Biochemical characterization of a rat oncofetal colonic antigen defined by a monoclonal antibody raised against gastric surface epithelium.
The 660 epitope was defined by a monoclonal antibody raised against rat gastric surface epithelium scrapings. This epitope, a marker of goblet cell differentiation, shows oncofetal behaviour in the colonic mucosa. We found that it co-purified with gastric mucin glycoproteins. We isolated rat gastric mucus glycoproteins using standard techniques: gastric scrapings in PBS were submitted to isopycnic density gradient centrifugation in CsCl in the presence of proteinase inhibitors. Fractions of relative density 1.4-1.45 with a high neutral sugar/protein ratio were chromatographed on an Ultrogel A4 column. According to the usual criteria, the high-molecular mass glycoproteins recovered in the excluded volume were purified mucins; when stained with periodic acid/Schiff reagent, they showed little migration on 4-15% gradient gel acrylamide electrophoresis. Serine+threonine+proline residues accounted for 35% of the total amino acids; the carbohydrate composition consisted of galactose, fucose, N-acetylgalactosamine and N-acetylglucosamine. These mucus glycoproteins carried the 660 epitope. After disulphide bond reduction, the remaining high-molecular-mass subunits were retained by the Ultrogel A4 column; amino acid and saccharide compositions were generally similar to those of the unreduced fraction. Trypsin digestion of the 660 epitope glycoprotein carrier did not modify its chromatographic and electrophoretic patterns, nor its chemical composition. The 660 epitope was still present after these treatments. However, trypsin digestion of subunits gave rise to smaller components that were retained by an Ultrogel A4 column. The saccharide composition of these fragments was unchanged, but the proportion of serine+threonine+proline residues rose to 46% of the total. These digested subunits had lost nearly all reactivity with monoclonal antibody 660. Our results fit well with the macromolecular model of Carlstedt, Lindgren and Sheehan [(1983) Biochem. J. 213, 427-435]: mucin glycoproteins are homopolymers of subunits assembled end-to-end via disulphide bonds into very large linear macromolecules. After disulphide bond reduction, proteolytic attack sites are uncovered and trypsin digestion results in glycopeptides bearing the typical oligosaccharidic units and with enhanced amounts of serine, threonine and proline, the characteristic amino acids of this hyperglycosylated region of the peptide core. These digested subunits have lost virtually all 660 epitope reactivity. We thus show that the 660 epitope, a determinant of a mucin molecule, is probably associated with the peptide core of the glycoprotein.
Decaens
C
C
CNRS URA 1343, Institut Curie, Bat. 110, Centre Universitaire, Orsay, France.
Nardelli
J
J
Bara
J
J
Burtin
P
P
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000951:Antigens, Neoplasm / Q000276:immunology*; D015153:Blotting, Western; D002850:Chromatography, Gel; D003106:Colon / Q000737:chemistry / Q000276:immunology*; D004220:Disulfides / Q000032:analysis / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004848:Epithelium / Q000276:immunology; D000939:Epitopes / Q000276:immunology; D005260:Female; D005753:Gastric Mucosa / Q000276:immunology*; D006023:Glycoproteins / Q000737:chemistry / Q000276:immunology* / Q000302:isolation & purification; D009093:Mucus / Q000737:chemistry / Q000276:immunology*; D010084:Oxidation-Reduction; D051381:Rats; D017208:Rats, Wistar; D014357:Trypsin / Q000737:chemistry
D000596:Amino Acids; D000911:Antibodies, Monoclonal; D000951:Antigens, Neoplasm; D004220:Disulfides; D000939:Epitopes; D006023:Glycoproteins; D014357:Trypsin