Transforming growth factor beta 1: importance of glycosylation and acidic proteases for processing and secretion.
The role of glycosylation of the transforming growth factor-beta 1 (TGF-beta 1) precursor was investigated by treating a transfected Chinese hamster ovary (CHO) cell line expressing high levels of recombinant TGF-beta 1 (TGF-beta 3-2000 cells) with a series of glycosylational inhibitors. Tunicamycin, a nucleoside antibiotic which prevents the formation of the dolichol intermediate necessary for oligosaccharide addition of the nascent polypeptide chain, appeared to block secretory exit and led to an increase in the cellular associated, nonglycosylated pro-TGF-beta 1 form. 1-Deoxymannojirimycin and swainsonine, inhibitors of the mannosidases I and II, respectively, blocked complete glycoprotein processing of the TGF-beta 1 precursor as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by sensitivity to glycosidases. However, the abnormal TGF-beta 1 polypeptides containing the altered carbohydrate side chains were secreted readily by the CHO cells. In contrast, inhibitors of the glucosidases at the first step in glycoprotein remodeling, 1-deoxynojirimycin and castanospermine, markedly inhibited secretion of the TGF-beta 1 polypeptides from transfected CHO cells. In all cases, these inhibitors did not appear to affect proteolytic processing of the TGF-beta 1 polypeptides. Furthermore, inhibitor treatment did not affect mannose-6-phosphorylation of the TGF-beta 1 polypeptides. These results suggest that glycosylation and early stage remodeling of oligosaccharide side chains are necessary for secretion of TGF-beta 1. Treatment of the transfected CHO cells with weak bases (NH4Cl and chloroquine), or a monovalent ionophore (monensin), prevented proteolytic processing of the TGF-beta 1 precursor indicating that cleavage occurs by proteases in an acidic cellular compartment.
Sha
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Department of Biochemistry, Medical College of Ohio, Toledo 43699.
Brunner
A M
AM
Purchio
A F
AF
Gentry
L E
LE
D000818:Animals; D016282:Aspartic Acid Endopeptidases; D015153:Blotting, Western; D050260:Carbohydrate Metabolism; D002460:Cell Line; D010450:Endopeptidases / Q000378:metabolism*; D006031:Glycosylation; D006863:Hydrogen-Ion Concentration; D008247:Lysosomes / Q000201:enzymology; D010766:Phosphorylation; D011498:Protein Precursors; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D011506:Proteins / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D013997:Time Factors; D016212:Transforming Growth Factor beta; D015291:Transforming Growth Factors / Q000378:metabolism*; D014415:Tunicamycin / Q000494:pharmacology
D011498:Protein Precursors; D011506:Proteins; D011994:Recombinant Proteins; D016212:Transforming Growth Factor beta; D014415:Tunicamycin; D015291:Transforming Growth Factors; D010450:Endopeptidases; D016282:Aspartic Acid Endopeptidases