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Transforming growth factor beta 1: importance of glycosylation and acidic proteases for processing and secretion
X Sha1, A M Brunner, A F Purchio
1Department of Biochemistry, Medical College of Ohio, Toledo 43699.
Abstract:
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.
Insights
Glycosylation is essential for the secretion of transforming growth factor-beta 1 (TGF-beta 1). Inhibitors blocking early glycoprotein remodeling prevent TGF-beta 1 secretion, highlighting its importance in cellular export.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Transforming growth factor-beta 1 (TGF-beta 1) is a crucial signaling protein.
- The role of glycosylation in TGF-beta 1 processing and secretion is not fully understood.
- Chinese hamster ovary (CHO) cells are a common system for recombinant protein production.
Purpose of the Study:
- To investigate the role of glycosylation in the secretion of TGF-beta 1.
- To determine how glycosylation inhibitors affect TGF-beta 1 precursor processing and release.
Main Methods:
- Treatment of transfected CHO cells expressing TGF-beta 1 with various glycosylation inhibitors (tunicamycin, 1-deoxymannojirimycin, swainsonine, 1-deoxynojirimycin, castanospermine).
- Analysis of TGF-beta 1 precursor forms using SDS-PAGE and glycosidase sensitivity.
- Assessment of protein secretion and proteolytic processing under inhibitor treatment.
Main Results:
- Tunicamycin blocked secretion and increased cellular, nonglycosylated TGF-beta 1.
- Mannosidase inhibitors (1-deoxymannojirimycin, swainsonine) allowed secretion of incompletely processed TGF-beta 1.
- Glucosidase inhibitors (1-deoxynojirimycin, castanospermine) significantly inhibited TGF-beta 1 secretion.
- Proteolytic processing and mannose-6-phosphorylation were unaffected by inhibitors.
Conclusions:
- Glycosylation and early oligosaccharide remodeling are critical for TGF-beta 1 secretion.
- Inhibitors of glucosidases specifically impair TGF-beta 1 secretion.
- Proteolytic cleavage of TGF-beta 1 occurs in an acidic compartment.