title: Formation of mitogenically active PDGF-B dimer does not require interchain disulfide bonds.
abstract: Platelet-derived growth factor (PDGF), a major mitogen for mesenchymal cells, is a disulfide-bonded dimer of two subunit polypeptides named A and B. All of the three possible dimeric forms, i.e. AA, BB, and AB, exist in nature. The dimeric structure has been presumed to be necessary for biological activity, since reduction of the dimer results in loss of activity and simultaneous conversion to monomeric form as determined by SDS-gel electrophoresis. However, reduction of the native molecule destroys intrachain, as well as interchain, disulfide bonds, and it is possible that the former rather than the latter are critical for proper conformation of the active protein. We show here that PDGF-B polypeptides in which all 8 cysteines or the 2nd, 4th, 5th, and 8th cysteines have been mutated to serines fail to form covalent dimers and possess dramatically less mitogenic activity than native PDGF-BB. Another mutant, PDGF-B(C2,4S), in which just the 2 cysteines involved in interchain disulfides were converted to serine, ran as a monomer on SDS-polyacrylamide gels as expected. Somewhat unexpectedly, however, the mitogenic activity of the PDGF-B(C2,4S) analog was similar to the activity of wild-type PDGF-BB disulfide-bonded dimer under physiological conditions. The activity of the analog was more sensitive to the effect of low pH than was the activity of wild-type PDGF-BB. Molecular weight analysis utilizing light scattering and sedimentation equilibrium demonstrated that the PDGF-B(C2,4S) analog exists as a noncovalent dimer at pH 4-7 but dissociates to a monomer at pH 2.5. Disulfide analysis of the mutant protein demonstrated that the intrachain disulfide bonds are the same as those formed in wild-type PDGF-BB homodimers. We conclude that proper formation of intrachain disulfide bonds is critical to maintaining the correct conformation of PDGF monomers, but that appropriately folded monomers can associate into active noncovalent dimers in the absence of interchain disulfide bonds. Interchain disulfide bonds thus appear to increase the stability of the PDGF dimer rather than being crucial to its existence.
authors.affiliation: Amgen Inc., Amgen Center, Thousand Oaks, California 91320.
authors.forename: W C
authors.initials: WC
authors.lastname: Kenney
authors.forename: M
authors.initials: M
authors.lastname: Haniu
authors.forename: A C
authors.initials: AC
authors.lastname: Herman
authors.forename: T
authors.initials: T
authors.lastname: Arakawa
authors.forename: V J
authors.initials: VJ
authors.lastname: Costigan
authors.forename: J
authors.initials: J
authors.lastname: Lary
authors.forename: D A
authors.initials: DA
authors.lastname: Yphantis
authors.forename: A R
authors.initials: AR
authors.lastname: Thomason
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000077214:Becaplermin; D002455:Cell Division / Q000187:drug effects*; D002460:Cell Line; D004220:Disulfides / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D046911:Macromolecular Substances; D008934:Mitogens / Q000494:pharmacology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D010982:Platelet-Derived Growth Factor / Q000096:biosynthesis / Q000737:chemistry* / Q000494:pharmacology*; D020574:Proto-Oncogene Proteins c-sis; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000494:pharmacology
chemical_list: D004220:Disulfides; D046911:Macromolecular Substances; D008934:Mitogens; D010446:Peptide Fragments; D010982:Platelet-Derived Growth Factor; D020574:Proto-Oncogene Proteins c-sis; D011994:Recombinant Proteins; D000077214:Becaplermin