Kenney W C, Haniu M, Herman A C, Arakawa T, Costigan V J, Lary J, Yphantis D A, Thomason A R
Amgen Inc., Amgen Center, Thousand Oaks, California 91320.
J Biol Chem. 1994 Apr 22;269(16):12351-9.
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.
血小板衍生生长因子（PDGF）是间充质细胞的主要促分裂原，是由两个名为A和B的亚基多肽通过二硫键连接而成的二聚体。三种可能的二聚体形式，即AA、BB和AB，在自然界中均存在。二聚体结构被认为是生物活性所必需的，因为通过SDS凝胶电泳测定，二聚体的还原会导致活性丧失并同时转化为单体形式。然而，天然分子的还原会破坏链内以及链间二硫键，并且有可能前者而非后者对于活性蛋白的正确构象至关重要。我们在此表明，所有8个半胱氨酸或第2、4、5和8个半胱氨酸已突变为丝氨酸的PDGF - B多肽无法形成共价二聚体，并且其促有丝分裂活性比天然PDGF - BB显著降低。另一个突变体PDGF - B(C2,4S)，其中仅参与链间二硫键的2个半胱氨酸被转化为丝氨酸，如预期的那样在SDS - 聚丙烯酰胺凝胶上以单体形式迁移。然而，有点出乎意料的是，在生理条件下，PDGF - B(C2,4S)类似物的促有丝分裂活性与野生型PDGF - BB二硫键连接的二聚体的活性相似。该类似物的活性比野生型PDGF - BB的活性对低pH的影响更敏感。利用光散射和沉降平衡进行的分子量分析表明，PDGF - B(C2,4S)类似物在pH 4 - 7时以非共价二聚体形式存在，但在pH 2.5时解离为单体。对突变蛋白的二硫键分析表明，链内二硫键与野生型PDGF - BB同型二聚体中形成的二硫键相同。我们得出结论，链内二硫键的正确形成对于维持PDGF单体的正确构象至关重要，但在没有链间二硫键的情况下，适当折叠的单体可以缔合形成活性非共价二聚体。因此，链间二硫键似乎增加了PDGF二聚体的稳定性，而不是其存在的关键。
