Site-directed mutagenesis of cysteine residues in the pro region of the transforming growth factor beta 1 precursor. Expression and characterization of mutant proteins.
Three cysteine residues are located in the pro region of the transforming growth factor beta 1 (TGF-beta 1) precursor at amino acid positions 33, 223, and 225. Previous studies (Gentry, L. E., Lioubin, M. N., Purchio, A. F., and Marquardt, H. (1988) Mol. Cell. Biol. 8, 4162-4168) with purified recombinant TGF-beta 1 (rTGF-beta 1) precursor produced by Chinese hamster ovary (CHO) cells revealed that Cys-33 can form a disulfide bond with at least 1 cysteine residue in mature TGF-beta 1, contributing to the formation of a 90-110-kDa protein. We now show that Cys-223 and Cys-225 form interchain disulfide bonds. Site-directed mutagenesis was used to change these Cys codons to Ser codons, and mutant constructs were transfected into COS cells. Analysis of recombinant proteins by immunoblotting showed that by substituting Cys-33 the 90-110-kDa protein is not formed, and thus, more mature dimer (24 kDa) is obtained, corresponding to a 3- to 5-fold increase in biological activity. Substitution of Cys-223 and/or Cys-225 resulted in near wild-type levels of mature TGF-beta 1. Furthermore, cells transfected with plasmid coding for Ser at positions 223 and 225 expressed only monomeric precursor proteins and released bioactive TGF-beta 1 that did not require acid activation, suggesting that dimerization of the precursor pro region may be necessary for latency.
Brunner
A M
AM
Oncogen, Seattle, Washington 98121.
Marquardt
H
H
Malacko
A R
AR
Lioubin
M N
MN
Purchio
A F
AF
D000818:Animals; D001426:Bacterial Proteins / Q000302:isolation & purification* / Q000378:metabolism; D001483:Base Sequence; D002460:Cell Line; D003062:Codon / Q000235:genetics; D003545:Cysteine; D004277:DNA, Single-Stranded / Q000302:isolation & purification* / Q000378:metabolism; D005796:Genes; D008969:Molecular Sequence Data; D009154:Mutation; D014176:Protein Biosynthesis; D011498:Protein Precursors; D011506:Proteins / Q000235:genetics* / Q000302:isolation & purification; D012329:RNA, Bacterial / Q000302:isolation & purification* / Q000378:metabolism; D014162:Transfection; D016212:Transforming Growth Factor beta; D015291:Transforming Growth Factors / Q000235:genetics*
D001426:Bacterial Proteins; D003062:Codon; D004277:DNA, Single-Stranded; D011498:Protein Precursors; D011506:Proteins; D012329:RNA, Bacterial; D016212:Transforming Growth Factor beta; C058681:msDNA; D015291:Transforming Growth Factors; D003545:Cysteine