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.
Highlights
Eight cysteine residues arefound in the primary structuorfe mature PDGF-A and PDGF-B monomers (Claesson-Welsh and Heldin, 1989).These residuesoccur in disulfide bonds,either as intrachain linkagesor as interchainbonds between two monomers comprising the native, dimeric forms
To generate large quantities of PDGF-BB for biological and out drastically reducing the biological activity (Giese et al, physicochemical studies, we have developed procedures for pro- 1987; Sauer and Donoghue, 1988), simultaneous mutation of ducing pure, active recombinant protein expressed in mammalian (Chinese hamster ovary) and bacterial (E. coli) host cells
In contrast to wild-type PDGF-BB dimer, the mutant These results show that theintrachain disulfide linkages in protein was highly labile to pepsin digestion
Summary
PDGF-B DimerDoes Not Require Interchain Disulfide Bonds four of the mutations eliminatedthe ability to transform fibro- of human PDGF-B This DNA was inserted into the bacterial expression blasts. The mutants were derivedby in vitro mutagenesis partial agonist activity in receptor binding and phosphoryla- of the DNA coding forthe 109-amino acid wild-type PDGF-B described tion assays Their studies did not determine, howewvheer,ther above. The peptides were eluted with a linear gradient a n d renders the protein unable to form significant amountosf dimer These results suggested two possibilities for dimer formation: 1) interchain disulfide bonds are essential for dimer formation; or 2) interchain disulfide bonadrse not essential for dimer formation,but intrachain disulfidebonds are required to from 5%B t o 40 or 60% B with a flow rate of 0.7 mumin.
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