Type Glycopeptides Research Articles

Mucin type glycopeptides: Synthesis of Core 2, Core 6 and F1-α building blocks and some unexpected reactions

Protected Core 2 and 6 and F1-α analogs were synthesized using the 4,6-diol of Tn-Ser/Thr and TF-Ser/Thr. Acetylation of glucosamine in acetic anhydride/pyridine resulted in an additional and unusual N-acetylation at the Fmoc protected amine of serine and threonine followed by β-elimination of the aglycons. Normal glucosamine acetylation was carried out at −20°C for 20 minutes to accomplish in synthesis of Core 2, Core 6 and F1-α as glycopeptide building blocks.

Enzymatic synthesis of a sialyl Lewis X dimer from egg yolk as an inhibitor of E-selectin

A dimeric sialyl Lewis X (SLe x) glycopeptide was synthesized enzymatically in three steps from an N-linked oligosaccharide prepared from egg yolk. Treatment of delipidated hen egg yolk with the protease Orientase and neuraminidase gave a dimeric N-acetyllactosamine-containing oligosaccharide linked to asparagine. Addition of sialic acid and fucose catalyzed by α-2,3-sialyltransferase and α-1,3-fucosyltransferase provided the dimeric SLe x, which was shown to be as active as monomeric SLe x as an inhibitor of E-selectin with IC 50 0.75 mM. The synthetic dimeric SLe x of the mucin type (i.e. SLe x linked to the 3- and 6-OH groups of Gal) is, however, about five times as active as the monomer. It is suggested that dimeric SLe x glycopeptides of the mucin type would be effective ligands for E-selectin.

Steric Control of N-Acetylgalactosamine In Glycosidic Bond Formation

N-Acetylgalactosamine, protected with a 4,6-cyclic acetal followed by selective acylation at 3-OH, provides an excellent donor for the synthesis of α-glycosides, particularly the cancer associated antigens such as Tn, TF, Sialyl-Tn and Sialyl-TF. This fast and efficient synthesis is easily adaptable for commercial production of mucin type glycopeptides with O-linked carbohydrate structures which are currently being investigated as vaccines against cancers.

Steric control of N-acetylgalactosamine in glycosidic bond formation

N-Acetylgalactosamine, protected with a 4,6-cyclic acetal followed by selective acylation at 3-OH, provides an excellent donor for the synthesis of α-glycosides, particularly the cancer associated antigens such as Tn, TF, Sialyl-Tn and Sialyl-TF. This fast and efficient synthesis is easily adaptable for commercial production of mucin type glycopeptides with O-linked carbohydrate structures which are currently being investigated as vaccines against cancers.

Stereoselective synthesis of a blood group A type glycopeptide present in human blood mucin

N,N- Dimethyl-O-(2- acetamido-2-deoxy-α- d-galactopyranosyl)-(1 → 3) -O-[(α- l-fucopyranosyl)-(1 → 2) ]-O-(β- d-galactopyranosyl)-(1 → 3) -O-( 2-acetamido-2-deoxy-α- d-galactopyranosyl)-(1 → 3)- l-serine , a core I glycotetraosyl peptide structure and a predominant substructure in complex glycan-glycoproteins present in human blood group A ovarian mucin, was synthesized for the first time. The title compound was synthetically accomplished via the following key manoeuvres: regio- and stereo-controlled construction of the α-GalNAc-(1 → 3)-Gal synthon, stereoselective glycosylation generating a α-GalN 3-(1 → 3)-Ser glycopeptide synthon and α-selective fucosylation towards an acceptor which was derived from glycosylation of the latter two synthons. An alternative route to that of the latter, to synthesize a fully protected equivalent of the title compound, involving the coupling of a α-GalNAc-(1 → 3)- β-Gal-(1 → 3)-GalN 3X synthon to an aglycon serine derivative, is described herein.

Cytosolic glycosidases: do they exist?

The substrate specificity of the alpha-D-mannosidases of rat liver lysosome and cytosol was examined using oligosaccharides of the oligomannosidic type. The hydrolysis products were characterized by 400 MHz 1H-NMR spectroscopy. Both catabolic pathways occur in ordered ways, but are quite different. In fact, the lysosomal pathway is a two-step process: the first step involves a Zn(2+)-independent alpha-1,2-mannosidase activity, whereas the second involves a Zn(2+)-dependent alpha-1,3- and alpha-1,6-mannosidase activity. The final product is the disaccharide Man(beta 1-4)GlcNAc. In contrast, the cytosolic pathway leads, in one step, to a unique hexasaccharide (Man5GlcNAc) which has the same structure as the polyprenolic intermediate synthesized on the cytosolic face of the rough endoplasmic reticulum during the biosynthesis of N-glycosylprotein glycans: Man(alpha 1-2)-Man(alpha 1-2)Man(alpha 1-3)[Man(alpha 1-6)] Man(beta 1-4)GlcNAc(beta 1-4)-GlcNAc(alpha)P-P-Dol. In addition, the enzymatic parameters of lysosome, endoplasmic reticulum and cytosol alpha-D-mannosidases are quite different. These results lead to the conclusion that the cytosol contains specific alpha-D-mannosidases which do not originate from lysosomes nor from endoplasmic reticulum. The discovery of cytosolic endo-N-acetyl-beta-D-glucosaminidase active on 'immature complex glycans' (glycopeptides of the oligomannosidic type and of the desialylated N-acetyllactosaminic type) as well as on the glycosyl-dolichol pyrophosphate intermediates allows us to hypothesize that these enzymes belong to a control system of N-glycosylprotein biosynthesis, their role being to destroy unfinished glycans. The fate of the formed oligosaccharide structures is discussed: are they destroyed by cytosolic or lysosomal exoglycosidases, or do they carry an 'oligosaccharin-like activity'?

Induction and Purification of Endo-β- N -Acetylglucosaminidase from Arthrobacter protophormiae Grown in Ovalbumin

Arthrobacter protophormiae produced a high level of extracellular endo-beta-N-acetylglucosaminidase when cells were grown in a medium containing ovalbumin. The enzyme was induced by the glycopeptide fraction of ovalbumin prepared by pronase digestion. Production of the enzyme was also induced by glycoproteins such as yeast invertase and bovine ribonuclease B but not by monosaccharides such as mannose, N-acetylglucosamine, and galactose. The enzyme was purified to homogeneity as demonstrated by polyacrylamide gel electrophoresis and has an apparent molecular weight of about 80,000. The enzyme showed a broad optimum pH in the range of pH 5.0 to 11.0. The enzyme hydrolyzed all heterogeneous ovalbumin glycopeptides, although the hydrolysis rates for hybrid type glycopeptides were very low. The substrate specificity of A. protophormiae endo-beta-N-acetylglucosaminidase was very similar to that of Endo-C(II) from Clostridium perfringens. Therefore, the enzyme induction by A. protophormiae seems to have a close relation to the substrate specificity of the enzyme.

Isolation and structural characterization of sialic-acid-containing glycopeptides of the O-glycosidic type from the urine of two patients with an hereditary deficiency in alpha-N-acetylgalactosaminidase activity.

Glycopeptides have been isolated from the urine of two patients, aged 5 and 6, with a new lysosomal storage disease characterized by a deficiency in alpha-N-acetylgalactosaminidase activity. Isolation of these glycopeptides was achieved using gel filtration and ion-exchange chromatography. Structural determination was done using one- and two-dimensional 500 MHz 1H-NMR spectroscopy and FAB mass spectrometry of native and derivatized glycopeptides. The following structures were inferred as being present: Glycopeptide A (up to 140 mg/l urine) (1)-(3) Neu5Ac alpha 2-3Gal beta 1-3 (Neu5Ac alpha 2-6)GalNAc alpha 1-R A1: R = Ser A2: R = Thr A3: R = Thr-Pro Glycopeptide B (up to 80 mg/l urine) (4)-(6) Neu5Ac alpha 2-3Gal beta 1-4GlcNAc beta 1-6 (Neu5Ac alpha 2-3-Gal beta 1-3) GalNAc alpha 1-R B1: R = Ser B2:R = Thr B3: R = Thr-Pro

Interactions of concanavalin A with asparagine-linked glycopeptides: formation of homogeneous cross-linked lattices in mixed precipitation systems.

We have previously shown that certain oligomannose and bisected hybrid type glycopeptides are bivalent for binding to concanavalin A (Con A) [Bhattacharyya, L., Ceccarini, C., Lorenzoni, P., & Brewer, C. F. (1987) J. Biol. Chem. 262, 1288-1293]. Each glycopeptide gives a quantitative precipitation profile with the protein which consists of a single peak that corresponds to the binding stoichiometry of glycopeptide to protein monomer (1:2). We have shown that the affinities of the primary and secondary sites of the glycopeptides influence their extent of precipitation with the lectin [Bhattacharyya, L., & Brewer, C. F. (1988) Eur. J. Biochem. (in press)]. In the present study, we demonstrate that equimolar mixtures of any two of the glycopeptides result in a quantitative precipitation profile which shows two protein peaks. Using radiolabeled glycopeptides, the precipitation profiles of the individual glycopeptides were determined. The results show that each glycopeptide forms its own precipitation profile with the protein which is independent of the profile of the other glycopeptide. For mixtures containing an equimolar ratio of two glycopeptides, the glycopeptide with lower affinity shows a precipitation maximum at a lower concentration than the one with higher affinity. However, this can be reversed by increasing the ratio of the lower affinity glycopeptide in the mixture. Thus, the relative precipitation maxima of the glycopeptides are determined by mass-action equilibria involving competitive binding of the two carbohydrates to the protein. These equilibria, in turn, are sensitive to the relative amounts and affinities of the carbohydrates at both their primary and secondary sites.(ABSTRACT TRUNCATED AT 250 WORDS)

Structural studies on the carbohydrate moieties of human liver α- l-fucosidase

α- l-Fucosidase was purified from human liver to apparent homogeneity and subjected to exhaustive digestion with Pronase. The resulting glycopeptides were isolated by gel filtration on Sephadex G-50 and further fractionated by Bio-Gel P-4 chromatography. Five glycopeptide fractions were obtained. The structures of the carbohydrate portions of all glycopeptide components were fully characterized by a combination of 500-MHz 1H NMR spectroscopy and carbohydrate composition analysis. Fraction I contained disialyl diantennary glycopeptides of the N-acetyllactosamine type. Fractions II and III contained predominantly mono(sialyl- N-acetyllactosaminyl) diantennary glycopeptides with the NeuAcα(2 → 6)Galβ(1 → 4)GlcNAcβ(1 → 2) branch attached to α(1 → 3)-linked Man in II and to α(1 → 6)-linked Man in III. The N-acetyllactosamine-type glycopeptides in fractions I to III have a small portion (10–15%) of their Asn-linked GlcNAc residues substituted by additional α(1 → 6)-linked Fuc. Also, a minor portion of the NeuAc residues appeared to be attached to Gal in α(2 → 3) rather than α(2 → 6) linkage. Fraction IV contained a mixture of larger-size oligomannoside-type glycopeptides with a variable number (6 to 9) of Man residues. Smaller-size oligomannoside-type glycopeptides were found in fraction V, containing 3 or 5 Man residues; a small portion (10%) of the Man 3GlcNAc 2Asn component appeared to contain in addition a Fuc residue in α(1 → 6) linkage to the Asn-bound GlcNAc. The overall ratio of oligomannoside-type to N-acetyllactosamine-type carbohydrate structures was found to be 5:4. This article is the first account of the complete characterization of the oligomannoside-type structures in α- l-fucosidase; furthermore, the occurrence in α- l-fucosidase of mono(sialyl- N-acetyllactosaminyl) structures, Fuc-containing oligosaccharides, and NeuAcα(2 → 3) linked to Gal are reported for the first time.

Formation of homogeneous cross-linked lattices between oligomannose type glycopeptides and concanavalin A

Certain oligomannose type glycopeptides have previously been shown to be bivalent for binding to concanavalin A, and to give quantitative precipitation profiles with the protein that consist of single peaks which correspond to the binding stoichiometry of glycopeptide to protein monomer (1:2) (Bhattacharyya, L., Ceccarini, C., Lorenzoni, P., and Brewer, C.F. (1987) J. Biol. Chem. 262, 1288-1293). In the present study, equimolar mixtures of two oligomannose type glycopeptides, a Man-6 and a Man-9 glycopeptide, gives a quantitative precipitation profile which shows two protein peaks. Each glycopeptide was radiolabelled with 3H or 14C, and the the precipitation profiles of the individual glycopeptides in the mixture determined. The results show that the radioactivity profile of the Man-6 glycopeptide corresponds to the first protein peak, while the radioactivity profile of the Man-9 glycopeptide corresponds to the second protein peak. The results indicate that each glycopeptide forms a unique homogeneous cross-linked lattice with the lectin which excludes the lattice of the other glycopeptide.

Concanavalin A interactions with asparagine-linked glycopeptides. The mechanisms of binding of oligomannose, bisected hybrid, and complex type carbohydrates

The affinity of concanavalin A (Con A) for simple saccharides has been known for over 50 years. However, the specificity of binding of Con A with cell-surface related carbohydrates has only recently been examined in detail. Brewer and coworkers [J Biol Chem (1986) 261:7306–10; J Biol Chem (1987) 262:1288–93; J Biol Chem (1987) 262:1294–99] have recently studied the binding interactions of a series of oligomannose and bisected hybrid type glycopeptides and complex type glycopeptides and oligosaccharides with Con A. The relative affinities of the carbohydrates were determined using hemagglutination inhibition measurements, and their modes of binding to the lectin examined by nuclear magnetic relaxation dispersion (NMRD) spectroscopy and quantitative precipitation analyses. The equivalence zones (regions of maximum precipitation) of the precipitin curves of Con A and the carbohydrates indicate that certain oligomannose and bisected hybrid type glycopeptides are bivalent for lectin binding. From the NMRD and precipitation data, two protein binding sites on each glycopeptide have been identified and characterized. Certain bisected complex type oligosaccharides also bind and precipitate Con A, while the corresponding nonbisected analogs bind but do not precipitate the protein. The precipitation data indicate that the bisected complex type oligosaccharides are also bivalent for lectin binding, while the nonbisected analogs are univalent. The NMRD and precipitation data are consistent with different mechanisms of binding of nonbisected and bisected complex type carbohydrates to Con A, including different conformations of the bound saccharides.

Application of the trichloroacetimidate method to the synthesis of glycopeptides of the mucin type containing a β- d-Gal p-(1→3)- d-Gal pNAc unit

Mucin-type O-glycopeptides containing the β- d-Gal p-(1→3)- d-Gal pNAc disaccharide core unit, which is also the T-antigenic determinant, were synthesized from d-galactose, 2-azido-2-deoxy- d-galactose, 2-azido-2-deoxylactose, and l-serine precursors by applying the trichloroacetimidate method. Thus, β- d-Gal p-(1→3)-α- d-Gal pNAc-(1→3)-Ser ( 1) and β- d-Galp-(1→4)-β- d-Glc pNAc-(1→6)-[β- d-Gal p-(1→3)]-α- d-Gal pNAc-(1→3)-Ser ( 2) were obtained. A protected precursor of 2 having free OH groups at C-4 and C-6 of the inner sugar unit is a valuable intermediate for the synthesis of further O-glycopeptides of this core-unit type.

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

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

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

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

3] Lectin-specific targeting of lysosomal enzymes to reticuloendothelial cells

The principles and methods used for enzymatic modification of the carbohydrate portion of glucocerebrosidase are similar to those performed by Ashwell and Morell, Stahl, and others. It is difficult to explain the lack of uptake of native enzyme through binding of the high-mannose type glycopeptide to Man/GlcNAc receptors since approximately 20% of the total oligosaccharides on the native enzyme are high mannose type. Possibly a requirement for multiple sites of attachment to the receptor is not met by a single high-mannose type oligosaccharide per molecule. Alternatively, the presence of complex type oligosaccharides on this enzyme, demonstrated by structural studies, may mask the mannose site and thus account for the poor uptake of native enzyme. The ability to successfully deglycosylate any protein or enzyme in order to specifically target a selected cell type requires that there be (1) an available source of pure enzyme; (2) specific exoglycosidases of high specific activity available either commercially or relatively easily purified; (3) chemical or biochemical means available for the testing of the product, preferably at each step; and (4) a means of separating the glycosidases used from the desired enzyme product. The characteristic and unique accumulation of glucocerebroside only in cells of the monocyte- histiocyte series, makes Gaucher's disease an excellent prototype for the study of enzyme replacement therapy. The principles demonstrated for the enzymatic deglycosylation of glucocerebrosidase may be applied to the cell-specific delivery of other glycoproteins as well. Other lysosomal diseases in which storage occurs in multiple cell types may be ameliorated by administration of macrophage-directed enzymes if, by so doing, storage material can be digested during the normal phagocytic turnover of senescent cells. Consideration of the kinetics of degradation and the structural features affecting the stability of enzymes in vivo are prerequisites to improving the bioengineering of targeted lysosomal enzymes. Animal and culture models have been developed for the study of glucocerebrosidase delivery to specific cell types and substrate degradation. Other studies have progressed toward a definition not only of the receptors at the plasma membrane involved in the internalization of exogenous enzyme, but also of internal receptors or properties of the lysosome responsible for intracellular protein trafficking. A complete understanding of the forces acting to direct endogenous or exogenously supplied enzyme to a given subcellular organelle will require a synthesis of experimental results from all areas of glycoprotein research.

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

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

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 α(1–6) arm of the core β-mannose residue involving a trimannosyl moiety, and another site on the α(1–3) arm of the core β-mannose residue involving an α(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.

D-Galactosyltransferase and its endogenous substrates in chick embryo fibroblasts transformed by rous sarcoma virus

UDP= d-galactose: 2-acetamido-2-deoxy-β- d-glucopyranosyl 4=β- d-galactosyltransferase (GalTase) activity was purified, from primary chick embryo fibroblast (CEF) transformed by a temperature-sensitive, Rous sarcoma virus mutant (CEF-RSV), by chromatography on an affinity resin prepared with monoclonal antibodies to GalTase. Cellular glycopeptides from CEF, as well as CEF-RSV, maintained at permissive (37°) [CEF-RSV (37°)] and nonpermissive temperatures (41°) [CEF-RSV (41°)], were solubilized and galactosylated in vitro by incubation with purified GalTase substrates, composed of at least six discrete complex glycopeptides having bi- to tetra-antennary structures. The glycopeptides isolated from transformed cells, CEF-RSV (37°), included the six types observed in nontransformed cells, but demonstrated alterations in their relative amounts, including an increase in the content of a glycopeptide containing 3 mannose and 4 glucosamine residues. Furthermore, two additional complex-type glycopeptides were isolated from CEF- but demonstrated alterations in their relative amounts, including an increase in the content of a glycopeptide containing 3 mannose and 4 glucosamine residues. Furthermore, two additional complex type glycopeptides were isolated from CEF-RSV (37°). These malignant transformation-related glycopeptides were partially characterized and found to represent tri- and tetra-antennary complex glycopeptides. Endogenous galactosylation appeared to have occurred in a branched, nonspecific manner in these transformed cell-derived glycopeptides. These findings indicate that transformed cells may contain a greater preponderance of more highly branched, complex oligosaccharides which are randomly galactosylated at non-reducing termini by cellular GalTase.

Evidence for unique homologous peptide sequences around the glycosylated seryl and threonyl residues in polysialoglycoproteins isolated from the unfertilized eggs of the Pacific salmon Oncorhynchus keta.

Structures of glycopeptides obtained by exhaustive Pronase digestion of high molecular weight (1.7 X 10(5)) salmon egg polysialoglycoprotein have been elucidated. Six principal glycopeptides isolated by gel chromatography and DEAE-Sephadex A-25 chromatography in the absence or presence of borate ion were analyzed for their carbohydrate and amino acid composition, as well as amino acid sequence, and found to be of two distinct types: glycotripeptides, Thr*-Ser*-Glu, and glycotetrapeptides, Thr*-Gly-Pro-Ser, where an asterisk indicates the amino acid residues to which either the Gal beta 1----3GalNAc or Fuc alpha 1----3GalNAc beta 1----3Gal beta 1----4Gal beta 1----3GalNAc chain is attached. Their final yield corresponds to 64% of the original desialylated glycoprotein. In view of the simple amino acid composition of salmon egg polysialoglycoprotein (molar ratio Asp2Thr2Ser3Glu1Pro1Gly1Ala3) and the result of alkaline beta-elimination indicating three carbohydrate units linked to two of two threonine and one of three serine residues, a unique primary structure comprising repetitive sequences of the above two types of glycopeptides, which are interspersed by short nonglycosylated peptides consisting of alanine and aspartic acid, has been proposed for the core protein. The molecular secondary ion mass spectra of underivatized glycopeptides were used to obtain their structural information. The anomeric configuration of the proximal sugar-peptide linkages was proven to be alpha by proton nuclear magnetic resonance spectroscopy. This is the first systematic reported study of O-glycosidically linked glycopeptides by these instrumental methods.