Fernández J A, Griffin J H, Chang G T, Stam J, Reitsma P H, Bertina R M, Bouma B N
Department of Molecular and Experimental Medicine, Scripps Research Institute, La Jolla, California 92037, USA.
Blood Cells Mol Dis. 1998 Jun;24(2):101-12; discussion 113. doi: 10.1006/bcmd.1998.0175.
Human protein S binds to C4b-binding protein (C4BP) both in plasma and in a system using purified proteins. Amino acid residues 420-434 of the first disulfide loop of the sex hormone binding globulinlike domain of protein S are involved in the interaction of protein S with C4BP. To define the involvement of specific polar amino acids within residues 420-434, we studied in parallel synthetic protein S peptides and recombinant protein S variants containing the same amino acid replacements, K423E, E424K, Q427E and K429E. Synthetic peptide analogs of peptide PSP-420 (residues 420-434) were assayed for binding C4BP and as inhibitors of complex formation. The PSP-420 peptide and the analogous peptide with the substitution E424K, but not the peptides containing the substitutions K423E and K429E, were able to bind C4BP. Recombinant proteins with mutations of K423E, Q427E and K429E showed reduced affinity for C4BP compared to plasma protein S, recombinant wild type protein S, or E424K-protein S. These results suggest that Lys-423, Gln-427 and Lys-429 of protein S are important for normal binding to C4BP. The anti-protein S monoclonal antibody LJ-56, raised against peptide PSP-420, recognizes only free protein S and inhibits complex formation with C4BP. Antibody LJ-56 recognized the E424K and Q427E peptides but not the K423E or K429E peptides. Similarly, the E424K and Q427E protein S mutants were recognized by LJ-56, whereas the K423E and K429E protein S mutants were not recognized. This suggests that both in the peptide PSP-420 and in protein S, Lys-423 and Lys-429 significantly contribute to binding to antibody LJ-56. These results demonstrate that protein S residues 423, 427 and 429, but not residue 424, are involved in binding to both the antibody LJ-56 and to C4BP. When peptides PSP 420 and SL-6 (residues 447-460) with carboxyterminal amide or carboxylate moieties were compared to their ability to inhibit C4BP-protein S complexation, PSP-420-amide was the most potent. This finding together with the other results described here supports the hypothesis that the residues 420 and 434 in protein S provides a major binding site for C4BP.
人蛋白S在血浆以及使用纯化蛋白的系统中均能与C4b结合蛋白（C4BP）结合。蛋白S的性激素结合球蛋白样结构域第一个二硫键环的420 - 434位氨基酸残基参与了蛋白S与C4BP的相互作用。为了确定420 - 434位残基中特定极性氨基酸的作用，我们同时研究了合成蛋白S肽段以及含有相同氨基酸替换（K423E、E424K、Q427E和K429E）的重组蛋白S变体。检测了肽段PSP - 420（420 - 434位残基）的合成肽类似物与C4BP的结合情况以及作为复合物形成抑制剂的作用。PSP - 420肽段以及具有E4K替换的类似肽段能够结合C4BP，而含有K423E和K429E替换的肽段则不能。与血浆蛋白S、重组野生型蛋白S或E424K - 蛋白S相比，具有K423E、Q427E和K429E突变的重组蛋白对C4BP的亲和力降低。这些结果表明，蛋白S的赖氨酸 - 423、谷氨酰胺 - 427和赖氨酸 - 429对于与C4BP的正常结合很重要。针对肽段PSP - 420产生的抗蛋白S单克隆抗体LJ - 56仅识别游离的蛋白S，并抑制与C4BP的复合物形成。抗体LJ - 56识别E424K和Q427E肽段，但不识别K423E或K429E肽段。同样，LJ - 56识别E424K和Q427E蛋白S突变体，而不识别K423E和K429E蛋白S突变体。这表明在肽段PSP - 420和蛋白S中，赖氨酸 - 423和赖氨酸 - 429对与抗体LJ - 56的结合有显著贡献。这些结果表明，蛋白S的423、427和429位残基，而非424位残基，参与了与抗体LJ - 56和C4BP的结合。当比较具有羧基末端酰胺或羧酸盐部分的肽段PSP 420和SL - 6（447 - 460位残基）抑制C4BP - 蛋白S复合物形成的能力时，PSP - 420 - 酰胺是最有效的。这一发现以及此处描述的其他结果支持了蛋白S中420和434位残基为C4BP提供主要结合位点的假说。
