A rapid and convenient method was established for analysis of the N-linked carbohydrate chains of glycoproteins on nitrocellulose sheets. Proteins were separated by polyacrylamide gel electrophoresis, transferred to nitrocellulose sheets, reacted with peroxidase-coupled lectins, and detected by color development of the enzyme reaction. Four glycoproteins having N-linked oligosaccharide chains were used as test materials: Taka-amylase A (which has a high-mannose-type chain), ovalbumin (high-mannose-type chains and hybrid-type chains), transferrin (biantennary chains of complex type), and fetuin (triantennary chains of complex type and O-linked-type chains). Concanavalin A interacted with Taka-amylase A, transferrin, and ovalbumin but barely interacted with fetuin. After treatment of the glycoproteins on a nitrocellulose sheet with endo-beta-N-acetylglucosaminidase H, transferrin reacted with concanavalin A but Taka-amylase A and ovalbumin did not. Wheat germ agglutinin interacted with Taka-amylase A but not ovalbumin; therefore, they were distinguishable from each other. Fetuin and transferrin were detected by Ricinus communis agglutinin or peanut agglutinin after removal of sialic acid by treatment with neuraminidase or by weak-acid hydrolysis. Erythroagglutinating Phaseolus vulgaris agglutinin detected fetuin and transferrin. Thus, the combined use of these procedures distinguished the four different types of N-linked glycoproteins. This method was also applied to the analysis of membrane glycoproteins from sheep red blood cells. The terminally positioned sugars of sialic acid, alpha-fucose, alpha-galactose, and alpha-N-acetylgalactosamine were also detected with lectins from Limulus polyphemus, Lotus tetragonolobus, Maclura pomifera, and Dolichos biflorus, respectively.     

Oligosaccharide structures present on asparagine-289 of recombinant human plasminogen expressed in a Chinese hamster ovary cell line

The oligosaccharide structures linked to Asn289 of a recombinant (r) variant (R561S) human plasminogen (HPg) expressed in Chinese hamster ovary (CHO) cells, after transfection of these cells with a plasmid containing the cDNA coding for the variant HPg, have been determined. Employing high-performance anion-exchange liquid chromatography mapping of the oligosaccharide units cleaved from the protein by glycopeptidase F, compared with elution positions of standard oligosaccharides, coupled with monosaccharide compositional determinations and analyses of sequential exoglycosidase digestions and specific lectin binding, we find that considerable microheterogeneity in oligosaccharide structure exists at this sole potential N-linked glycosylation site on HPg. A variety of high-mannose structures, as well as bi-, tri-, and tetraantennary complex-type carbohydrate, has been found, in relative amounts of 1-25% of the total oligosaccharides. The complex-type structures contain variable amounts of sialic acid (Sia), ranging from 0 to 5 mol/mol of oligosaccharide in the different glycan structures. Neither hybrid-type molecules, N-acetylglucosamine bisecting oligosaccharides, nor N-acetyllactosaminyl-repeat structures were found to be present in the complex-type carbohydrate pool in observable amounts. Of interest, a significant portion of the Sia exists an outer arm structures in an (alpha 2,6) linkage to the penultimate galactose, a novel finding in CHO cell-directed glycosylation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of the N- and O-linked oligosaccharides in glycoproteins synthesized by Schistosoma mansoni schistosomula

This report describes the structural analyses of the O- and N-linked oligosaccharides contained in glycoproteins synthesized by 48-hr-old Schistosoma mansoni schistosomula. Schistosomula were prepared by mechanical transformation of cercariae and were then incubated in media containing either [2-3H] mannose, [6-3H]glucosamine, or [6-3H]galactose to metabolically radiolabel the oligosaccharide moieties of newly synthesized glycoproteins. Analysis by SDS-polyacrylamide gel electrophoresis and fluorography demonstrated that many glycoproteins were metabolically radiolabeled with the radioactive mannose and glucosamine precursors, whereas few glycoproteins were labeled by the radioactive galactose precursor. Glycopeptide were prepared from the radiolabeled glycoproteins by digestion with pronase and fractionated by chromatography on columns of concanavalin A-Sepharose and pea lectin-agarose. The structures of the oligosaccharide chains in the glycopeptides were analyzed by a variety of techniques. The major O-linked sugars were not bound by concanavalin A-Sepharose and consisted of simple O-linked monosaccharides that were terminal O-linked N-acetylgalactosamine, the minor type, and terminal O-linked N-acetylglucosamine, the major type. The N-linked oligosaccharides were found to consist of high mannose- and complex-type chains. The high mannose-type N-linked chains, which were bound with high affinity by concanavalin A-Sepharose, ranged in size from Man6GlcNAc2 to Man9GlcNAc2. The complex-type chains contained mannose, fucose, N-acetylglucosamine, and N-acetylgalactosamine. No sialic acid was present in any metabolically radiolabeled glycoproteins from schistosomula.     

Analysis of the oligosaccharides on rat androgen-binding protein using serial lectin chromatography

The microheterogeneity seen when rat androgen-binding protein (rABP) is analyzed by two-dimensional polyacrylamide gel electrophoresis is attributable, at least in part, to the differential glycosylation of a single promoter. Further insight into the chemical nature of the oligosaccharide units on rABP was obtained by serial lectin chromatography. When rABP was chromatographed on immobilized Concanavalin A (Con-A), it was fractionated into three classes: (1) one that did not bind to the lectin (about 44% of the rABP), (2) one that was bound and could be eluted with 10 mM 1-O-methyl alpha-D-glucopyranoside (glucoside), about 34%, and (3) one that could be eluted with 0.5 M methyl alpha-D-mannopyranoside (mannoside), about 23%. Binding to Con-A indicates the presence of asparagine-linked oligosaccharides. Chromatography of the glucoside-eluted peak on lentil lectin (LcH) indicated that the rABP in that fraction contained a fucose residue on the chitobiose core. Chromatography of the mannoside-eluted peak on wheat germ agglutinin (WGA) indicated the presence of rABP with high mannose- (44%) and hybrid-type (56%) glycans attached. Chromatography on Ricinus communis I (RCA-I) lectin indicated a species containing galactosylated complex-type oligosaccharide chains. Treatment of rABP forms with exoglycosidases confirmed the presence of externally disposed fucose, sialic acid, mannose, and galactose residues. LcH chromatography indicated that about 30% of the rABP that did not bind to Con-A possessed triantennary oligosaccharides with fucose on the chitobiose core. About 28% of the rABP was retarded when it was chromatographed on Phaseolus vulgaris E lectin, suggesting the presence of bisected biantennary chains with terminal galactose residues. We were unable to detect rABP species with serine- or threonine-linked oligosaccharide chains in this fraction. Other forms of rABP in the nonretained fraction of Con-A were not resolved. Western blotting did not reveal major differences in relative molecular weight (Mr) among the rABP species; some differences in the ratio of the heavy to the light subunit of the molecule were detectable.     

Oligosaccharides of the Hazelhurst vesicular stomatitis virus glycoprotein are more extensively processed in Rous sarcoma virus-transformed baby hamster kidney cells

Because of the extensive oligosaccharide heterogeneity of the membrane glycoprotein (G) from the Hazelhurst strain of vesicular stomatitis virus, this virus has been used as a specific intracellular probe of altered protein glycosylation in Rous sarcoma virus-transformed versus normal baby hamster kidney cells. Over 70% of G protein from virus released from the transformed cells had acidic-type oligosaccharides at both glycosylation sites, compared to less than 50% from the corresponding normal host cells. The remaining G protein contained an acidic-type oligosaccharide at one site and an endo-beta-N-acetylglucosaminidase H-sensitive oligosaccharide at the other. The major endoglycosidase-sensitive species were sialylated hybrid-type (NeuNAc-Gal-GlcNAc-Man5GlcNAc2-Asn) from the transformed and neutral-type (Man5-6GlcNAc2-Asn) from the normal host cells. The degree of branching of the acidic-type oligosaccharides was not increased in the transformed cells (approx. 80% biantennary for viral G protein from both cell types). At a reduced growth temperature (24 versus 37 degrees C), the G protein oligosaccharides were more extensively processed in both cell types (approximately 85-95% of G protein contained acidic-type structures at both sites), even though the level of viral protein synthesis and virus release was decreased. Essentially all of the minor, endoglycosidase-sensitive oligosaccharides on mature viral G protein were sialic acid-containing hybrid-type structures. At 24 degrees C the branching of the acidic-type oligosaccharides was increased in the virus released from the transformed cells versus normal cells.     

Structural characterization of the oligosaccharides of a human monoclonal anti-lipopolysaccharide immunoglobulin M

The oligosaccharide side chains of a human anti-lipopolysaccharide IgM produced by a human-human-mouse heterohybridoma were analyzed at each of its five conserved N-glycosylation sites. This antibody also has a potential sixth N-glycosylation site in the variable region of its heavy chain which is not glycosylated. The oligosaccharides were released by digestion with various endo- and exoglycosidases and analyzed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry and fluorophore-assisted carbohydrate electrophoresis. The antibody has various complex- and hybrid-type oligosaccharide structures at Asn 171, various sialylated complex-type oligosaccharides at Asn 332 and 395, and high-mannose-type oligosaccharides at Asn 402 and 563. Of note is the presence in this human IgM of oligosaccharides containing N-glycolylneuraminic acid and N-acetylneuraminic acid in the ratio of 98:2 as determined using anion- exchange chromatography. Furthermore, we observed oligosaccharide structures containing Gal alpha (1,3)Gal that have not been reported as components of human glycoproteins.      

Detailed structural analysis of asparagine-linked oligosaccharides of the nicotinic acetylcholine receptor from Torpedo californica.

The structures of the major oligosaccharide moieties of the nicotinic acetylcholine receptor (AcChoR) protein from Torpedo californica have been reported [Nomoto, H., Takahashi, N., Nagaki, Y., Endo, S., Arata, Y. and Hayashi, K. (1986) Eur. J. Biochem. 157, 233-242] to be high-mannose types. Here we report detailed analyses of the structures of the remaining oligosaccharides in this receptor. The sialylated oligosaccharides released by glycopeptidase (almond) digestion were separated according to the number of sialic acid residues using high-performance anion-exchange chromatography with pulsed amperometric detection. After removal of sialic acid from each fraction, the resulting neutral oligosaccharides were separately pyridylaminated and were analyzed by a combination of sequential exoglycosidase digestion and HPLC, then identified on a two-dimensional sugar map. The structures of two desialylated pyridylamino-oligosaccharides were further analyzed by high-resolution proton NMR. Each oligosaccharide was composed of species containing varying numbers of sialic acids. The desialylated complex-type oligosaccharides of AcChoR consisted of ten, eight and one different biantennary, triantennary and tetraantennary oligosaccharide, respectively. The biantennary oligosaccharides were divided into two groups; oligosaccharides with fucose at the proximal N-acetylglucosamine (six varieties) and oligosaccharides without fucose (four varieties). Each group consisted of species differing in the number of terminal galactose residues. The major component of the biantennary oligosaccharides had two galactose residues at the non-reducing termini. The terminal alpha-galactose residue(s) linked to C3 of beta-galactose were found in the fucose-containing biantennary oligosaccharides (two varieties). The triantennary oligosaccharides were also divided into two groups; oligosaccharides with (four varieties) and without (four varieties) besecting N-acetylglucosamine. These groups were composed of species differing in the number of terminal galactose residues. The major component of the triantennary oligosaccharides was fully galactosylated with three galactose residues. An unusual group, Gal beta 1-3GlcNAc, was present in low levels in the triantennary oligosaccharides. In contrast, the tetraantennary oligosaccharide was composed of only one species, which is fully galactosylated with four galactose residues.     

Intracellular processing of apolipoprotein J precursor to the mature heterodimer.

Circulating apolipoprotein J (apoJ) is a 70 kDa glycoprotein comprised of disulfide-linked alpha and beta subunits derived from a single precursor. Post-translational modifications that occur prior to apoJ secretion were assessed, with specific focus on carbohydrate type, the timing of proteolytic cleavage, and the importance of glycosylation on the cleavage and secretion processes. ApoJ was initially resolved as a single chain, intracellular precursor of 58 kDa which contained N-linked oligosaccharide but no O-linked oligosaccharide. The precursor was converted to an intracellular 70 kDa glycoprotein, which became the major intracellular form of apoJ prior to secretion. Maturation of the 58 kDa precursor involved conversion of high-mannose carbohydrate to complex-type carbohydrate containing sialic acid, as well as intracellular cleavage to yield alpha and beta subunits. This cleavage event occurred at a late stage of carbohydrate modification, most likely in the trans-Golgi or a post-Golgi compartment. The maturation and secretion of apoJ occurred rapidly, with a half-time of 30-35 min. Tunicamycin treatment of cells resulted in an unglycosylated doublet comprised of one single chain and one cleaved form of apoJ. The unglycosylated apoJ species were secreted rapidly with a half-time of 20 min. Both cleavage and secretion were independent of glycosylation.     

Fucosylated hybrid-type N-glycans on the secreted human epidermal growth factor receptor from swainsonine-treated A431 cells

N-Glycans linked to the human secreted form of epidermal growth factor receptor were isolated from A431 cells after swainsonine treatment. Analysis of the oligosaccharides by (1)H NMR spectroscopy and mass spectrometry shows the presence of oligomannose- and (alpha2-3)-sialylated hybrid-type glycans. The major hybrid-type oligosaccharide chains are fucosylated at the Asn-bound GlcNAc residue. Smaller amounts of the hybrid-type structures are also fucosylated at peripheral GlcNAc residues, constituting the sialyl-Le(x) antigen. No complex-type glycans are found, suggesting the absence of alpha-mannosidase III. An assay for alpha-mannosidase III on the A431 cells in the absence and presence of 6 microM swainsonine shows that Man(5)GlcNAc(2) is not converted into Man(3)GlcNAc(2), thereby confirming that these cells do not contain alpha-mannosidase III activity.     

O- and N-glycosylation lead to different molecular mass forms of human monocyte interleukin-6

The biosynthesis and secretion of human interleukin-6 (IL-6) was studied in monocyte cultures stimulated with endotoxin. After labeling with [35S]methionine and immunoprecipitation with a specific antiserum one major (24 kDa) and four minor (27.5, 23.3, 22.5 and 21.8 kDa) molecular mass forms of IL-6 could be found in the cells and media. Incubation of monocyte media with sialidase and subsequently with endo-alpha-N-acetylgalactosaminidase, which cleaves Gal(beta 1-3)Gal-NAc from serine or threonine, led to the formation of only two forms of IL-6 with apparent molecular masses of 25 and 21.8 kDa. The latter had an electrophoretic mobility indistinguishable from that of 125I-labeled recombinant human IL-6. The results suggest that human monocyte IL-6 carries O-glycosidically bound carbohydrates with a Gal(beta 1-3)Gal-NAc core to which only sialic acid is bound. Differences in O-glycosylation are the major cause for the molecular heterogeneity of IL-6. A small part of IL-6 (27.5 kDa form) is in addition N-glycosylated. Incubation of monocytes with tunicamycin and 1-deoxymynnojirimycin and treatment of IL-6 with endoglucosaminidase H suggested that the 27.5 kDa form of IL-6 carries at least one N-linked complex-type oligosaccharide chain.     

Oligosaccharides of the glycoprotein of rabies virus.

The number of oligosaccharide side chains on rabies virus glycoprotein (G-protein) was investigated. Analysis of glycopeptides obtained by protease digestion of desialated G-protein revealed three discrete glycopeptides. Comparison of the protease digestion products from desialated and from untreated G-protein indicated a heterogeneity among the glycopeptides in the sialic acid content. Two major tryptic glycopeptides were isolated from desialated rabies virus G-protein and analyzed after protease digestion; one contained two oligosaccharide side chains and the other contained a single oligosaccharide side chain.     

Formation of homogeneous carbohydrate-lectin cross-linked precipitates from mixtures of D-galactose/N-acetyl-D-galactosamine-specific lectins and multiantennary galactosyl carbohydrates.

Quantitative precipitation studies have shown that the Man/Glc-specific lectin concanavalin A (ConA) forms homogeneous (homopolymeric) cross-linked precipitates with individual asparagine-linked oligomannose and bisected hybrid-type glycopeptides in the presence of binary mixtures of the carbohydrates [Bhattacharyya, L., Khan, M. I. & Brewer, C. F. (1988) Biochemistry 27, 8762-8767]. The results indicate that the ConA-glycopeptide precipitates are highly organized cross-linked lattices that are unique for each carbohydrate. Using similar techniques, the present study shows that the Gal-specific lectins from Erythrina indica and Ricinus communis (agglutinin I) form homogeneous cross-linked complexes with individual carbohydrates in binary mixtures of triantennary and tetraantennary complex-type oligosaccharides with terminal Gal residues. Conversely, binary mixtures of Gal/GalNAc-specific lectins from E. indica, Erythrina cristagalli, Erythrina flabelliformis, R. communis, soybean (Glycine max), and Wistaria floribunda (tetramer) in the presence of a naturally occurring or synthetic branched-chain oligosaccharide with terminal GalNAc or Gal residues provide evidence for the formation of separate cross-linked lattices between each lectin and the carbohydrate. The present results therefore demonstrate the formation of homogeneous lectin-carbohydrate cross-linked lattices in (a) a mixture of branched-chain complex-type oligosaccharides in the presence of a specific Gal/GalNAc-binding lectin, and (b) a mixture of lectins with similar physicochemical and carbohydrate binding properties in the presence of an oligosaccharide. These findings show that lectin-carbohydrate cross-linking interactions provide a high degree of specificity which may be relevant to their biological functions as receptors.     

Biosynthesis and glycosylation of the human complement regulatory protein decay-accelerating factor

The biosynthesis and oligosaccharide structure of the human complement regulatory glycoprotein decay-accelerating factor (DAF) were studied in erythrocytes and cell lines. Initial information relative to carbohydrate moieties of DAF was obtained by enzymatic digestions. The 74,000 Mr erythrocyte DAF was lowered 3000 by endoglycosidase F, whereas endoglycosidase H had no effect, indicating one N-linked complex-type unit. Treatment with endo-alpha-N-acetylgalactosaminidase to remove O-linked oligosaccharides resulted in a 48,000 Mr molecule (67% of the Mr shift being due to sialic acid), which decreased to 45,000 Mr after sequential endoglycosidase F treatment. To additionally define the oligosaccharide structure and identify precursors in biosynthetic pathways, DAF was studied in the HL-60 cell line differentiated by vitamin D toward monocytes. Pulse-chase experiments with [35S]methionine revealed a precursor species of 43,000 Mr that underwent an early post-translational modification to a 46,000 Mr intermediate, and subsequently was chased into a mature species of 80,000 Mr that aligned with 125I surface-labeled DAF from these cells. All three forms of DAF were approximately 3000 lower in Mr in the presence of tunicamycin. The two lower Mr DAF species were sensitive to endoglycosidases F and H but not to neuraminidase or endo-alpha-N-acetylgalactosaminidase. In summary, DAF is synthesized as a 43,000 Mr precursor species containing one N-linked high-mannose unit. Before entering the central region of the Golgi, it is converted to a 46,000 Mr species by an as yet unknown post-translational modification. The 46,000 Mr form is converted to the 74,000 Mr (erythrocyte) or 80,000 Mr (leukocyte) membrane form of DAF by the addition of multiple, sialylated O-linked oligosaccharide chains (responsible for the large electrophoretic mobility shift) and conversion of the single N-linked high-mannose unit to a complex-type structure. The cell-specific Mr variation between red and white blood cells arises during this post-translational modification from the 46,000 Mr biosynthetic intermediate to the mature DAF species expressed on the cell surface.