N-terminal sequence extension in the glycosylated forms of human pancreatic stone protein. The 5-oxoproline N-terminal chain is O-glycosylated on the 5th amino acid residue.

The pancreatic stone protein isolated from human calculi (PSP) derives from the immunoreactive protein forms detected in human pancreatic juice (PSP S2-5) through the tryptic cleavage of the Arg-11-Ile-12 bond. Among the eleven amino acids of the PSP S2-5 N-terminal extension Z-E-A-Q-T-E-L-P-Q-A-R, the first residue is an oxoproline and the fifth, a threonine, bears the single carbohydrate chain of the protein molecules. Variations in the glycan chain composition account for the differences in the Mr of PSP S2-5. The PSP S2-5 forms are very soluble in aqueous solutions between the pH values 5.0-9.0, whereas the proteolysated form is scarcely soluble.

The glycan moiety of human pancreatic lithostathine. Structure characterization and possible pathophysiological implications.

Lithostathine, also known as pancreatic stone protein, pancreatic thread protein or regenerating protein, is a glycoprotein which is normally found in the exocrine pancreas, whereas in other tissues it appears either only under pathological conditions, such as Alzheimer's disease (brain), cancer (colon) or during regeneration (endocrine pancreas). In the latter case, it has been shown recently that it acts as a growth factor which stimulates islet regeneration. Little is known about its glycan moiety, which conceivably might be involved in this tissue specificity and pathophysiological characteristics. Therefore we isolated the major oligosaccharide chains of human pancreatic lithostathine and determined their sequences by means of NMR analysis. We obtained eleven different glycoforms and we were able to determine the sequence of seven of them. They all were from the same site of glycosylation (Thr5) and displayed the same core 2 structure: GlcNAc(beta 1-6)[Gal(beta 1-3)]GalNAc alpha-. They ranged in size from 4 to 9 sugar residues. Elongation was found to proceed from a common tetrasaccharidic core: Gal(beta 1-4)GlcNAc(beta 1-6)[Gal(beta 1-3)]GalNAc-ol through N-acetyllactosamine units. The non-reducing ends of some oligosaccharides carry the antigenic determinant H, with presence of external Fuc linked only in (alpha 1-2) to Gal. All the glycans, except one, carry a sialic acid in (alpha 2-3) linkage to Gal, with one disialylated form which displays a supplementary (alpha 2-6) linkage. These findings are consistent with the polymorphism of the protein, shown by means of SDS gel electrophoresis and isoelectric focusing, either in its native form or after enzymic processing. Moreover, sialylation seems to protect to some extent the Arg11-Ile12 bond from in situ hydrolysis, thus preventing the harmful precipitation of the C-terminal polypeptide in the pancreatic ducts.

Isolation of the major O-glycosidically linked oligosaccharides obtained by alkaline borohydride degradation of human meconium glycoproteins.

Neutral and acidic oligosaccharides derived from human meconium glycoproteins by alkaline borohydride degradation have been separated by high-performance liquid chromatography on a Micro-Pak anion-exchange column. In each class, oligosaccharides were purified by normal-phase (neutral and acidic oligosaccharides) and reversed-phase (neutral oligosaccharides) chromatography. Effective separations of neutral oligosaccharides and acidic oligosaccharides were achieved.

Human pre-alpha-inhibitor: isolation from a by-product of industrial scale plasma fractionation and structural analysis of its H3 heavy chain.

Pre-alpha-inhibitor (P alpha I) is a serine proteinase inhibitor from human plasma. It comprises bikunin (BK) responsible for antiprotease activity, covalently linked to a heavy chain H3. Here we describe its isolation from a side fraction of an industrial preparation of plasma clotting factors. By using a highly specific polyclonal antiserum prepared from rabbit immunized with a H3P polypeptide obtained in a bacterial expression system, we were able to identify the fractions containing P alpha I. Then, taking advantage of the differential affinity of the members of the inter-alpha-inhibitor family (I alpha I) for heparin-Sepharose and blue-Sepharose, we isolated P alpha I. Its specific antitryptic activity was 580 IU/g, higher than that of I alpha I: 420 IU/g. Its M(r), determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis, with or without prior reduction, was 130,000. Its peptide chains were identified by N-terminal sequencing. The H3 heavy chain was isolated from P alpha I by alkaline dissociation and anion-exchange chromatography. Its electrophoretic mobility was compared to that of the HI and H2 heavy chains of I alpha I. In reducing conditions, it was quite similar to that of H2 (M(r) 85,000) but clearly different from that of H1 (M[r] 78,000). Thus, the so-determined apparent M(r) of H3 was overestimated since its molecular mass determined by MALDI-TOF was 74,100. This result agrees with the proposed structure for H3. Indeed, by carbohydrate analysis and PNGase F digestion, we demonstrate that the two potential N-glycosylation sites present in the core-protein (theoretical mass: 69,454) are really occupied by two N-glycans, probably of biantennary type.

Glycosylation pattern of human inter-alpha-inhibitor heavy chains.

Human inter-alpha-inhibitor (IalphaI) is a plasma serine-proteinase inhibitor. It consists of three polypeptide chains covalently linked by a glycosaminoglycan chain: a light chain named bikunin carrying the anti-proteinase activity and two heavy chains, H1 and H2, which exhibit specific properties, e.g. they interact with hyaluronan thus stabilizing the extracellular matrix. In this study, using matrix-assisted laser desorption ionization-time-of-flight MS and amino acid sequencing of tryptic peptides, we provide a detailed analysis of the glycosylation pattern of both heavy chains. H1 carries two complex-type N-glycans of predominantly biantennary structure linked to asparagine residues at positions 256 and 559 respectively. In contrast, the oligosaccharides attached to H2 are a complex-type N-glycan in the N-terminal region of the protein (Asn64) and three to four type-1 core-structure O-glycans mono- or di-sialylated, clustered in the C-terminal region. We propose that these O-glycans might function as a recognition signal for the H2 heavy chain. The biological implications of this hypothesis, notably for the biosynthetic pathway of IalphaI, are discussed.

Sd(a)-antigen-like structures carried on core 3 are prominent features of glycans from the mucin of normal human descending colon.

This paper describes structural characterization by NMR, MS and degradative studies of mucin glycans from normal human descending colon obtained freshly at autopsy. The saccharides were mainly based on core 3 (GlcNAcbeta1-3GalNAc). Among the terminal saccharide determinants Sd(a)/Cad-antigen-like structures were prominent, and Lewis x, sialyl Lewis x and sulphated Lewis x were found as minor components, whereas blood group H and A antigenic determinants were absent. The saccharides were markedly different from those of mucins from colon cancers or colon cancer cell lines analysed so far, in which cores 1 and 2 are prominent features, and in which various other terminal determinants have been found, but not Sd(a)/Cad.

Comparative study of glycophorin A derived O-glycans from human Cad, Sd(a+) and Sd(a-) erythrocytes.

Glycophorin A was purified from the erythrocyte membranes of blood group Cad, Sd(a+) and Sd(a-) donors and the oligosaccharide alditols, obtained after alkaline borohydride degradation, separated by h.p.l.c. on an alkylamine silica gel column, were characterized by sugar analysis. Structure determination of the major acid components by methylation analysis, g.l.c.-m.s. and 1H-n.m.r. indicated that the three blood group Cad red cells under study (samples Cad., Bui. and Des.) carry the same pentasaccharide GalNAc(beta 1-4)[NeuAc(alpha 2-3)]Gal(beta 1-3)[NeuAc(alpha 2-6)]GalNAc -ol(Cad determinant) but in different amounts. This pentasaccharide, however, was absent from glycophorin A of Sd(a+) and Sd (a-) donors, suggesting that the Sda determinant is not associated with glycophorins. It was calculated that glycophorin A from the original Cad donor (Cad.) carries about 12 O-glycosidically linked pentasaccharide chains per molecule whereas only 2-3 of these chains were present in the samples from the two other unrelated Cad individuals (Bui. and Des.) It is well known from quantitative agglutination studies that the proportion of red cells which can be agglutinated by the Dolichos biflorus lectin varies from one Cad blood sample to another. Some are completely agglutinated (Cad. donor) whereas others are only partially agglutinated (Bui. and Des. donors) suggesting that some red cells might not carry the Cad determinants. From the results presented above and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis studies it is suggested that Cad red cells from Bui. and Des. do not carry a mixture of glycophorin A molecules with or without the Cad pentasaccharides but a spectrum of glycoprotein molecules with varying amounts of Cad determinants.

Chondroitin sulphate covalently cross-links the three polypeptide chains of inter-alpha-trypsin inhibitor.

Inter-alpha-trypsin inhibitor (ITI) is a tight complex of three different proteins: bikunin and two heavy chains H1 and H2. In order to demonstrate that the three chains are covalently linked by a chondroitin sulphate chain as previously proposed [Enghild, J. J., Salvesen, G., Hefta, S. A., Thogersen, I. B., Rutherford, S. and Pizzo, S. V. (1991) J. Biol. Chem. 266, 747-751], ITI was extensively digested with thermolysin and the glycosaminoglycan-containing fragment was isolated from the digest by ion-exchange chromatography. Its peptide structural determination and mass spectrometry analysis both provide evidence that the different peptide chains constituting ITI are associated by the new cross-link described as the protein-glycosaminoglycan-protein cross-link.

Decreased faecal exoglycosidase activities identify a subset of patients with active Crohn's disease.

1. alpha 1-Proteinase inhibitor (alpha 1-antitrypsin) is excreted in a deglycosylated form (M(r) 38,000) in the faeces of healthy subjects and in patients with quiescent Crohn's disease. By contrast, in most patients with active Crohn's disease, alpha 1-proteinase inhibitor is excreted in a glycosylated form (M(r) 51,000). 2. Faecal extracts containing deglycosylated alpha 1-proteinase inhibitor are able to deglycosylate alpha 1-proteinase inhibitor by an exoglycosidic process. Conversely, we demonstrate that in faecal extracts from patients excreting glycosylated alpha 1-proteinase inhibitor, glycosidase activities, such as N-acetyl-beta-glucosaminidase (EC 3.2.1.30), alpha-mannosidase (EC 3.2.1.24) and particularly beta-galactosidase (EC 3.2.1.23), are strongly decreased. 3. Degradation of glycosidases by proteases could not explain the decreased glycosidase activity in these faecal extracts. 4. Our data suggest that a modification of the bacterial colonic flora (or of its metabolic activity) occurs in most patients with active Crohn's disease and could be responsible for an impaired colonic salvage of carbohydrates.

Oligosaccharide structures of mucins secreted by the human colonic cancer cell line CL.16E.

Cl.16E, a stably differentiated clonal derivative of the human colonic cancer cell line HT29, was used to investigate the structure of oligosaccharide chains of mucins in colonic cancer. Secretory mucins were purified by equilibrium density gradient centrifugation in CsCl. Oligosaccharide side chains were isolated after beta-elimination. Compositional analysis of oligosaccharide-alditols performed after purification by gel filtration on a Bio-gel P-6 column showed 1) that GalNAc residues were located exclusively at the reducing ends of the chains, and 2) that fucose was absent from the preparation. Oligosaccharide-alditols were separated by high performance liquid chromatography (HPLC) on quaternary amine packings into a minor neutral fraction representing about 6.5% by weight of released oligosaccharides and four acidic fractions. Two acidic fractions, namely FI and FII encompassing mono- and disialylated structures, respectively, and containing 78% of total oligosaccharide alditols, were separated by HPLC. Structural determinations were carried out using methylation analysis, 1H NMR spectroscopy, and fast atom bombardment-mass spectrometry. Twelve oligosaccharide structures were determined which ranged in size from 3 to 8 residues. These oligosaccharides were based on core types 1, 2, and 4. Elongation of oligosaccharide chains was terminated by addition of sialic acid in alpha 2-3 linkage to Gal beta 1-3R and to Gal beta 1-4R residues. The predominant structure was a hexasaccharide (fraction FII-4). This contrasts with normal colonic mucins whose oligosaccharides were previously found to be based on core 3 structures and carry sialic acids in alpha (2-6) linkage to Gal beta 1-3R, to Gal beta 1-4R, and to GalNAc alpha-R (Podolsky, D.K. (1985) J. Biol. Chem. 260, 8262-8271; Podolsky, D.K. (1985) J. Biol. Chem. 260, 15510-15515). Collectively our findings suggest that Cl.16E colon cancer cells are able to synthesize mucin oligosaccharides of gastric type whose elongation is truncated by premature sialylation.

Sulfated lewis X determinants as a major structural motif in glycans from LS174T-HM7 human colon carcinoma mucin.

This article describes oligosaccharide structures of mucin isolated from nude mouse xenograft tumors produced by LS174T-HM7 cells, a subline of the human colon carcinoma LS174T with higher metastatic tendency and higher mucin production. A striking feature of the oligosaccharides of the LS174T-HM7 xenograft tumor mucin was a predominance of sulfated Lewis X determinants: HSO3-Galbeta1-4(Fucalpha1-3)GlcNAc. In addition to one previously known saccharide with one sulfated Lewis X determinant, the HM7 xenograft tumor mucin contained multiple novel structures containing one, two, or three sulfated Lewis X determinants. This determinant, known to act as a selectin ligand, has been found previously in minor saccharide components of human milk as well as mucins, but never before as a predominant structure in one mucin source.

Purification and characterization of the MUC1 mucin-type glycoprotein, epitectin, from human urine: structures of the major oligosaccharide alditols.

The MUC1 glycoprotein, epitectin, a component of the human bladder epithelium, was purified from human urine. Sedimentation equilibrium analysis and gel filtration using polysaccharide or protein standards revealed a polydisperse preparation with molecular weights ranging from about 0.9 to 1.3 x 10(6). This suggests that in the native state epitectin exists as aggregates of three or four monomer units of 350-400 kDa. Epitectin was found to have significant affinity to hexyl-, octyl- or phenyl agarose indicating that hydrophobic interactions and possibly carbohydrate-carbohydrate interactions may be responsible for the self-association. Chemical and enzymic deglycosylation of [125I]-labeled urine epitectin and metabolically labeled H.Ep.2 epitectin resulted in extremely polydisperse products. The buoyant densities of epitectin purified from urine and H.Ep.2 cells were found to be 1.39-1.40 g ml(-1), suggesting that the total carbohydrate content of these preparations is not significantly different. The O-linked saccharides of epitectin were fractionated by HPLC and analyzed by permethylation and FAB-MS. The neutral saccharides from both sources contain three common structures, namely Gal1 --> 3GalNAc, GlcNAc1 --> 6 (Gal1 --> 3) GalNAc and Gal1 --> 4GlcNAc --> 6 (Gal1 --> 3)GalNAc. The sialic acid of urine epitectin consisted entirely of N-acetylneuraminic acid. The two sources of epitectin, in vitro labeled on sialic acid, were found to have the same sialyl oligosaccharides but in different proportions. Metabolic labeling and N-glycanase susceptibility experiments firmly established the presence of N-linked saccharides in epitectin as minor components. The remarkable similarities in the total carbohydrate content, the carbohydrate composition and structures of saccharides between epitectin from urine, a non-malignant source, and H.Ep.2 cells is surprising in view of the prevailing view that MUC1 glycoproteins of cancer cells are underglycosylated compared to those produced by non-malignant cells.

Structures of O-glycosidically linked oligosaccharides isolated from human meconium glycoproteins.

The structure of the major O-glycosidically linked neutral and acidic oligosaccharides isolated from human meconium glycoproteins were established. Neutral and acidic oligosaccharides were released by alkaline borohydride treatment, purified by Biogel P-6 and fractionated by high-performance liquid chromatography. This approach resulted in 50 neutral and 30 acidic oligosaccharides. The present study reports the primary structural analysis of five neutral oligosaccharides, ten monosialylated oligosaccharides, one monosialylated monosulfated oligosaccharide and three disialylated oligosaccharides, by permethylation, fast-atom-bombardment mass spectrometry analysis and 400-MHz 1H-NMR spectroscopy. The following structure have not been described previously: (formula; see text) The intestinal glycoproteins of human meconium are characterized as high molecular mass compounds with numerous carbohydrate chains of the mucin type. These mucins are a rich source of carbohydrate structures which express multiple blood group activities and occur as membrane-associated antigens, recognized by hybridoma antibodies [1-4]. A previous study [5] described the structure of fifteen free oligosaccharides derived from catabolism of O- and N-glycans accumulating in new born meconium. In the same group [6] the research has been extended to glycoasparagines with the description of thirteen of them by high resolution 1H-NMR spectroscopy analysis. From O-glycosidically linked glycoproteins, further studies [7] established the structures of nine major monosaccharides to tetrasaccharides obtained after mild acid hydrolysis and base-borohydride degradation from meconium samples of group O secretors. These oligosaccharides were derived from meconium glycopeptides which had been depleted of I- and i-antigen activities. Recently [8], neutral and acidic oligosaccharide-alditols obtained by alkaline borohydride degradation of human meconium glycoproteins have been separated by HPLC on an anion-exchange column. The neutral fraction was further purified by normal-phase and reversed-phase chromatography while acidic fractions were fractionated only by normal-phase chromatography. Thus, we have isolated several low molecular mass oligosaccharides of different size and composition and also isomers which vary in linkage position or anomer configuration. Using methylation analysis, 1H-NMR spectroscopy and fast-atom-bombardment mass spectrometry (FAB-MS), we propose the primary structure for 19 major oligosaccharides.

Studies on the structures of the Tm, Sj, M1, Can, Sext and Hu blood group antigens.

The Glycophorins (GPs = sialoglycoproteins) in erythrocyte membranes from various Black individuals, some of which exhibit the M1, Can, Sj, Tm, Sext and/or Hu antigens, and several Caucasian donors, including pooled fetal red cells, were studied. Using agglutination inhibition assays with GP fractions, GP fragments and chemically modified GPs as well as trypsin treatment of intact red cells, the antigens defined by anti-M1, anti-M+M1, anti-Can and anti-Tm sera were found to be located on the N-terminal tryptic peptide (T2, residues 1-31) of the major GP (GP A = MN sialoglycoprotein). Evidence was obtained that the N-terminal amino-acid residue, NeuNAc and/or (a) different sugar residue(s) are involved in the antigens. Amino-acid sequence and composition analyses excluded an amino-acid exchange within the N-terminal region (residues 1-31) of GP A. Carbohydrate analyses revealed the attachment of GlcNAc residues (up to about five, dependent on the strength of the above-mentioned antigens) to O-glycosidically linked oligosaccharides within the N-terminal portion (residues 1-31) of GP A. As judged from the carbohydrate compositions of peptides, the alteration of the O-glycosidic oligosaccharides is associated with a slight increase of the Gal and Fuc contents and a slight decrease of the NeuNAc level. Analyses of small, secondary cyanogen bromide and V8 proteinase peptides from the N-terminal region of GP A from Blacks, Caucasians and Caucasian fetal cells suggest that the variable attachment of small quantities of GlcNAc (about 0.03 to about 0.2 residues per peptide molecule) accounts, at least in part, for the polymorphisms detected by anti-Can and the original anti-Tm (serum Sheerin). Remarkably, the GlcNAc-containing O-glycosidic oligosaccharides occur only in small quantities, or not all at, within the positions 32-61 of GP A and the glycosylated domains of GP B and GP C.(ABSTRACT TRUNCATED AT 400 WORDS)

The heavy chains of human plasma inter-alpha-trypsin inhibitor: their isolation, their identification by electrophoresis and partial sequencing. Differential reactivity with concanavalin A.

Inter-alpha-trypsin inhibitor (ITI) is a complex protein made up of a light chain so-called bikunin and two heavy chains (apparent Mr values 96000 and 86000 in SDS/PAGE in non-reducing conditions). By sequence analysis, we clearly identified those two components as H1 and H2, respectively. We demonstrate that alkaline treatment (50mM NaOH during 5 min at room temperature) as well as chondroitinase digestion both lead to the dissociation of ITI. The conditions used for alkaline treatment were previously reported for cleavage of the covalent linkage between bikunin and H3 inside pre-alpha-trypsin inhibitor (Enghild et al. (1991) J. Biol. Chem. 266, 747-751). Carbohydrate analysis of the two heavy chains isolated by ion-exchange chromatography suggests the presence of complex-type N-glycans in both H1 and H2 and that of O-glycans in H2. H1 is eluted from Con-A Sepharose by alpha-methylmannoside, in agreement with the existence of at least one biantennary glycan chain. In contrast, H2 remains strongly bound to this support when submitted to the same conditions. Therefore this binding does not depend on carbohydrates. The capacity of H2 to develop such interactions is discussed with regard to the unusual bindings likely to exist between the different peptide chains constituting ITI.

Antibacterial activity of glycosylated and phosphorylated chromogranin A-derived peptide 173-194 from bovine adrenal medullary chromaffin granules.

Recently, we have isolated from bovine chromaffin granules and identified two natural peptides possessing antibacterial activity: secretolytin (chromogranin B 614-626) and enkelytin (proenkephalin-A 209-237). Here, we characterize a large natural fragment, corresponding to chromogranin A 79-431, that inhibits growth of both Gram-positive and Gram-negative bacteria. The aim of the present work was to determine the shortest active peptide located in the 79-431 chromogranin A region. Three peptides, which shared the same 173-194 chromogranin A sequence (YPGPQAKEDSEGPSQGPASREK) but differed in post-translational modifications, including O-glycosylation and tyrosine phosphorylation, were isolated. A detailed study using microsequencing and mass spectrometry allowed us to correlate their antibacterial activity with these post-translational modifications. The chromogranin A precursor fragment (79-431) and the active glycosylated and phosphorylated peptides were, respectively, named prochromacin and chromacin (P, G, and PG for phosphorylated, glycosylated, and phosphorylated-glycosylated form).

Phosphorylation and O-glycosylation sites of human chromogranin A (CGA79-439) from urine of patients with carcinoid tumors.

Because of their water-soluble properties, chromogranins (CGs) and chromogranin-derived fragments are released together with catecholamines from adrenal chromaffin cells during stress situations and can be detected in the blood by radiochemical and enzyme assays. It is well known that chromogranins can serve as immunocytochemical markers for neuroendocrine tissues and as a diagnostic tool for neuroendocrine tumors. In 1993, large CGA-derived fragments have been shown to be excreted into the urine in patients with carcinoid tumors and the present study deals with the characterization of the post-translational modifications (phosphorylation and O-glycosylation) located along the largest natural CGA-derived fragment CGA79-439. Using mild proteolysis of peptidic material, high performance liquid chromatography, sequencing, and mass spectrometry analysis, six post-translational modifications were detected along the C-terminal CGA-derived fragment CGA79-439. Three O-linked glycosylation sites were located in the core of the protein on Thr163, Thr165, and Thr233, consisting in di-, tri-, and tetrasaccharides. Three phosphorylation sites were located in the middle and C-terminal domain, on serine residues Ser200, Ser252, and Ser315. These modified sites were compared with sequences of others species and discussed in relation with the post-translational modifications that we have reported previously for bovine CGA.

Permanent exposure of mucin-secreting HT-29 cells to benzyl-N-acetyl-alpha-D-galactosaminide induces abnormal O-glycosylation of mucins and inhibits constitutive and stimulated MUC5AC secretion.

Previous work has shown that treatment of HT-29 methotrexate (MTX) cells with benzyl-N-acetyl-alpha-D-galactosaminide results in profound changes in mucin oligosaccharide chains. To analyse in depth the effect of this drug, we first determined the structure of mucin oligosaccharide chains synthesized by HT-29 MTX cells and the changes induced by permanent drug exposure. Mucins from untreated cells contained nine monosialylated structures (core types 1, 2, 3 and 4) and four disialylated structures (types 1, 2 and 4). Core 1 structures predominated, in particular NeuAcalpha2-3Galbeta1-3GalNAc-ol. Exposure of HT-29 MTX cells to benzyl-N-acetyl-alpha-D-galactosaminide from days 2-21 resulted in a decrease in intracellular mucins and both their sialic acid and galactose content, and an increased T (Galbeta1-3GalNAcalpha-O-Ser/Thr) and Tn (GalNAcalpha-O-Ser/Thr) antigenicity. A 3-fold increase in both Galbeta1-3GalNAc alpha2, 3-sialyltransferase activity and mRNA expression was detected. At the ultrastructural level, T-antigen was not detectable in mucin droplets in control cells, but was strongly expressed in intracytoplasmic vesicles in treated cells. In these cells, MUC1 and MUC3 transcripts were up-regulated, whereas MUC2, MUC5B and MUC5AC were down-regulated. Furthermore, constitutive and secretagogue-induced MUC5AC secretion was reduced and no mucus layer was detected. In conclusion, benzyl-N-acetyl-alpha-D-galactosaminide induces abnormal O-glycosylation and altered regulation of MUC5AC secretion.