Calliope Capon - Academia.edu (original) (raw)

Papers by Calliope Capon

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

Journal of Biological Chemistry, 1992

Cl.16E, a stably differentiated clonal derivative of the human colonic cancer cell line HT29, was... more 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.

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

Biochemical Journal, 1985

Glycophorin A was purified from the erythrocyte membranes of blood group Cad, Sd(a+) and Sd(a-) d... more 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 sa...

Research paper thumbnail of Sda-antigen-like structures carried on core 3 are prominent features of glycans from the mucin of normal human descending colon

Biochemical Journal, 2001

This paper describes structural characterization by NMR, MS and degradative studies of mucin glyc... more 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 (GlcNAcβ1-3GalNAc). Among the terminal saccharide determinants Sda/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 Sda/Cad.

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

Biochemical Journal, 1998

Previous work has shown that treatment of HT-29 methotrexate (MTX) cells with benzyl-N-acetyl-α-d... more Previous work has shown that treatment of HT-29 methotrexate (MTX) cells with benzyl-N-acetyl-α-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 NeuAcα2–3Galβ1–3GalNAc-ol. Exposure of HT-29 MTX cells to benzyl-N-acetyl-α-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 (Galβ1–3GalNAcα-O-Ser/Thr) and Tn (GalNAcα-O-Ser/Thr) antigenicity. A 3-fold increase in both Galβ1–3GalNAc α2,3-sialyltransferase activity and mRNA expression was detected. At the ultrastructural level, T-an...

Research paper thumbnail of Glycosylation pattern of human inter-α-inhibitor heavy chains

Biochemical Journal, 1998

Human inter-α-inhibitor (IαI) is a plasma serine-proteinase inhibitor. It consists of three polyp... more Human inter-α-inhibitor (IαI) 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 m...

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

Journal of Chromatography B: Biomedical Sciences and Applications, 1988

Neutral and acidic oligosaccharides derived from human meconium glycoproteins by alkaline borohyd... more 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.

Research paper thumbnail of Structural determination of O-glycans by tandem mass spectrometry

Methods in molecular biology (Clifton, N.J.), 2006

Nano-electrospray ionization quadrupole time-of-flight mass spectrometry (nanoESI-Q-TOF-MS) provi... more Nano-electrospray ionization quadrupole time-of-flight mass spectrometry (nanoESI-Q-TOF-MS) provides a sensitive means for mapping and sequencing underivatized O-glycans. This chapter describes fragmentation rules of O-glycans by ESI-MS/MS and provides a series of diagnostic ions relevant for the determination of the core type, position, and linkage of fucose, sialic acid, and sulphate residues, as well as information on type I or II chains. Positive-ion mode gives information about core type, linkage, and position of fucose residues. Negative-ion mode can be applied for differentiation between isomeric molecules and for analysis of sulphated or sialylated glycans. The current technology successfully determines the sequence of underivatized oligosaccharides in complex mixtures and provides a significant step toward the goal of characterizing all aspects of carbohydrate structure using a single instrument.

Research paper thumbnail of Diagnostic ions for the rapid analysis by nano-electrospray ionization quadrupole time-of-flight mass spectrometry of O-glycans from human mucins

Rapid communications in mass spectrometry : RCM, 2004

Nano-electrospray ionization quadrupole time-of-flight mass spectrometry (nanoESI-Q-TOFMS) was us... more Nano-electrospray ionization quadrupole time-of-flight mass spectrometry (nanoESI-Q-TOFMS) was used for sensitive mapping and sequencing of underivatized oligosaccharide alditols obtained from human mucins. Using subnanomolar amounts of oligosaccharides previously analyzed by nuclear magnetic resonance (NMR), series of diagnostic ions relevant to the structural characterization of O-glycans were deduced. Determination of the core type as well as positions and partial linkages of fucose residues could be readily obtained from the dominant [M+Na](+) ions. Differentiation of isomeric structures and glycosidic linkages were defined by the characteristic cross-ring (0,2)A-type cleavages in the negative ion mode. Tandem (MS/MS) mass spectra of [M-H](-) ions from sialylated or sulfated O-glycans revealed information concerning the position and linkage of such residues. These fragmentation rules were further applied in the structural determination of glycans from human colonic mucins. All t...

Research paper thumbnail of 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 ... more 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؋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 [ 125 I]-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 Gal1P P3GalNAc, GlcNAc1P P6 (Gal1P P3) GalNAc and Gal1P P4 GlcNAcP P6 (Gal1P P3)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.

Research paper thumbnail of Studies on the Structures of the Tm, Sj, M1, Can, Sext and Hu Blood Group Antigens

Biological Chemistry Hoppe-Seyler, 1991

Research paper thumbnail of Monosaccharide Composition of Mucins

Glycoprotein Methods and Protocols, 2000

Research paper thumbnail of Chondroitin sulphate covalently cross-links the three polypeptide chains of inter-alpha-trypsin inhibitor

European Journal of Biochemistry, 1994

Research paper thumbnail of The Heavy Chains of Human Plasma Inter-α-Trypsin Inhibitor: Their Isolation, Their Identification by Electrophoresis and Partial Sequencing. Differential Reactivity with Concanavalin A

Biological Chemistry Hoppe-Seyler, 1992

Trypsininhibitor (ITI) ist ein komplexes Protein, das aus einer leichten Kette, dem sogenannten B... more Trypsininhibitor (ITI) ist ein komplexes Protein, das aus einer leichten Kette, dem sogenannten Bikunin besteht sowie aus zwei schweren Ketten mit den apparenten M r-Werten 96000 und 86000 (in der SDS/PAGE unter nicht reduzierenden Bedingungen). Durch Sequenzanalyse konnten wir diese beiden Komponenten klar als HI und H 2 identifizieren. Wir zeigen, daß sowohl alkalische Behandlung (50mM NaOH 5 min bei Raumtemperatur) wie Chondroitase-Verdauung zur Dissoziation von ITI führen. Die für die Alkalibehandlung verwendeten Bedingungen wurden bereits für die Spaltung der kovalenten Bindung zwischen Bikunin und H 3 im Prä-atrypsininhibitor mitgeteilt (Enghild et al. (1991) /. Biol. Chem. 266, 747-751).

Research paper thumbnail of The Glycan Moiety of Human Pancreatic Lithostathine. Structure Characterization and Possible Pathophysiological Implications

European Journal of Biochemistry, 1995

Lithostathine, also known as pancreatic stone protein, pancreatic thread protein or regenerating ... more 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.

Research paper thumbnail of Structures of O-glycosidically linked oligosaccharides isolated from human meconium glycoproteins

European Journal of Biochemistry, 1989

The structure of the major 0-glycosidically linked neutral and acidic oligosaccharides isolated f... more The structure of the major 0-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 live neutral oligosaccharides, ten monosialylated oligosaccharides, one monosialylated monosulfated oligosaccharide and three disialylated oligosaccharides, by permethylation, fast-atom-bombardment mass spectrometry analysis and 400-MHz 'H-NMR spectroscopy. The following structure have not been described previously: F 11-1-6 : NeuAc(a2-6) \ I B GalNAc-ol Gal(Pl-3)GlcNAc(p1-3) F 11-2-1 : F 11-2-4 : F 11-2-7 : NeuAc(a2-6) \ \ ,Gal(p1-4) GalNAc-ol \ 3Gal(p1-4) GalNAc-ol HS03 / \ I / I FUC (al-3) NeuAc(a2-6) \ GalNAc-ol NeuAc(a2-6) NeuAc(a2-3)Gal(pl-4) \ \ GlcNAc(P1-3) / Fuc(a1-3) ____ The intestinal glycoproteins of human meconium are characterized as high molecular 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 [I-41. A previous study [5] described the structure of fifteen free oligosaccharides derived from catabolism of 0-and N-glycans

Research paper thumbnail of Expression of a Core 3 Disialyl-Lex Hexasaccharide in Human Colorectal Cancers: A Potential Marker of Malignant Transformation in Colon

Journal of Proteome Research, 2009

Cancer-associated alterations in cell surface and secreted glycoproteins have been catalogued for... more Cancer-associated alterations in cell surface and secreted glycoproteins have been catalogued for many years but many of the studies of alterations in mucin carbohydrate have relied on histochemical or immunohistochemical methods, with little direct chemical analysis. In this study, we analyzed the O-glycosylation pattern of MUC2 glycoprotein isolated from colorectal carcinomas, transitional mucosa and resection margins from three patients with blood group A, B and O, respectively. After alkaline borohydride treatment, the released oligosaccharides were structurally characterized by nanoESI Q-TOF tandem mass spectrometry without prior fractionation or derivatization. As expected, we found an increased expression of sialyl-Tn antigen in the colonic cancer mucins. A more interesting feature was the increased expression of a core 3 sialyl-Le x hexasaccharide, NeuAcR2-3Gal 1-4(FucR1-3)GlcNAc 1-3(NeuAcR2-6)GalNAc in tumor, which appeared to compete with its sulfo-Le x counterpart in normal tissue, SO3-3Gal 1-4(FucR1-3)GlcNAc 1-3(NeuAcR2-6)GalNAc. This antigen, whose structure was confirmed by NMR experiments, is based on a core 3 glycan and may be a potential marker for the malignant transformation of colonic cells. Unexpectedly, most of the glycans recovered in normal and carcinomas extracts were based on a sialylated core 3, GlcNAc 1-3(NeuAcR2-6)GalNAcol. Moreover, the pattern of glycosylation was very similar between mucins isolated from each sample, the main differences related to the level of expression of the major oligosaccharides. The data obtained in this investigation may have value for future screening studies on colorectal cancer.

Research paper thumbnail of Evidence of Regio-specific Glycosylation in Human Intestinal Mucins

Journal of Biological Chemistry, 2003

Mucin glycans were isolated from different regions of the normal human intestine (ileum, cecum, t... more Mucin glycans were isolated from different regions of the normal human intestine (ileum, cecum, transverse and sigmoid colon, and rectum) of two individuals with ALe b blood group. A systematic study of the monosaccharides and oligosaccharide alditols released by reductive ␤-elimination from mucins was performed using gas chromatography, matrix-assisted laser desorption ionization time-of-flight mass spectrometry, and nuclear magnetic resonance spectroscopy techniques. Important variations were observed in the mucin-associated oligosaccharide content with an increasing gradient of sialic acid from the ileum to the colon associated with a reverse gradient of fucose. Moreover, a comparative study of the Sda/Cad and ABH blood group determinants along the gastrointestinal tract showed the same reverse distribution in the two kinds of antigens. In addition, besides their heterogeneity, sialic acids presented considerable variations in the degree of O-acetylation in relation to glycan sialylation level. These data are discussed in view of recent concepts suggesting that the oligosaccharide composition of the gut constitutes a varied ecosystem for microorganisms that are susceptible to adapt there and possess the specific adhesion system and specific enzymes able to provide a carbohydrate nutrient. Mucins are very high molecular weight glycoproteins secreted by mucosae or some exocrine glands into the lumen of the respiratory, gastrointestinal, and reproductive tracts (1). They consist of a protein backbone with hundreds of carbohydrate side chains of varying lengths, sequences, compositions, and anomeric linkages. A distinguishing feature of mucins is that they contain O-linked oligosaccharides, i.e. the sugar chains are attached to the peptide backbone via a O-glycosidic linkage between a Ser or Thr residue and a GalNAc 1 residue.

Research paper thumbnail of Sulfated Lewis X Determinants as a Major Structural Motif in Glycans from LS174T-HM7 Human Colon Carcinoma Mucin

Journal of Biological Chemistry, 1997

This article describes oligosaccharide structures of mucin isolated from nude mouse xenograft tum... more 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: HSO 3-Gal␤1-4(Fuc␣1-3)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.

Research paper thumbnail of Antibacterial Activity of Glycosylated and Phosphorylated Chromogranin A-derived Peptide 173-194 from Bovine Adrenal Medullary Chromaffin Granules

Journal of Biological Chemistry, 1996

Recently, we have isolated from bovine chromaffin granules and identified two natural peptides po... more 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 (YPGPQAKED-SEGPSQGPASREK) 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 posttranslational 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).

Research paper thumbnail of Phosphorylation and O-Glycosylation Sites of Human Chromogranin A (CGA79-439) from Urine of Patients with Carcinoid Tumors

Journal of Biological Chemistry, 1998

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

Journal of Biological Chemistry, 1992

Cl.16E, a stably differentiated clonal derivative of the human colonic cancer cell line HT29, was... more 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.

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

Biochemical Journal, 1985

Glycophorin A was purified from the erythrocyte membranes of blood group Cad, Sd(a+) and Sd(a-) d... more 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 sa...

Research paper thumbnail of Sda-antigen-like structures carried on core 3 are prominent features of glycans from the mucin of normal human descending colon

Biochemical Journal, 2001

This paper describes structural characterization by NMR, MS and degradative studies of mucin glyc... more 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 (GlcNAcβ1-3GalNAc). Among the terminal saccharide determinants Sda/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 Sda/Cad.

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

Biochemical Journal, 1998

Previous work has shown that treatment of HT-29 methotrexate (MTX) cells with benzyl-N-acetyl-α-d... more Previous work has shown that treatment of HT-29 methotrexate (MTX) cells with benzyl-N-acetyl-α-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 NeuAcα2–3Galβ1–3GalNAc-ol. Exposure of HT-29 MTX cells to benzyl-N-acetyl-α-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 (Galβ1–3GalNAcα-O-Ser/Thr) and Tn (GalNAcα-O-Ser/Thr) antigenicity. A 3-fold increase in both Galβ1–3GalNAc α2,3-sialyltransferase activity and mRNA expression was detected. At the ultrastructural level, T-an...

Research paper thumbnail of Glycosylation pattern of human inter-α-inhibitor heavy chains

Biochemical Journal, 1998

Human inter-α-inhibitor (IαI) is a plasma serine-proteinase inhibitor. It consists of three polyp... more Human inter-α-inhibitor (IαI) 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 m...

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

Journal of Chromatography B: Biomedical Sciences and Applications, 1988

Neutral and acidic oligosaccharides derived from human meconium glycoproteins by alkaline borohyd... more 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.

Research paper thumbnail of Structural determination of O-glycans by tandem mass spectrometry

Methods in molecular biology (Clifton, N.J.), 2006

Nano-electrospray ionization quadrupole time-of-flight mass spectrometry (nanoESI-Q-TOF-MS) provi... more Nano-electrospray ionization quadrupole time-of-flight mass spectrometry (nanoESI-Q-TOF-MS) provides a sensitive means for mapping and sequencing underivatized O-glycans. This chapter describes fragmentation rules of O-glycans by ESI-MS/MS and provides a series of diagnostic ions relevant for the determination of the core type, position, and linkage of fucose, sialic acid, and sulphate residues, as well as information on type I or II chains. Positive-ion mode gives information about core type, linkage, and position of fucose residues. Negative-ion mode can be applied for differentiation between isomeric molecules and for analysis of sulphated or sialylated glycans. The current technology successfully determines the sequence of underivatized oligosaccharides in complex mixtures and provides a significant step toward the goal of characterizing all aspects of carbohydrate structure using a single instrument.

Research paper thumbnail of Diagnostic ions for the rapid analysis by nano-electrospray ionization quadrupole time-of-flight mass spectrometry of O-glycans from human mucins

Rapid communications in mass spectrometry : RCM, 2004

Nano-electrospray ionization quadrupole time-of-flight mass spectrometry (nanoESI-Q-TOFMS) was us... more Nano-electrospray ionization quadrupole time-of-flight mass spectrometry (nanoESI-Q-TOFMS) was used for sensitive mapping and sequencing of underivatized oligosaccharide alditols obtained from human mucins. Using subnanomolar amounts of oligosaccharides previously analyzed by nuclear magnetic resonance (NMR), series of diagnostic ions relevant to the structural characterization of O-glycans were deduced. Determination of the core type as well as positions and partial linkages of fucose residues could be readily obtained from the dominant [M+Na](+) ions. Differentiation of isomeric structures and glycosidic linkages were defined by the characteristic cross-ring (0,2)A-type cleavages in the negative ion mode. Tandem (MS/MS) mass spectra of [M-H](-) ions from sialylated or sulfated O-glycans revealed information concerning the position and linkage of such residues. These fragmentation rules were further applied in the structural determination of glycans from human colonic mucins. All t...

Research paper thumbnail of 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 ... more 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؋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 [ 125 I]-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 Gal1P P3GalNAc, GlcNAc1P P6 (Gal1P P3) GalNAc and Gal1P P4 GlcNAcP P6 (Gal1P P3)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.

Research paper thumbnail of Studies on the Structures of the Tm, Sj, M1, Can, Sext and Hu Blood Group Antigens

Biological Chemistry Hoppe-Seyler, 1991

Research paper thumbnail of Monosaccharide Composition of Mucins

Glycoprotein Methods and Protocols, 2000

Research paper thumbnail of Chondroitin sulphate covalently cross-links the three polypeptide chains of inter-alpha-trypsin inhibitor

European Journal of Biochemistry, 1994

Research paper thumbnail of The Heavy Chains of Human Plasma Inter-α-Trypsin Inhibitor: Their Isolation, Their Identification by Electrophoresis and Partial Sequencing. Differential Reactivity with Concanavalin A

Biological Chemistry Hoppe-Seyler, 1992

Trypsininhibitor (ITI) ist ein komplexes Protein, das aus einer leichten Kette, dem sogenannten B... more Trypsininhibitor (ITI) ist ein komplexes Protein, das aus einer leichten Kette, dem sogenannten Bikunin besteht sowie aus zwei schweren Ketten mit den apparenten M r-Werten 96000 und 86000 (in der SDS/PAGE unter nicht reduzierenden Bedingungen). Durch Sequenzanalyse konnten wir diese beiden Komponenten klar als HI und H 2 identifizieren. Wir zeigen, daß sowohl alkalische Behandlung (50mM NaOH 5 min bei Raumtemperatur) wie Chondroitase-Verdauung zur Dissoziation von ITI führen. Die für die Alkalibehandlung verwendeten Bedingungen wurden bereits für die Spaltung der kovalenten Bindung zwischen Bikunin und H 3 im Prä-atrypsininhibitor mitgeteilt (Enghild et al. (1991) /. Biol. Chem. 266, 747-751).

Research paper thumbnail of The Glycan Moiety of Human Pancreatic Lithostathine. Structure Characterization and Possible Pathophysiological Implications

European Journal of Biochemistry, 1995

Lithostathine, also known as pancreatic stone protein, pancreatic thread protein or regenerating ... more 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.

Research paper thumbnail of Structures of O-glycosidically linked oligosaccharides isolated from human meconium glycoproteins

European Journal of Biochemistry, 1989

The structure of the major 0-glycosidically linked neutral and acidic oligosaccharides isolated f... more The structure of the major 0-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 live neutral oligosaccharides, ten monosialylated oligosaccharides, one monosialylated monosulfated oligosaccharide and three disialylated oligosaccharides, by permethylation, fast-atom-bombardment mass spectrometry analysis and 400-MHz 'H-NMR spectroscopy. The following structure have not been described previously: F 11-1-6 : NeuAc(a2-6) \ I B GalNAc-ol Gal(Pl-3)GlcNAc(p1-3) F 11-2-1 : F 11-2-4 : F 11-2-7 : NeuAc(a2-6) \ \ ,Gal(p1-4) GalNAc-ol \ 3Gal(p1-4) GalNAc-ol HS03 / \ I / I FUC (al-3) NeuAc(a2-6) \ GalNAc-ol NeuAc(a2-6) NeuAc(a2-3)Gal(pl-4) \ \ GlcNAc(P1-3) / Fuc(a1-3) ____ The intestinal glycoproteins of human meconium are characterized as high molecular 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 [I-41. A previous study [5] described the structure of fifteen free oligosaccharides derived from catabolism of 0-and N-glycans

Research paper thumbnail of Expression of a Core 3 Disialyl-Lex Hexasaccharide in Human Colorectal Cancers: A Potential Marker of Malignant Transformation in Colon

Journal of Proteome Research, 2009

Cancer-associated alterations in cell surface and secreted glycoproteins have been catalogued for... more Cancer-associated alterations in cell surface and secreted glycoproteins have been catalogued for many years but many of the studies of alterations in mucin carbohydrate have relied on histochemical or immunohistochemical methods, with little direct chemical analysis. In this study, we analyzed the O-glycosylation pattern of MUC2 glycoprotein isolated from colorectal carcinomas, transitional mucosa and resection margins from three patients with blood group A, B and O, respectively. After alkaline borohydride treatment, the released oligosaccharides were structurally characterized by nanoESI Q-TOF tandem mass spectrometry without prior fractionation or derivatization. As expected, we found an increased expression of sialyl-Tn antigen in the colonic cancer mucins. A more interesting feature was the increased expression of a core 3 sialyl-Le x hexasaccharide, NeuAcR2-3Gal 1-4(FucR1-3)GlcNAc 1-3(NeuAcR2-6)GalNAc in tumor, which appeared to compete with its sulfo-Le x counterpart in normal tissue, SO3-3Gal 1-4(FucR1-3)GlcNAc 1-3(NeuAcR2-6)GalNAc. This antigen, whose structure was confirmed by NMR experiments, is based on a core 3 glycan and may be a potential marker for the malignant transformation of colonic cells. Unexpectedly, most of the glycans recovered in normal and carcinomas extracts were based on a sialylated core 3, GlcNAc 1-3(NeuAcR2-6)GalNAcol. Moreover, the pattern of glycosylation was very similar between mucins isolated from each sample, the main differences related to the level of expression of the major oligosaccharides. The data obtained in this investigation may have value for future screening studies on colorectal cancer.

Research paper thumbnail of Evidence of Regio-specific Glycosylation in Human Intestinal Mucins

Journal of Biological Chemistry, 2003

Mucin glycans were isolated from different regions of the normal human intestine (ileum, cecum, t... more Mucin glycans were isolated from different regions of the normal human intestine (ileum, cecum, transverse and sigmoid colon, and rectum) of two individuals with ALe b blood group. A systematic study of the monosaccharides and oligosaccharide alditols released by reductive ␤-elimination from mucins was performed using gas chromatography, matrix-assisted laser desorption ionization time-of-flight mass spectrometry, and nuclear magnetic resonance spectroscopy techniques. Important variations were observed in the mucin-associated oligosaccharide content with an increasing gradient of sialic acid from the ileum to the colon associated with a reverse gradient of fucose. Moreover, a comparative study of the Sda/Cad and ABH blood group determinants along the gastrointestinal tract showed the same reverse distribution in the two kinds of antigens. In addition, besides their heterogeneity, sialic acids presented considerable variations in the degree of O-acetylation in relation to glycan sialylation level. These data are discussed in view of recent concepts suggesting that the oligosaccharide composition of the gut constitutes a varied ecosystem for microorganisms that are susceptible to adapt there and possess the specific adhesion system and specific enzymes able to provide a carbohydrate nutrient. Mucins are very high molecular weight glycoproteins secreted by mucosae or some exocrine glands into the lumen of the respiratory, gastrointestinal, and reproductive tracts (1). They consist of a protein backbone with hundreds of carbohydrate side chains of varying lengths, sequences, compositions, and anomeric linkages. A distinguishing feature of mucins is that they contain O-linked oligosaccharides, i.e. the sugar chains are attached to the peptide backbone via a O-glycosidic linkage between a Ser or Thr residue and a GalNAc 1 residue.

Research paper thumbnail of Sulfated Lewis X Determinants as a Major Structural Motif in Glycans from LS174T-HM7 Human Colon Carcinoma Mucin

Journal of Biological Chemistry, 1997

This article describes oligosaccharide structures of mucin isolated from nude mouse xenograft tum... more 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: HSO 3-Gal␤1-4(Fuc␣1-3)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.

Research paper thumbnail of Antibacterial Activity of Glycosylated and Phosphorylated Chromogranin A-derived Peptide 173-194 from Bovine Adrenal Medullary Chromaffin Granules

Journal of Biological Chemistry, 1996

Recently, we have isolated from bovine chromaffin granules and identified two natural peptides po... more 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 (YPGPQAKED-SEGPSQGPASREK) 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 posttranslational 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).

Research paper thumbnail of Phosphorylation and O-Glycosylation Sites of Human Chromogranin A (CGA79-439) from Urine of Patients with Carcinoid Tumors

Journal of Biological Chemistry, 1998