Characterization of mucin glycoprotein-specific translation products from swine and human trachea, pancreas and colon.
RNA was isolated from cultured swine trachea epithelial cells and mucus-secreting tumor cell lines from human pancreas, lung and colon by extraction with guanidine isothiocyanate. Poly(A)+mRNA rich fractions were purified by repeated chromatography on oligo (dT)-cellulose columns and they were translated in a cell-free rabbit reticulocyte system. Translation products labelled with 35S-methionine were isolated by immunoprecipitation with specific antibodies to the polypeptide chains of mucin glycoproteins and they were analyzed by SDS-PAGE and fluorography. A single principal polypeptide band of 67 kDa was found in all cases when the immunoprecipitates were washed with buffer containing bovine serum albumin and unlabeled deglycosylated mucin glycoprotein. The intensity of the 67 kDa band decreased when unlabeled deglycosylated mucin glycoprotein was added to the translation mixture before immunoprecipitation. Affinity purified monospecific antibodies elicited against chemically deglycosylated polypeptide chains of purified mucin glycoproteins from human and swine trachea and Cowper's gland were all equally effective in immunoprecipitating the 67 kDa translation product. Monospecific antibodies directed against the glycosylated and unglycosylated regions of the polypeptide chain yielded single bands with a molecular size of 67 kDa in each case. Peptide profiles obtained by digestion of the 67 kDa translation product with S. aureus V-8 protease were identical to those obtained with deglycosylated human and swine trachea mucin glycoproteins. These studies clearly demonstrate that the translation product of swine trachea and human lung, colon and pancreatic mucin glycoprotein gene is a single polypeptide chain of 67 kDa. The relative size and properties of the translation products synthesized with poly (A)+RNA isolated from mucus-secreting cells derived from three different tissues are similar to those of mucin glycoproteins purified directly from mucus secretions of human and swine trachea epithelium.
Sangadala
S
S
Department of Biochemistry, University of Georgia, Athens 30602.
Wallace
P
P
Mendicino
J
J
D000818:Animals; D000918:Antibody Specificity; D002478:Cells, Cultured; D003106:Colon / Q000737:chemistry*; D003470:Culture Media / Q000737:chemistry; D006031:Glycosylation; D006801:Humans; D006868:Hydrolysis; D009077:Mucins / Q000235:genetics / Q000302:isolation & purification*; D010179:Pancreas / Q000737:chemistry*; D010455:Peptides / Q000276:immunology / Q000302:isolation & purification; D011061:Poly A / Q000302:isolation & purification; D011233:Precipitin Tests; D014176:Protein Biosynthesis; D012313:RNA / Q000302:isolation & purification; D012697:Serine Endopeptidases; D013552:Swine; D014132:Trachea / Q000737:chemistry*
D003470:Culture Media; D009077:Mucins; D010455:Peptides; D011061:Poly A; D012313:RNA; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase