Site-directed mutagenesis of glycosylation sites in the transforming growth factor-β1 (TGFβ1) and TGFβ2 (414) precursors and of cysteine residues within mature TGFβ1: effects on secretion and bioactivity

Abstract

The transforming growth factor-β1 (TGFβ1) and -β2 (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 TGFβ2 (414) precursor and at residues 82, 136, and 176 for the TGFβ1 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 TGFβ1 precursor. We now show that the major site of M-6-P addition within the TGFβ2 (414) precursor is at Asn241, the third glycosylation site. To determine the importance of N-linked glycosylation to the secretion of TGFβ1 and -β2, 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 TGFβ activity. Substitution of Asn (241) of the TGFβ2 (414) precursor resulted in an 82% decrease in secreted TGFβ2 bioactivity. Mutation at Asn72 resulted in a 44% decrease, while mutation at Asn140 was without effect. Elimination of all three glycosylation sites resulted in undetectable levels of TGFβ2. -These results were compared with similar mutations made in the cDNA encoding the TGFβ1 precursor. Mutagenesis of the two M-6-P-containing sites (Asn82 and Asn136) resulted in an 83% decrease in secreted TGFβ1; replacement of Asn82 and Asn136 with Ser individually resulted in 85% and 42% decreases in activity, respectively. Substitution of Asn176 with Ser was without effect, while substitution of all three sites of glycosylation resulted in undetectable levels of TGFβ1 activity, similar to the results obtained with TGFβ2. The nine Cys residues within the mature region of TGFβ1 were mutated to serine, and their effects on TGFβ1 secretion were evaluated. Mutation of most Cys residues resulted in undetectable levels of TGFβ1 protein or activity in conditioned medium. Mutation of Cys (355) led to the secretion of inactive TGFβ1 monomers, suggesting that this residue is either directly involved in dimer formation or required for correct interchain disulfide bond forma-tion.