Characterization of mucin glycoprotein-specific translation products from swine and human trachea,pancreas and colon |
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Authors: | Sreedhara Sangadala Peggy Wallace Joseph Mendicino |
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Institution: | (1) Department of Biochemistry, University of Georgia, 30602 Athens, GA, USA |
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Abstract: | Summary 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 stydies 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.Abbreviations TFMS
Trifluoromethanesulfonic acid
- SDS
Sodium Dodecyl Sulfate
- PAGE
Polyacrylamide Gel Electrophoresis
- GalNAc
N-Acetylgalactosamine
- HTMG
Human Trachea Mucin Glycoprotein
- deHTMG
deglycosylated Human Trachea Mucin Glycoprotein
- STMG
Swine Trachea Mucin Glycoprotein
- deSTMG
deglycosylated Swine Trachea Mucin Glycoprotein
- CCMG
Cowper's Gland Mucin Glycoprotein
- deCGMG
deglycosylated Cowper's Gland Mucin Glycoprotein
- HPMG
Pancreatic Mucin Glycoprotein from BxPC-3 cells
- HCMG
Colon Mucin Glycoprotein from SW 403 cells
- HLMG
Human Lung Mucin Glycoprotein from A-549 cells
- STMG+deSTMG–
antibodies which bind to immobilized STMG but do not bind to immobilized deSTMG
- deSTMG+STMG–
antibodies which bind to immobilized deSTMG but do not bind to immobilized STMG
- STMG+deSTMG+
antibodies which bind to both STMG and deSTMG
- HTMG+deHTMG–
antibodies which bind to immobilized HTMG but do not bind to immobilized deHTMG
- deHTMG+HTMG–
antibodies which bind to immobilized deHTMG but do not bind to immobilized HTMG
- HTMG+deHTMG+
Antibodies which bind to both HTMG and deHTMG |
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Keywords: | mRNA respiratory mucin glycoproteins translation products deglycosylated mucin glycoproteins |
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