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Structural basis of the heterogeneity of asparagine-linked oligosaccharides of a Waldenstrom's macroglobulinemia immunoglobulin M
Abstract
The extent of glycans heterogeneity in a pathological human immunoglobulin M ZAJ has been studied on oligosaccharides released by hydrazinolysis from the purified glycoprotein. After reduction with NaB3H4, asparagine-linked carbohydrate chains were separated by affinity chromatography on concanavalin A-Sepharose into oligomannosidic and N-acetyllactosaminic types. Glycans of the oligomannosidic type were further fractionated by HPLC and those of the N-acetyllactosamine type by preparative high-voltage electrophoresis. The primary structure of the main oligosaccharides was investigated on the basis of micro-methylation analysis, mass spectrometry and sequential exo-glycosidase digestion. Glycans of the oligomannosidic type varied in size from Man5GlcNAc2 to Man9GlcNAc2. N-Acetyllactosaminic glycans were found of the biantennary, bisected-biantennary and triantennary types. They presented a higher degree of heterogeneity due to the presence of a variable number of NeuAc and fucose residues. The new structures we report here were in addition to the major biantennary one we previously described on the basis of methylation analysis and 500 MHz 1H-NMR spectroscopy (Cahour, A., Debeire, P., Hartmann, L., Montreuil, J., Van Halbeek, H. and Vliegenthart, J.F.G. (1984) FEBS Lett. 170, 343-349): NeuAc(alpha 2-6)Gal(beta 1-4)GlcNAc(beta 1-2)Man(alpha 1-3)[Gal(beta 1-4)Glc-NAc(beta 1-2)Man(alpha 1-6)]Man(beta 1-4)]Glc-NAc(beta 1-4) [Fuc(alpha 1-6)]GlcNAc.
The pentameric 71-domain structure of human and mouse immunoglobulin M (IgM) was investigated by synchrotron X-ray solution scattering and molecular graphics modelling. The radii of gyration RG of human IgM Quaife and its Fc5, IgM-S, Fab'2 and Fab fragments were determined as 12.2 nm, 6.1 nm, 6.1 nm, 4.9 nm and 2.9 nm in that order. The RG values were similar for mouse IgM P8 and its Fab'2 and Fab fragments, despite the presence of an additional carbohydrate site. The IgM scattering curves, to a nominal resolution of 5 nm, were compared with molecular graphics models based on published crystallographic alpha-carbon co-ordinates for the Fab and Fc structures of IgG. Good curve fits for Fab were obtained based on the crystal structure of Fab from IgG. A good curve fit was obtained for Fab'2, if the two Fab arms were positioned close together at their contact with the C mu 2 domains. The addition of the Fc fragment close to the C mu 2 domains of this Fab'2 model, to give a planar structure, accounted for the scattering curve of IgM-S. The Fc5 fragment was best modelled by a ring of five Fc monomers, constrained by packing considerations and disulphide bridge formation. A position for the J chain between two C mu 4 domains rather than at the centre of Fc5 was preferred. The intact IgM structure was best modelled using a planar arrangement of these Fab'2 and Fc5 models, with the side-to-side displacement of the Fab'2 arms in the plane of the IgM structure. All these models were consistent with hydrodynamic simulations of sedimentation data. The solution structure of IgM can therefore be reproduced quantitatively in terms of crystallographic structures for the fragments of IgG. Putative Clq binding sites have been identified on the C mu 3 domain. These would become accessible for interaction with Clq when the Fab'2 arms move out of the plane of the Fc5 disc in IgM, that is, a steric mechanism exposing pre-existing Clq sites. Comparison with a solution structure for Clq by neutron scattering shows that two or more of the six globular Clq heads in the hexameric head-and-stalk structure are readily able to make contacts with the putative Clq sites in the C mu 3 domains of free IgM if if the Clq arm-axis angle in solution is reduced from 40 degrees-45 degrees to 28 degrees. This could be the trigger for Cl activation.
A stereocontrolled synthetic route to a glycotetraoside, allyl O-(3,4,6-tri-O-benzyl-2-deoxy-2-phthalimido-beta-D-glucopyranosyl)-(1--- -4)-O- (3,6-di-O-allyl-2-O-benzyl-beta-D-mannopyranosyl)-(1----4)-O-3, 6-di-O-benzyl-2-deoxy-2-phthalimido-beta-D-glucopyranosyl)-(1----4)-3-O- benzyl- 2-deoxy-6-O-p-methoxy-phenyl-2-phthalimido-beta-D-glucopyranoside, an important intermediate for the synthesis of "bisected" complex type glycans of glycoproteins has been established by employing two glycosyl donors, 3,4,6-tri-O-benzyl-2-deoxy-2-phthalimido-beta-D-glucopyranosyl trichloroacetimidate and 4-O-acetyl-3,6-di-O-allyl-2-O-benzyl-alpha-D-mannopyranosyl bromide, and a glycosyl acceptor, allyl O-(3,6-di-O-benzyl-2-deoxy-2-phthalimido-beta-D-glucopyranosyl)-(1----4) -3-O- benzyl-2-deoxy-6-O-p-methoxyphenyl-2-phthalimido-beta-D-glucopyranoside.
Purified rat liver UDP-GlcNAc:alpha-D-mannoside beta 1-2 N-acetylglucosaminyltransferase II (Bendiak, B., and Schachter, H. (1987) J. Biol. Chem. 262, 5775-5783) has been characterized kinetically, and its substrate specificity and inhibition characteristics have been determined. Kinetic data indicate an ordered, or largely ordered sequential mechanism, with UDP-GlcNAc binding prior to the acceptor. The minimal acceptor structure required for full activity is: (Formula: see text) The acceptor molecule must have a terminal Man alpha 1-6 residue, and a terminal GlcNAc beta 1-2Man alpha 1-3 branch to display any activity, but does not require the reducing GlcNAc residue, as the enzyme was about 50% as active after reduction of this residue to N-acetylglucosaminitol. Additional residues (Gal beta 1-4 on the GlcNAc beta 1-2Man alpha 1-3 arm, or a bisecting GlcNAc beta 1-4 on the beta-Man residue) abolish catalytic activity. These results suggest a rigid order in the biosynthesis of all N-linked complex oligosaccharides (bisected and nonbisected bi-, tri-, and tetraantennary), since the enzyme must act to completion prior to the action of either UDP-Gal:GlcNAc beta 1-4 galactosyltransferase or N-acetylglucosaminyltransferase III to make such structures. Inhibition studies with nucleotides, sugars, nucleotide-sugars, and their respective analogues revealed that analogues of UDP and UTP, in which the hydrogen at the 5 position of the uracil was substituted with -CH3, bromine, or mercury (as the mercaptide) were good reversible inhibitors of the enzyme, whereas substitution at other sites lessened the inhibitory potency, usually to a large degree.
The activity of N-acetylglucosaminyltransferase III, which adds a "bisecting" GlcNAc in beta 1,4 linkage to the beta-linked Man of the core of Asn-linked oligosaccharides (Narasimhan, S. (1982) J. Biol. Chem. 257, 10235-10242), was determined in hepatic nodules of rats initiated by administration of a single dose of carcinogen 1,2-dimethylhydrazine.2HCl (100 mg/kg, intraperitoneal) 18 h after partial hepatectomy and promoted by feeding a diet supplemented with 1% orotic acid for 32-40 weeks. N-Acetylglucosaminyltransferase III was assayed using glycopeptide GlcNAc beta 1,2Man alpha 1,6(GlcNAc beta 1,2Man alpha 1,3)Man beta 1, 4GlcNAc beta 1,4GlcNAc-Asn as substrate and, as enzyme sources, microsomal membranes of the hepatic nodules, surrounding liver, regenerating liver, and age- and sex-matched control liver. The nodules had significant N-acetylglucosaminyltransferase III activity (0.78-2.18 nmol GlcNAc transferred/h/mg of protein), while the surrounding liver, the regenerating liver (24 h after partial hepatectomy), and the control liver had negligible activity (0.02-0.03 nmol/h/mg of protein). Product isolated from a large scale N-acetylglucosaminyltransferase III incubation with hepatic nodules as enzyme source showed the presence of the bisecting GlcNAc residue by 500 MHz proton NMR spectroscopy. Concomitant with the appearance of N-acetylglucosaminyltransferase III activity in the preneoplastic nodules, the activities of N-acetylglucosaminyltransferase I and II were decreased in these membranes when compared to those from surrounding liver, regenerating liver, and control liver. These results suggest that N-acetylglucosaminyltransferase III is induced at the preneoplastic stage in liver carcinogenesis promoted by orotic acid and are consistent with the reported presence of bisecting GlcNAc residues in the Asn-linked oligosaccharides of rat and human hepatoma gamma-glutamyl transpeptidase and their absence in enzyme from normal liver of rats and humans (Kobata, A., and Yamashita, K. (1984) Pure Appl. Chem. 56, 821-832).
We investigated the carbohydrate structure of the third component of complement (C3) newly synthesized by cultured rat hepatocytes. When the cells were incubated with [3H]mannose, [3H]galactose or [3H]glucosamine, these radioactive precursors were incorporated only into the alpha subunit of C3, demonstrating that only the alpha subunit contains oligosaccharide chains. [3H]Mannose-labelled C3 was purified from the culture medium by immunoaffinity chromatography. Oligosaccharides prepared by Pronase digestion and strong alkaline hydrolysis were separated into two fractions by Bio-Gel P-2 chromatography (Fractions I and II). The two fractions were analysed by concanavalin A-Sepharose chromatography, ion-exchange high-performance liquid chromatography, and Bio-Gel P-4 gel filtration before and after sequential exoglycosidase digestions. It was found that Fraction I contained two complex type oligosaccharide chains, (NeuAc)2(Gal)2(GlcNAc)2(Man)3(GlcNAc)2 and (NeuAc)3(Gal)3(GlcNAc)3(Man)3(GlcNAc)2, and Fraction II contained the high-mannose type, consisting mainly of (Man)8(GlcNAc)2. Taken together with the carbohydrate composition of rat serum C3, the results suggest that rat C3 has one high-mannose type oligosaccharide chain and two complex type chains in the alpha subunit, which is different from the proposal for human C3.
Human plasma low-density lipoproteins were purified by flotation followed by gel filtration. The protein moiety of the lipoproteins, apolipoprotein-B, which was detected by polyacrylamide gel electrophoresis as the only protein component, contained 4.4% (by weight) carbohydrate. Glycopeptides liberated from apolipoprotein-B by pronase were fractionated by affinity chromatography on concanavalin-A–Sepharose. The results indicated that high-mannose glycopeptides interacting strongly with the lectin comprise about 37% of the total monosaccharides of apolipoprotein-B. Thus, as compared to the total serum glycoproteins having about 5% of their monosaccharides in high-mannose glycopeptides, low-density lipoproteins are relatively enriched in these structures amounting up to about 10% of the total high-mannose oligosaccharides in serum. The rest of the carbohydrates in low-density lipoproteins are suggested to be mainly biantennary acidic oligosaccharides interacting weakly with concanavalin A.
We analysed the oligosaccharides of a human IgM produced by a human-human-mouse hybridoma at each of its five conserved heavy chain glycosylation sites. Consistent with previous reports, this IgM possesses sialylated oligosaccharides at Asn171, Asn332 and Asn395, and high-mannose-type oligosaccharides at Asn402. In contrast to previous reports for human IgMs, we find that Asn563 is not occupied by oligosaccharide on perhaps 25% of IgM heavy chains, while occupied Asn563 sites contain both high-mannose-type and sialylated oligosaccharides. These latter results are consistent with the glycosylation at Asn563 previously reported for the mouse MOPC 104E IgM. We demonstrate that both the human hybridoma IgM and the mouse MOPC 104E IgM are mixtures of pentamers and hexamers, raising the possibility that the unique findings concerning the glycosylation at Asn563 in this study and the previous study of the MOPC 104E IgM could be related, at least in part, to the different packing requirements of the hexameric geometry and the accessibility of oligosaccharides in the hexameric geometry for processing to complex type. In addition, we used high-pH anion-exchange (HPAE) chromatography, neutral anion-exchange chromatography, fluorophore-assisted carbohydrate electrophoresis and Western blots to compare the oligosaccharide compositions of the human hybridoma IgM, pooled human serum IgM and two mouse monoclonal IgMs (MOPC 104E and TEPC 183). Of note is the presence of N-glycolylneuraminic acid (NeuGc) and N-acetylneuraminic acid (NeuAc) at a 2:1 ratio in the oligosaccharides of the human hybridoma IgM. The presence of both NeuGc and NeuAc complicates the interpretation of HPAE chromatographs.
The complete structure of the oligosaccharide moiety of J chain isolated from a Waldenströms macroglobulin Wa has been established. The oligosaccharide is present in three forms differing in the amount of sialic acid and designated J-A, J-B, and J-C. The structure and proportion of each of these is: formula : (see text) : Removal of the oligosaccharide moiety with glycosidases results in an increased mobility of J chain in sodium dodecyl sulfate polyacrylamide gel electrophoresis corresponding to a shift in apparent molecular weight from 23,500 to 19,500. The preparation and utilization of iodinated glycopeptides for sequence analysis is presented.
Clone 15B cells, derived from Chinese hamster ovary cells and deficient in a specific UDP-N-acetylglucosamine:glycoprotein N-acetylglucosaminyltransferase activity, synthesize glycoproteins with altered oligosaccharide units. Glycopeptides prepared from these glycoproteins contain large quantities of a glycopeptide with the composition (Man)5(GlcNAc)2-Asn whereas parent cells have only small amounts of this glycopeptide. The structure of the glycopeptide was determined by the combination of methylation analysis, acetolysis, Smith periodate degradation, and alpha- and beta-mannosidase digestion. Its complete structure is Manalpha 1 leads to 6[Manalpha1 leads to 3]-Manalpha1 leads to 6[Manalpha 1 leads to 3]-Manbeta1 leads to 4 GlcNAcbeta1 leads to 4 GlcNAc leads to Asn-peptide. The structures of two other glycopeptides found in smaller quantities in clone 15B but not detected in the parent cells were determined and are Manalpha 1 leads to 6 [Manalpha 1 leads to 3]-Manalpha1 leads to 6Manbeta 1 leads to 4 GlcNAcbeta 1 leads to 4GlcNAc-Asn-peptide and Manalpha 1 leads to 3 Manalpha 1 leads to 6[Manalpha 1 leads to 3] Manbeta 1 leads to 4GlcNAcbeta 1 leads to 4GlcNAc-Asn-peptide. It is proposed that the (Man)5(GlcNAc)2-Asn unit is the physiologic acceptor for the particular N-acetylglucosaminyltransferase which is deficient in clone 15B cells and that this reaction is necessary for complex oligosacchari-e biosynthesis.
Seven glycopeptide fractions, representing five different glycopeptides, were isolated from a γM-immunoglobulin obtained from a patient with Waldenström's macroglobulinemia. Two of the glycopeptides contained only mannose and N-acetylglucosamine residues in the molar ratio of 7:2, while the other three glycopeptides contained sialic acid, galactose, mannose, N-acetylglucosamine, and fucose in the molar ratio of 1:2:3 to 4:4 to 4.5:0.5. The structure of the oligosaccharide portion of four of the glycopeptides was investigated as follows. Sequential enzymatic degradation of the oligosaccharide chains with purified glycosidases helped to determine the sequences of the sugars. The linkages between the sugars were established by serial periodate oxidation and by methylation of the glycopeptides, followed by identification of the alditol acetate derivatives of the methylated sugars by gas chromatography and mass spectrometry. The methylation data demonstrated the existence of microheterogeneity in one of the oligosaccharide units which contains only mannose and N-acetylglucosamine residues. Microheterogeneity was also shown to occur in the outer branches of the more complex oligosaccharide units which lacked a full complement of fucose and sialic acid residue. Partial structures are proposed for four of the glycopeptides.
The well-known heterogeneity of normal and pathological immunoglobulins M was investigated in a study involving the characterization of their carbohydrate moieties. Oligosaccharide units were released from the native molecule by hydrazinolysis, and they were fractionated by affinity chromatography on a concanavalin A-Sepharose column to yield separate N-acetyl-lactosaminic-type and oligomannosidic-type structures. Further identification of these oligosaccharides was attempted by t.l.c. on silica gel and by determination of their monosaccharide compositions. A comparative study of the oligosaccharide units belonging to each population of immunoglobulin M was possible. Similarities were found in the occurrence of both types of oligosaccharide structures, and, in addition, a common double heterogeneity could be demonstrated for N-acetyl-lactosaminic-type structures: they could be resolved by affinity chromatography into bi-, tri- and tetra-antennary structures, and they also showed differences in N-acetylneuraminic acid content. Though some variations were observed in the exact composition of the oligosaccharide units within each population, it was possible to consider a representative oligosaccharide-unit composition of normal immunoglobulin M as a standard for comparison. On this basis a predominance of multi-antennary structures was observed in the more glycosylated pathological immunoglobulins M (10% carbohydrate content), whereas oligomannosidic structures were increased in pathological immunoglobulins M with a lower content of carbohydrates (7%). These variations are thought to reflect differences in the biosynthetic processing pathway of the carbohydrate units of the pathological immunoglobulins M or the enhanced expression of a molecular clone.
Dans 5 cas de mannosidose rencontrés pour la première fois en France, nous avons isolé et étudié les structures de plusieurs oligosaccharides riches en mannose excrétés dans les urines. Trois d'entre eux avaient été décrits antérieurement par Norden et al.: α-d-Manp-(1 → 3) β-d-Manp-(1 → 4) d-GlcNAcp; α-d-Manp-(1 → 2) α-d-Manp-(1 → 3) β-d-Manp-(1 → 4) d-GlcNAc et α-d-Manp-(1 → 2) α-d-Manp-(1 → 2) α-d-Manp-(1 → 3) β-d-Manp-(1 4) d-GlcNAcp. Toutefois, nous avons isolé 4 nouveaux oligosaccharides excrétés en plus faible quantité qui répondent aux structures suivantes: α-d-Manp-(1 → 3) [α-d-Manp-(1 → 2) α-d-Manp-(1 → 2) α-d-Manp-(1 → 6)] β-d-Manp-(1 → 4) GlcNAc; α-d-Manp-(1 → 2) α-d-Manp-(1 → 3) [α-d-Manp-(1 → 2) α-d-Manp-(1 → 2) α-d-Manp-(1 → 6)] β-d-Manp-(1 → 4) GlcNAcp; [α-d-Manp-(1 → 2)4 α-d-Manp-(1 → 3) [α-d-Manp-(1 → 6)] β-d-Manp-(1 → 4) GlcNAcp and [α-d-Manp-(1 → 2)5 α-d-Manp-(1 → 3) [α-d-Manp-(1 → 6)] β-d-Manp-(1 → 4) GlcNAcp. Ces structures se rattachent à celles des glycannes de «type oligomannosidique qui existent dans de nombreuses glycoprotéines. Toutes présentent un seul résidu de N-acétylglucosamine en position réductrice terminale et renforcent ainsi l'hypothèse de Kobata et al. et de Montreuil et al. que le catabolisme des glycannes liés N-glycosidiquement à la fraction protéique commence par l'action du β-endo-N-acétylglucosaminidase.
Earlier studies on the oligomannoside-type carbohydrate chain linked to Asn-563 of a human IgM protein from blood plasma of a patient (Du) with Waldenström's macroglobulinemia (Jouanneau, J. and Bourillon, R. (1979) Biochem. Biophys. Res. Commun. 91, 1057–1061) showed the occurrence of an unusual core structure. It was suggested that only one N-acetylglucosamine residue was present in stead of two. However, reinvestigation of this glycopeptide sample by 500-MHz 1H-NMR spectroscopy lent no support for an unusual core structure. On the contrary, the NMR spectrum shows all spectral features characteristic for an N,N′-diacetylchitobiose unit. Moreover, it revealed a heterogeneity in both the carbohydrate and the peptide moiety. The structure of the major component is the following¶: Besides, larger glycopeptides are present having one or two additional mannose residues α(1→2)-linked to terminal residues and/or in the above structure. It should be noted that for this sample, containing only 25 nmoles of glycopeptide, the microheterogeneity of the carbohydrate chain could excellently be revealed by 500-MHz 1H-NMR spectroscopy.
In previous papers were described 2 glycopeptides (Carbohydrate → Asn-Lys and Ser Asn ← Carbohydrate) isolated from pronase digest of human asialo serotransferrin, and it was demonstrated that in both glycopeptides the glycans were bound through a 4 N (2 acetamido 2 deoxy β D glucopyranosyl) L asparagine linkage. Evidence that the 2 glycans proceed from two different parts of the polypeptide chain was demonstrated by determination of amino acid sequences of tryptic and chymotryptic glycopeptides: glycan I is conjugated in the N terminal part of the peptide chain and corresponds to the dipeptide Asn-Lys, whereas glycan II is conjugated in the C terminal part and corresponds to the dipeptide Ser-Asn. In this paper, the authors report the primary structure of glycans I and II determined by application of different methods: exhaustive methylation, mild hydrolysis, acetolysis, hydrazinolysis as well as use of specific glycosidases.
A quantitative gas-liquid chromatographic method has been developed for the analysis of carbohydrates. The method involves the conversion of free or covalently bound monosaccharides into their O-methyl glycosides and the analysis of the glycosides as their trifluoroacetate derivatives. The procedure has been tested on standard monosaccharides, glycolipids and glycoproteins. The results show that all commonly occurring pentoses, hexoses, hexosamines, N-acetylhexosamines and sialic acids are accurately determined by the use of mesoinositol as the internal standard.
N-glycosidically-linked glycans released by hydrazinolysis of human factor VIII/von Willebrand factor (FVIII/vWf) were separated by high-voltage electrophoresis. Five fractions were obtained, one of them representing 60% of the total amount of the N-glycosidically-linked glycans of FVIII/vWf. On the basis of the carbohydrate composition, methylation analysis and 500 MHz 1H-NMR spectroscopy, we describe the primary structure of this major glycan which is of the monosialylated and monofucosylated biantennary N-acetyllactosaminic type.
The carbohydrate chains of the pathological human immunoglobulins M from two patients with Waldenström's macroglobulinemia were released by hydrazinolysis. The N-acetyllactosamine-type glycans were obtained by affinity chromatography on concanavalin A and fractionated by high-voltage paper electrophoresis. The primary structure of the major compounds was elucidated on the basis of carbohydrate analysis, methylation analysis, including mass-spectrometry, and 500 MHz 1H-NMR spectroscopy. For both patients, this appeared to be a monosialyl monofucosyl biantennary structure; the compounds differed by the presence of an intersecting N-acetylglucosamine residue.
The asialoglycopeptides obtained from secretory immunoglobulins A from human milk have been separated by gel filtration and affinity chromatography on Concanavalin A-Sepharose and Lens culinaris agglutinin-Sepharose columns. Their structures have been determined by sugar analysis, methylation studies including mass spectrometry and 500-MHz 1H-NMR spectroscopy. The glycans are of the biantennary N-acetyllactosamine type differing in their degree of extension by fucosyl-N-acetyllactosamine residues. The overall structures of the glycopeptides are as follows:
Most of the asialoglycospeptide structures possess an intersecting GlcNAc residue; they are suggested to be located on the α chain of the secretory immunoglobulins A of human milk. The non-interesected structures probably occur on the secretory piece. The methodology applied to the structural analysis adequately coped with the exteremely high degree of heterogeneity shown by the structures.
Earlier studies on the oligomannoside-type carbohydrate chain linked to Asn-563 of a human IgM protein from blood plasma of a patient (Du) with Waldenström's macroglobulinemia (Jouanneau, J. and Bourillon, R. (1979) Biochem. Biophys. Res. Commun. 91, 1057-1061) showed the occurrence of an unusual core structure. It was suggested that only one N-acetylglucosamine residue was present in stead of two. However, reinvestigation of this glycopeptide sample by 500-MHz 1H-NMR spectroscopy lent no support for an unusual core structure. On the contrary, the NMR spectrum shows all spectral features characteristic for an N,N′-diacetylchitobiose unit. Moreover, it revealed a heterogeneity in both the carbohydrate and the peptide moiety. The structure of the major component is the following¶ ¶ All sugars mentioned possess the D-configuration; all amino acids mentioned possess the L-configuration.: {A figure is presented} Besides, larger glycopeptides are present having one or two additional mannose residues α(1→2)-linked to terminal residues B and/or C in the above structure. It should be noted that for this sample, containing only 25 nmoles of glycopeptide, the microheterogeneity of the carbohydrate chain could excellently be revealed by 500-MHz 1H-NMR spectroscopy.
Seven kinds of asparagine-linked oligosaccharides were bound to the Fc region of a human immunoglobulin D(NIG-65). The oligosaccharides quantitatively released from four species of glycopeptides by digestion with almond glycopeptidase, were separated by Bio-Gel p-4 column chromatography and were purified further by thin-layer chromatography. The sugars were identified with GC-MS following the permethylation of respective oligosaccharide. To Asn-68 (NIG-65 Fc numbering (1)), two kinds of high-mannose-type oligosaccharides were bonded. To Asn-159, a kind of hybride-type and two kinds of bisected complex-type oligosaccharides were attached. From Asn-210, four kinds of bisected complex-type oligosaccharides were isolated.
Tryptic glycopeptide T1 of human glycophorin A [Tomita, M., & Marchesi, V. T. (1975) Proc. Natl. Acad. Sci. U.S.A. 72, 2964-2968] was subjected to hydrazinolysis. After N-acetylation, the complex-type oligosaccharides were isolated by gel filtration. The major neutral oligosaccharide (2000 molecular weight) was purified by a combination of ion-exchange and gel-permeation chromatography. Treatments with endo- and exo-glycosidases, periodate oxidation, and methylation analysis indicated that the major neutral oligosaccharide has the following structure: Gal beta 1 leads to -4GlcNAc beta 1 leads to 2Man alpha 1 leads to 6(GlcNAc beta 1 leads to 4)(Gal beta 1 leads to -4GlcNAc beta 1 leads to 2Man alpha 1 leads to 3)Man beta 1 leads to 4GlcNAc beta 1 leads to 4-(Fuc alpha 1 leads to 6)GlcNAc. This oligosaccharide was retained by Ricinus communis agglutinin-Sepharose and retarded by Sepharose 4B coupled with erythroagglutinating E4 isolectin from Phaseolus vulgaris. Retention by concanavalin A-Sepharose was observed only after treatment of the oligosaccharide with beta-galactosidase.
The mass spectra of 52 partially methylated and acetylated methyl glycosides of galactose, mannose, glucose, and N-acetylglucosamine have been determined. Each derivative was identified on the basis of its gas-liquid chromatography retention time and mass spectra. The analysis of methyl ethers obtained by methanolysis of fully methylated glycans of α1-acid glycoprotein is described as an application of the method.
The structures of the oligosaccharides present in the mouse immunoglobulin M secreted by the plasma cell tumor MOPC 104E have been determined making use of glycopeptides derived from purified unlabeled immunoglobulin and from [3H]mannose and [14C]glucosaminelabeled immunoglobulin M. The glycopeptides were fractionated on columns of Bio-Gel P-6 and concanavalin A-Sepharose and high mannose type oligosaccharides were released from glycopeptide with clostridial endo-β-N-acetylglucosaminidase CII. MOPC 104E immunoglobulin M was shown to contain four complex-type and one high mannose type oligosaccharide units per heavy chain. The glycopeptides with complex oligosaccharides were separated on concanavalin A-Sepharose into two classes with structures containing either two or three outer branches with the sequence ± NGNAα2 → 6Galβ1 → 4GlcNAcβ1 → attached to the 2 position or to the 2 and 4 positions of mannose in a core with the structure Manαl → 6(Manα1 → 3)Manβ1 → 4GlcNAcβ1 → 4(Fucα1 → 6)GlcNAc → Asn. The high mannose oligosaccharides liberated by endo-β-N-acetylglucosaminidase CII varied in size from Man8GlcNAc to Man5GlcNAc. A Man6GlcN Ac with the structure Manα → 6(Manα1 → 3)Manα1 → 6(Manα1 α 2Manα1 → 3)Manβ1 → 4GlcNAc was the predominant species. Some high mannose oligosaccharide was resistant to endo-β-N-acetylg]ucosaminidase CII release and apparently has a “hybrid” or atypical structure.
The anomeric proton (H-1) chemical shifts of D-mannopyranosides in aqueous solution are affected both by the aglycon and by substitution of the ring [Lee, Y. C., & Ballou, C. E. (1965) Biochemistry 4, 257]. We have examined the 1H NMR spectra for a variety of linear and branched mannooligosaccharides and have assigned the H-1 resonances to the component sugars. The chemical shifts, which range from delta 4.76 to 5.36, provide information regarding the linkages, sequences, and anomeric configurations of mannose residues in an oligomer. Thus, 1H NMR spectroscopy can complement enzymatic hydrolysis, methylation analysis, and acetolysis for the structural characterization of oligosaccharides. Furthermore, small structural differences between otherwise identical oligosaccharides are often accompanied by long-range chemical shift changes for the anomeric protons. Because sugars three or more residues away from the structural alteration can be affected, the changes must reflect conformational differences. We have placed emphasis on the mannose-rich oligosaccharides from glycoproteins, particularly those produced by endo-beta-N-acetylglucosaminidase digestion. Two mannose-rich glycopeptides were isolated from a monoclonal human IgM and their positions of origin on the polypeptide chain were determined. The oligosaccharides were released with endo-beta-N-acetylglucosaminidase and fractionated into several size classes. Our structural studies show that each glycopeptide possessed a unique set of oligosaccharides, in agreement with a recent report [Chapman, A. & Kornfeld, R. (1979) J. Biol. Chem. 254, 816]. The NMR spectra were particularly valuable in detecting and quantitating isomeric fragments not observed previously, and our results suggest a modification of the scheme presented by Chapman and Kornfeld for the processing of mannose-rich IgM oligosaccharides.
THE ammoniacal silver nitrate spray 1 used for the detection of sugars has several disadvantages; to those mentioned by Partridge 2 should be added the necessity for very careful control of the heating step, particularly important in laboratories lacking special apparatus. We have introduced modifications, based on a test given by Feigl 3 for reducing sugars, which eliminate the heating step, and in which the reagents are applied in organic solvents, thus removing the danger of migration of the sugar spots. The method has been in use for more than a year, and has proved easy to handle and extremely reliable.