Site-Directed Mutagenesis of Glycosylation Sites in the Transforming Growth Factor-p1 (TGFPI) and TGFP2 (414) Precursors and of Cysteine Residues within Mature TGFPI: Effects on Secretion and Bioactivity

The transforming growth factor-p1 (TGFPl) and -p2 (414) precursors both contain three predicted sites of N-linked glycosylation within their pro regions. These are located at amino acid residues 72, 140, and 241 for the TGFP2 (414) precursor and at residues 82, 136, and 176 for the TGFBl precursor; both proteins contain mannose-6-phosphate (M-6-P) residues. The major sites of M-6-P addition are at Asn (82) and Asn (136), the first two sites of glycosylation, for the TGFBI precursor. We now show that the major site of M-6-P addition within the TGFP2 (414) precursor is at Asr?, the third glycosylation site. To determine the importance of N-linked glycosylation to the secretion of TGFBl and -82, site-directed mutagenesis was used to change the Asn residues to Ser residues; the resulting DNAs were transfected into COS cells, and their supernatants were assayed for TGFP activity. Substitution of Asn (241) of the TGFB2 (414) precursor resulted in an 82% decrease in secreted TGFPP bioactivity. Mutation at Asn7’ resulted in a 44% decrease, while mutation at Asn14’ was without effect. Elimination of all three glycosylation sites resulted in undetectable levels of TGFPP. These results were compared with similar mutations made in the cDNA encoding the TGFBI precursor. Mutagenesis of the two M-6-P-containing sites (Asns2 and Asn’36) resulted in an 83% decrease in secreted TGFPI; replacement of Asn@ and Asn’36 with Ser individually resulted in 85% and 42% decreases in activity, respectively. Substitution of Asn’76 with Ser was without effect, while substitution of all three sites of glycosylation resulted in undetectable levels