title: Site-directed mutagenesis of glycosylation sites in the transforming growth factor-beta 1 (TGF beta 1) and TGF beta 2 (414) precursors and of cysteine residues within mature TGF beta 1: effects on secretion and bioactivity.
abstract: The transforming growth factor-beta 1 (TGF beta 1) and -beta 2 (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 TGF beta 2 (414) precursor and at residues 82, 136, and 176 for the TGF beta 1 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 TGF beta 1 precursor. We now show that the major site of M-6-P addition within the TGF beta 2 (414) precursor is at Asn241, the third glycosylation site. To determine the importance of N-linked glycosylation to the secretion of TGF beta 1 and -beta 2, 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 TGF beta activity. Substitution of Asn (241) of the TGF beta 2 (414) precursor resulted in an 82% decrease in secreted TGF beta 2 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 TGF beta 2. These results were compared with similar mutations made in the cDNA encoding the TGF beta 1 precursor. Mutagenesis of the two M-6-P-containing sites (Asn82 and Asn136) resulted in an 83% decrease in secreted TGF beta 1; 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 TGF beta 1 activity, similar to the results obtained with TGF beta 2. The nine Cys residues within the mature region of TGF beta 1 were mutated to serine, and their effects on TGF beta 1 secretion were evaluated. Mutation of most Cys residues resulted in undetectable levels of TGF beta 1 protein or activity in conditioned medium. Mutation of Cys (355) led to the secretion of inactive TGF beta 1 monomers, suggesting that this residue is either directly involved in dimer formation or required for correct interchain disulfide bond formation.
authors.affiliation: Bristol-Myers Squibb Pharmaceutical Research Institute, Seattle, Washington 98121.
authors.forename: A M
authors.initials: AM
authors.lastname: Brunner
authors.forename: M N
authors.initials: MN
authors.lastname: Lioubin
authors.forename: H
authors.initials: H
authors.lastname: Marquardt
authors.forename: A R
authors.initials: AR
authors.lastname: Malacko
authors.forename: W C
authors.initials: WC
authors.lastname: Wang
authors.forename: R A
authors.initials: RA
authors.lastname: Shapiro
authors.forename: M
authors.initials: M
authors.lastname: Neubauer
authors.forename: J
authors.initials: J
authors.lastname: Cook
authors.forename: L
authors.initials: L
authors.lastname: Madisen
authors.forename: A F
authors.initials: AF
authors.lastname: Purchio
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D006224:Cricetinae; D003545:Cysteine; D006031:Glycosylation; D008360:Mannosephosphates / Q000032:analysis; D016297:Mutagenesis, Site-Directed; D011498:Protein Precursors / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000494:pharmacology; D015183:Restriction Mapping; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000096:biosynthesis / Q000235:genetics* / Q000494:pharmacology
chemical_list: D008360:Mannosephosphates; D011498:Protein Precursors; D011994:Recombinant Proteins; D016212:Transforming Growth Factor beta; C027693:mannose-6-phosphate; D003545:Cysteine