Binding characteristics of an anti-Siaalpha2-6GalNAcalpha-Ser/Thr (sialyl Tn) monoclonal antibody (MLS 132).

To determine the epitopic structure for an anti-Siaalpha2-6GalNAcalpha-Ser/Thr (anti-sialyl Tn) monoclonal antibody, MLS 132, ovine submaxillary mucin (OSM) was digested with the combination of trypsin and thermolysin and the digest fractionated by immunoaffinity column chromatography and HPLC. From tryptic digest, a major glycopeptide designated as T3 was obtained as an immunoaffinity column-bound fraction. On solid-phase radioimmunoassay, it was found that T3 exhibited strong immunoreactivity with MLS 132. On treatment with thermolysin, T3 was converted into about 50 fragments, as found on fractionation by HPLC. Several of them were strongly immunoreactive and had the same amino acid sequence, i.e. Phe-Ser*-Gly-Glu-Thr*-Ser*-Thr*-Thr*-Val-Ile-Ser*-Gly-Thr*-Asn-Val, where asterisks denote the sites of attachment of carbohydrate. Of these, one was fully sialylated, the others having one Ser or Thr with unsialylated GalNAc attached. Results of analyses of the carbohydrate attached in these glycopeptides led us to postulate that a cluster composed of four sialyl Tn antigens is the essential epitopic structure for MLS 132.     

Expression of the Tn antigen on T-lymphoid cell line Jurkat.

Expression of the Tn antigen on a T-lymphoid cell line, Jurkat, was investigated using an anti-Tn monoclonal antibody, MLS 128. Immunoprecipitation or immunoaffinity chromatography of a lysate of Jurkat cells led to the isolation of a 120 kDa glycoprotein carrying the Tn antigen. This glycoprotein and leukosialin (CD43) were indistinguishable on SDS-PAGE and as to immunoreactivity with MLS 128. Leukosialin from an erythroid cell line, K562, exhibited no reactivity with MLS 128 despite that this leukosialin has several GalNAc alpha-Ser(Thr) structures. Pulse-chase experiments with the Jurkat leukosialin showed that newly synthesized leukosialin acquired the antigenecity after a lag of about 30 min, whereas incorporation of GalNAc into the leukosialin occurred earlier. These results indicate that the Tn antigen is expressed on leukosialin and that its epitopic structure is more complex than GalNAc alpha-Ser(Thr).     

A monoclonal antibody directed to Tn antigen

A murine monoclonal antibody, MLS 128, that was assigned to an anti-Tn antibody has been established by immunizing mice with human colonic cancer cells (LS 180). MLS 128 bound to mucin glycopeptides from LS 180 cells and their asialo forms to the same extent as well as to ovine submaxillary mucin (OSM) and asialo OSM. Special non-sialylated GalNAc residue(s) attached to a certain peptide region in the antigens seems to be involved in the binding since N-acetylgalactosaminidase treatment of the antigen abolished the binding and pronase digestion diminished the binding markedly.     

A monoclonal antibody that recognizes a cluster of a disaccharide, NeuAc alpha(2----6)GalNAc, in mucin-type glycoproteins

The structure of an epitopic carbohydrate recognized by a monoclonal antibody, MLS 102, was determined. A disaccharide, NeuAc alpha (2----6)GalNAc, the major prosthetic group of ovine submaxillary mucin (OSM) and related synthetic glycosides, NeuAc alpha(2----6)GalNAc alpha----Ser, NeuAc alpha(2----6)GalNAc beta----Ser, and NeuAc alpha (2----6)GalNAc beta----propyl, reacted with MLS 102 to similar extents, but the reaction was considerably weaker compared to that of OSM. This difference in reactivity could be ascribed to the occurrence of a cluster of the disaccharide on OSM. Purification of MLS 102-reactive antigens from a Triton X-100 extract of LS 180 cells by means of immunoaffinity chromatography gave mucin fractions (cMLS 102 antigen) with an OSM-like domain. Correlation between the content of the disaccharide, NeuAc alpha(2----6)GalNAc, in mucins and their reactivity with MLS 102 was observed.     

Short synthetic glycopeptides successfully induce antibody responses to carcinoma-associated Tn antigen.

Glycopeptides containing a tumor-associated carbohydrate antigen (mono-, tri- or hexa-Tn antigen) as a B-cell epitope and a CD4+ T-cell epitope (PV: poliovirus or TT: tetanus toxin) were prepared for immunological studies. Several Tn antigen residues [FmocSer/Thr (alpha-GalNAc)-OH] were successively incorporated into the peptide sequence with unprotected carbohydrate groups. The tri- and hexa-Tn glycopeptides were recognized by MLS128, a Tn-specific monoclonal antibody. The position of the tri-Tn motif in the peptide sequence and the peptide backbone itself do not alter its antigenicity. As demonstrated by both ELISA and FACS analysis, the glycopeptides induced high titers of anti-Tn antibodies in mice, in the absence of a carrier molecule. In addition, the generated antibodies recognized the native Tn antigen on cancer cells. The antibody response obtained with a D-(Tn3)-PV glycopeptide containing three alpha-GalNAc-D-serine residues is similar that obtained with the Tn6-PV glycopeptide. These results demonstrate that short synthetic glycopeptides are able to induce anticancer antibody responses.     

The monosaccharide composition and sequence of the carbohydrate moiety of human serum low density lipoproteins.

Human serum low density lipoprotein (d = 1.027-1.045) was delipidated with organic solvents and the apoprotein digested with thermolysin. The digest was fractionated by gel filtration and DEAE-cellulose chromatography. Two glycopeptides were obtained. One of the glycopeptides (GP-I) contained 2 residues of N-acetylglucosamine and 6 residues of mannose per mole of the glycopeptide, while the other contained 2 sialic acid, 5 mannose, 2 galactose, and 3 N-acetylglucosamine residues per mole of glycopeptide. The results of sequential enzymatic digestion with purified glycosidases, periodate oxidation, and partial acid hydrolysis lead us to propose the following sturctures for the two glycopeptides: (see article). These glycopeptides represent at least 50% of the carbohydrate moiety of LDL.     

Analysis of the fine specificity of Tn-binding proteins using synthetic glycopeptide epitopes and a biosensor based on surface plasmon resonance spectroscopy

Using synthetic Tn (GalNAc-O-Ser/Thr) glycopeptide models and a biosensor based on surface plasmon resonance spectroscopy we have determined that isolectin B4 from Vicia villosa (VVLB4) binds to one Tn determinant whereas the anti-Tn monoclonal antibodies 83D4 and MLS128 require at least two Tn residues for recognition. When an unglycosylated amino acid is introduced between the Tn residues, both antibodies do not bind. MLS128 affinity was higher on a glycopeptide with three consecutive Tn residues. These results indicate that Tn residues organized in clusters are essential for the binding of these antibodies and indicate a different Tn recognition pattern for VVLB4.     

Monoclonal antibodies toward different Tn-amino acid backbones display distinct recognition patterns on human cancer cells. Implications for effective immuno-targeting of cancer

The Tn antigen (GalNAcα-O-Ser/Thr) is a well-established tumor-associated marker which represents a good target for the design of anti-tumor vaccines. Several studies have established that the binding of some anti-Tn antibodies could be affected by the density of Tn determinant or/and by the amino acid residues neighboring O-glycosylation sites. In the present study, using synthetic Tn-based vaccines, we have generated a panel of anti-Tn monoclonal antibodies. Analysis of their binding to various synthetic glycopeptides, modifying the amino acid carrier of the GalNAc(*) (Ser* vs Thr*), showed subtle differences in their fine specificities. We found that the recognition of these glycopeptides by some of these MAbs was strongly affected by the Tn backbone, such as a S*S*S* specific MAb (15G9) which failed to recognize a S*T*T* or a T*T*T* structure. Different binding patterns of these antibodies were also observed in FACS and Western blot analysis using three human cancer cell lines (MCF-7, LS174T and Jurkat). Importantly, an immunohistochemical analysis of human tumors (72 breast cancer and 44 colon cancer) showed the existence of different recognition profiles among the five antibodies evaluated, demonstrating that the aglyconic part of the Tn structure (Ser vs Thr) plays a key role in the anti-Tn specificity for breast and colon cancer detection. This new structural feature of the Tn antigen could be of important clinical value, notably due to the increasing interest of this antigen in anticancer vaccine design as well as for the development of anti-Tn antibodies for in vivo diagnostic and therapeutic strategies.     

Coexpression of cancer-associated carbohydrate antigens,Tn and sialyl Tn

The expression of cancer-associated antigens, Tn and sialyl Tn, was examined using monoclonal antibodies, MLS 128 and MLS 102, recognizing these two antigens, respectively. A cell lysate from a human carcinoma cell line, LS 180 cells, was analysed by Western blotting using these two antibodies. Three glycoprotein bands were discernible with each antibody, of which two, corresponding to 250 and 210 kDa, were reactive with both the antibodies. LS 180 cells were metabolically labelled with³H-glucosamine and then the lysate from these cells was applied to two immunoaffinity columns. Sixty-five per cent of the Tn antigenic glycoproteins, based on radioactivity, bound to the MLS 102 affinity column. On the other hand, 45% of the sialyl Tn antigenic glycoproteins bound to the MLS 128 affinity column. These results indicate that some Tn and sialyl Tn antigens were expressed on the same polypeptide chains.The presence of non-sialylated GalNAc residues on the polypeptide chain with many Sia-GalNAc residues appears to be due to the incapability of three consecutive moieties of GalNAc-Ser/Thr to accept sialic acid.Abbreviations PSMF phenylmethylsulfonyl fluoride - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis - GalNAc N-acetylgalactosamine - Sia sialic acid     

The incorporation of glucose into alpha-1,4-glucan proteins of bovine retina membranes.

—Incubation of bovine retina membranes with UDP-[¹⁴C]glucose resulted in the incorporation of [¹⁴C]glucose into endogenous α-1, 4-glucan proteins. The transferring system was concentrated in membranes that floated at 0.94 and 1.10m -sucrose when centrifuged in a discontinuous sucrose density gradient and was almost absent in the rod outer segment (ROS) and the 100, 000 g supernatant fractions. The glucan proteins labelled by incubation with the radioactive sugar nucleotide at micromolar concentrations were distinguished in two fractions by their solubilities in trichloroacetic acid (TCA): glucan protein-I (GP-I), insoluble in TCA, and glucan protein-II (GP-II), soluble in TCA and precipitable by ethanol from the TCA soluble fraction. GP-I and GP-II were precipitated by trichloroacetic acid-phosphotungstic acid (TCA-PTA). A third fraction, glucan protein-III (GP-III) was found when incubations were carried out with UDP-[¹⁴C]glucose at millimolar instead of micromolar concentrations. GP-III was soluble in TCA and in TCA-PTA and precipitable by ethanol from the TCA soluble fraction. GP-II was excluded from a Sephadex G-200 column and showed a greater size than GP-I in a Sepharose 2B column. The radioactive residues obtained from the glucan proteins after digestion with pronase were totally included in a Sephadex G-25 column and were of a greater size than the labelled residues released with salivary α-amylase. Only radioactive maltose was found after a-amylase treatment. When membranes containing labelled GP-I and GP-II were incubated with unlabelled UDP-glucose at millimolar concentrations, GP-I was converted into GP-II and GP-III was formed.     

On the oligosaccharide moiety of angiotensin-converting enzyme.

Two glycopeptide fractions in a pronase digest of rabbit pulmonary angiotensin-converting enzyme were resolved by gel filtration. GP-I, the minor component (～1 mole/mol enzyme) contained mannose, galactose, glucose $\text{N}$-acetylglucosamine, $\text{N}$-acetylgalactosamine and sialic acid in an approximate molar ratio of 1:5:3:4:1:2 and molar equivalents of aspartic acid, threonine and serine. GP-II, the major oligosaccharide unit (～ 12 moles/mol enzyme, ～ 90% of total carbohydrate), contained fucose, mannose, galactose, $\text{N}$-acetylglucosamine, sialic acid and aspartic acid in a molar ratio of 1:4:4:4:1:1. Although accounting for about one-quarter of the weight of the enzyme, GP-II did not compete with the intact glycoprotein for binding to goat antienzyme antibodies. Some structural features of GP-II were deduced by periodate oxidation and digestion with various glycosidases.     

Surface plasmon resonance and NMR analyses of anti Tn-antigen MLS128 monoclonal antibody binding to two or three consecutive Tn-antigen clusters

Tn-antigens are tumour-associated carbohydrate antigens that are involved in metastatic processes and are associated with a poor prognosis. MLS128 monoclonal antibody recognizes the structures of two or three consecutive Tn-antigens (Tn2 or Tn3). Since MLS128 treatment inhibits colon and breast cancer cell growth [Morita, N., Yajima, Y., Asanuma, H., Nakada, H., and Fujita-Yamaguchi, Y. (2009) Inhibition of cancer cell growth by anti-Tn monoclonal antibody MLS128. Biosci. Trends 3, 32-37.], understanding the interaction between MLS128 and Tn-clusters may allow us to the development of novel cancer therapeutics. Although MLS128 was previously reported to have specificity for Tn3 rather than Tn2, similar levels of Tn2/Tn3 binding were unexpectedly observed at 37°C. Thus, thermodynamic analyses were performed via surface plasmon resonance (SPR) using synthetic Tn2- and Tn3-peptides at 10, 15, 20, 25 and 30°C. SPR results revealed that MLS128's association constants for both antigens were highly temperature dependent. Below 25°C MLS128's association constant for Tn3-peptide was clearly higher than that for Tn2-peptide. At 30°C, however, the association constant for Tn2-peptide was higher than that for Tn3-peptide. This reversal of affinity is due to the sharp increase in K(d) for Tn3. These results were confirmed by NMR, which directly measured MLS128-Tn binding in solution. This study suggested that thermodynamic control plays a critical role in the interaction between MLS128/Tn2 and MLS128/Tn3.     

The Composition of Saccharogenic Amylase from Rhizopus javanicus and the Isolation of Glycopeptides

Saccharogenic amylase from Rhizopus javanicus sp. 3–46 was known to be a glycoprotein which contained 27 residues of mannose and 4 residues of N-acetylglucosamine per mole of the saccharogenic amylase. Attempts have been made to obtain glycopeptides from the saccharogenic amylase. Three glycopeptides, GP-I-a, GP-I-b and GP-II, were separated from a Pronase digest of heat-denatured saccharogenic amylase by gel filtration on Sephadex G-50 and chromatography on DEAE-Sephadex A-25. GP-I-a contained asparagine, glycine, mannose and N-acetylglucosamine in a molar ratio of 1: 1: 6: 2. GP-I-b contained asparagine, threonine, mannose and N-acetylglucosamine in a molar ratio of 1: 1: 9:2. GP-II consisted of threonine, serine, proline, alanine and mannose in a molar ratio of 6: 2: 2: 2: 12.     

Structural heterogeneity in the core oligosaccharide of the S-layer glycoprotein from Aneurinibacillus thermoaerophilus DSM 10155.

The surface layer glycoprotein of Aneurinibacillus thermoaerophilus DSM 10155 has a total carbohydrate content of 15% (by mass), consisting of O-linked oligosaccharide chains. After proteolytic digestion of the S-layer glycoprotein byPronase E and subsequent purification of the digestion products by gel permeation chromatography, chromatofocusing and high-performance liquid chromatography two glycopeptide pools A and B with identical glycans and the repeating unit structure -->4)-alpha-l-Rha p -(1-->3)-beta-d- glycero -d- manno -Hep p -(1--> (Kosma et al., 1995b, Glycobiology, 5, 791-796) were obtained. Combined evidence from modified Edman-degradation in combination with liquid chromatography electrospray mass-spectrometry and nuclear magnetic resonance spectroscopy revealed that both glycopeptides contain equal amounts of the complete core structure alpha-l-Rha p -(1-->3)-alpha-l-Rha p -(1-->3)-beta-d-Gal p NAc-(1-->O)-Thr/Ser and the truncated forms alpha-l-Rha p -(1-->3)-beta-d-Gal p NAc-(1-->O)-Thr/Ser and beta-d-Gal p NAc-(1-->O)-Thr/Ser. All glycopeptides possessed the novel linkage types beta-d-Gal p NAc-(1-->O)-Thr/Ser. The different cores were substituted with varying numbers of disaccharide repeating units. By 300 MHz proton nuclear magnetic resonance spectroscopy the complete carbohydrate core structure of the fluorescently labeled glyco-peptide B was determined after Smith-degradation of its glycan chain. The NMR data confirmed and complemented the results of the mass spectroscopy experiments. Based on the S-layer glycopeptide structure, a pathway for its biosynthesis is suggested.     

Development and characterization of an antibody directed to an α-N-acetyl-D-galactosamine glycosylated MUC2 peptide

In an attempt to raise anti-Tn antibodies, an -N-acetyl-D-galactosamine glycosylated peptide based on the tandem repeat of the intestinal mucin MUC2 was used as an immunogen. The MUC2 peptide (PTTTPISTTTMVTPTPTPTC) was glycosylated in vitro using concentrated -N-acetylgalactosaminyltransferases activity from porcine submaxillary glands which resulted in the incorporation of 8–9 mol of Ga/NAc. Rabbits and mice developed specific anti-MUC2-GalNAc glycopeptide antibodies and no detectable anti-Tn antibodies. Anti-glycopeptide antibodies did not show reactivity with the unglycosylated MUC2 peptide or with other GalNAc glycosylated peptides. A mouse monoclonal antibody (PMH1) representative of the observed immune response was generated and its immunohistological reactivity analysed in normal tissues. PMH1 reacted similarly to other anti-MUC2 peptide antibodies. However, in some cells the staining was not restricted to the supranuclear area but extended to the entire cytoplasm. In addition, PMH1 reacted with purified colonic mucin by Western blot analysis suggesting that PMH1 reacted with some glycoforms of MUC2. The present work presents a useful approach for development of anti-mucin antibodies directed to different glycoforms of individual mucins.     

'Wave-type' structure of a synthetic hexaglycosylated decapeptide: A part of the extracellular domain of human glycophorin A

The three-dimensional structure of a glycopeptide, His-Thr*-Ser*-Thr*-Ser*-Ser*-Ser*-Val-Thr-Lys, with 2-acetamido-2-deoxy--D-galactose (GalNAc) residues linked to six adjacent amino acids from Thr-10 to Ser-15, was studied by NMR spectroscopy and molecular dynamics (MD) simulations. The hexaglycosylated decapeptide is part of the extracellular domain of human glycophorin A and shows an extended structure of the peptide backbone due to O-glycosylation. Furthermore, each GalNAc residue exhibits one and only one NOE contact from the NHAc proton to the backbone amide proton of the amino acid that the sugar is directly bound to. This indicates a strong preference for the orientation of all GalNAc residues towards the N-terminus. NOE build-up curves were used to determine 42 inter-proton distances that, in connection with angles of the peptide backbone obtained from 3J-coupling constants, resulted in constraints for a MD simulation in water. The NMR data and the MD simulations show a preference for an extended backbone structure. The GalNAc residues are located alternatingly on opposite sides of the backbone and reduce the flexibility of the peptide backbone. The conformation of the molecule is relatively rigid and shows a 'wave-type' 3D structure of the peptide backbone within the glycosylation cluster. This new structural element is also supported by the unusual CD spectrum of the glycopeptide.     

Analysis of Tn antigenicity with a panel of new IgM and IgG1 monoclonal antibodies raised against leukemic cells

CD175 or Tn antigen is a carbohydrate moiety of N-acetylgalactosamine (GalNAc)α1-O- linked to the residue of amino acid serine or threonine in a polypeptide chain. Despite the chemical simplicity of the Tn antigen, its antigenic structure is considered to be complex and the clear determinants of Tn antigenicity remain poorly understood. As a consequence, a broad variety of anti-Tn monoclonal antibodies (mAbs) have been generated. To further investigate the nature and complexity of the Tn antigen, we generated seven different anti-Tn mAbs of IgM and IgG classes raised against human Jurkat T cells, which are Tn-positive due to the low activity of T-synthase and mutation in specific chaperone Cosmc. The binding analysis of anti-Tn mAbs with the array of synthetic saccharides, glycopeptides and O-glycoproteins revealed unexpected differences in specificities of anti-Tn mAbs. IgM mAbs bound the terminal GalNAc residue of the Tn antigen irrespective of the peptide context or with low selectivity to the glycoproteins. In contrast, IgG mAbs recognized the Tn antigen in the context of a specific peptide motif. Particularly, JA3 mAb reacted to the GSPP or GSPAPP, and JA5 mAb recognized specifically the GSP motif (glycosylation sites are underlined). The major O-glycan carrier proteins CD43 and CD162 and isoforms of CD45 expressed on Jurkat cells were precipitated by anti-Tn mAbs with different affinities. In summary, our data suggest that Tn antigen-Ab binding capacity is determined by the peptide context of the Tn antigen, antigenic specificity of the Ab and class of the immunoglobulin. The newly generated anti-Tn IgG mAbs with the strong specificity to glycoprotein CD43 can be particularly interesting for the application in leukemia diagnostics and therapy.     

The oncofetal structure of human fibronectin defined by monoclonal antibody FDC-6. Unique structural requirement for the antigenic specificity provided by a glycosylhexapeptide

Previously, monoclonal antibody FDC-6 was established, which defines a structure specific for fibronectins isolated from fetal and malignant cells and tissues. The presence of the FDC-6-defined structure at type III connecting segment (III CS) is characteristic of oncofetal fibronectin (onf-FN), and its absence is characteristic of normal fibronectin (nor-FN) (Matsuura, H., and Hakomori, S. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 6517-6521). Hepatoma fibronectin was sequentially digested by various proteases, followed by subsequent chromatography on an FDC-6 affinity column and reverse-phase columns at each step of digestion. A single strongly active glycosylhexapeptide (glycopeptide 1) and an inactive glycosylpentapeptide (glycopeptide 3) were isolated from glycopeptide A containing 35 amino acid residues. The minimum essential structure required for the FDC-6 activity was found to be a hexapeptide sequence Val-Thr-His-Pro-Gly-Tyr having NeuAc alpha 2----3Gal beta 1----3GalNAc or its core (Gal beta 1----3GalNAc or GalNAc) linked at threonine. Various synthetic peptides including the Val-Thr-His-Pro-Gly-Tyr sequence and a glycopeptide having the Val-Thr-His-Pro-Gly pentapeptide with the same glycosylation at threonine were all inactive. Elimination of sialic acid slightly increased the activity, and subsequent elimination of galactose did not alter the activity; however, removal of the Gal beta 1----3GalNAc residue by endo-alpha-N-acetylgalactosaminidase from desialylated glycopeptide A resulted in total inactivation of the reactivity with FDC-6 antibody. Thus, a single glycosylation at a defined threonine residue of the III CS region may induce conformational changes in the peptide to form the specific oncofetal epitope recognized by FDC-6 antibody. This finding opens the possibility that a number of other oncofetal epitopes consist of a peptide and a common O-linked carbohydrate and that the combination produces a conformation specific to cancer or to a stage of development.     

Partial purification and characterization of an endo-alpha-N-acetylgalactosaminidase from the culture of medium of Diplococcus pneumoniae.

The culture medium of Diplococcus pneumoniae contains enzymic activity that cleaves Galbeta1 leads to 3GalNAc from desialized human erythrocyte membrane glycoprotein. The enzyme was purified 180-fold by ammonium sulfate fractionation, gel filtration through a Sephadex G-200 column, and DEAE A-25 Sephadex chromatography. The purified enzyme liberates Galbeta1 leads to 3GalNAc from glycopeptides and glycoproteins with Galbeta1 leads to 3GalNAcalpha1 leads to Ser and Thr moieties. The optimum pH of this enzyme is 6.0. Using glycopeptides obtained by trypsin digestion of human erythrocyte membrane glycoprotein as a substrate, a Km of 0.20 mM (on the basis of the amount of Galbeta1 leads to 3GalNAc residues) was obtained. So far, the enzyme appears to have a strict specificity for Galbeta1 leads to 3GalNAcalpha1 leads to Ser and Thr structures, because no oligosaccharides larger than trisaccharides were liberated from porcine submaxillary mucin.     

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.