| Abstract: |
The precursor for transforming growth factor-α (TGF-α) is a membrane glycoprotein that can establish contact with epidermal growth factor/TGF-α receptors on adjacent cells or can be cleaved to release TGF-α that diffuses into the medium. Cleavage of pro-TGF-α occurs at Ala/Leu-Ala/Leu-Ala-Val-Val sites located at each end of the mature TGF-α sequence. To characterize the cleavage process of pro-TGF-α and the role of glycosylation in this process, we have introduced a pro-TGF-α expression vector in wild type Chinese hamster ovary (CHO) cells and in the mutant CHO cell clone ldlD that has a reversible defect in protein glycosylation. Analysis of metabolically labeled and cell surface-labeled products immunoprecipitated with antibodies directed against the extracellular TGF-α sequence and the cytoplasmic pro-TGF-α C-terminal domain shows that cleavage of pro-TGF-α in wild type CHO cells occurs in two steps. Both processing steps occur after pro-TGF-α reaches the cell surface. In the first step, pro-TGF-α rapidly (t( 1/2 ) = 30 min) loses the amino-terminal segment that precedes the TGF-α sequence. In the second step, pro-TGF-α is cleaved at the carboxyl terminus of the TGF-α sequence releasing this factor into the medium. This second step is slow (t( 1/2 ) = 2 h). The action of pancreatic elastase added to CHO-TGF-α cells mimics the first step but not the second one. Synthesis, cell surface exposure, rate of cleavage, and generation of bioactive TGF-α in ldlD-TGF-α cells are not markedly affected by the lack of N-acetylgalactosamine-dependent protein O-glycosylation or galactose-dependent glycan chain modification. The results indicate that, despite their similarity in amino acid sequence, the two cleavage sites that flank TGF-α may be processed with different kinetics which can lead to retention of pro-TGF-α on the cell surface. |
