Kim S W, Quinn-Allen M A, Camp J T, Macedo-Ribeiro S, Fuentes-Prior P, Bode W, Kane W H
Division of Hematology, Departments of Medicine and Pathology, Duke University Medical Center, Durham, North Carolina 27710, USA.
Biochemistry. 2000 Feb 29;39(8):1951-8. doi: 10.1021/bi992256r.
We have previously determined that the C2-domain of human factor V (residues 2037-2196) is required for expression of cofactor activity and binding to phosphatidylserine (PS)-containing membranes. Naturally occurring factor V inhibitors and a monoclonal antibody (HV-1) recognized epitopes in the amino terminus of the C2-domain (residues 2037-2087) and blocked PS binding. We have now investigated the function of individual amino acids within the C2-domain using charge to alanine mutagenesis. Charged residues located within the C2-domain were changed to alanine in clusters of 1-3 mutations per construct. In addition, mutants W2063A, W2064A, (W2063, W2064)A, and L2116A were constructed as well. The resultant 30 mutants were expressed in COS cells using a B-domain deleted factor V construct (rHFV des B). All mutants were expressed efficiently based on the polyclonal antibody ELISA. The charged residues, Arg(2074), Asp(2098), Arg(2171), Arg(2174), and Glu(2189) are required for maintaining the structural integrity of the C2-domain of factor V. Four of these residues (Arg(2074), Asp(2098), Arg(2171), and Arg(2174)) correspond to positions in the factor VIII C-type domains that have been identified as point mutations in patients with hemophilia A. The epitope for the inhibitory monoclonal antibody HV-1 has been localized to Lys(2060) through Glu(2069) in the factor V C2-domain. The epitope for the inhibitory monoclonal antibody 6A5 is composed of amino acids His(2128) through Lys(2137). The PS-binding site in the factor V C2-domain includes amino acid residues Trp(2063) and Trp(2064). This site overlaps with the epitope for monoclonal antibody HV-1. These factor V C2-domain mutants should provide valuable tools for further defining the molecular interactions responsible for factor V binding to phospholipid membranes.
我们之前已经确定，人凝血因子V的C2结构域（第2037 - 2196位氨基酸残基）对于辅因子活性的表达以及与含磷脂酰丝氨酸（PS）的膜的结合是必需的。天然存在的凝血因子V抑制剂和一种单克隆抗体（HV - 1）识别C2结构域氨基末端（第2037 - 2087位氨基酸残基）的表位，并阻断PS结合。我们现在使用电荷到丙氨酸诱变研究了C2结构域内单个氨基酸的功能。C2结构域内的带电荷残基以每个构建体1 - 3个突变的簇的形式突变为丙氨酸。此外，还构建了突变体W2063A、W2064A、(W2063, W2064)A和L2116A。使用缺失B结构域的凝血因子V构建体（rHFV des B）在COS细胞中表达所得的30个突变体。基于多克隆抗体ELISA，所有突变体均高效表达。带电荷残基Arg(2074)、Asp(2098)、Arg(2171)、Arg(2174)和Glu(2189)对于维持凝血因子V的C2结构域的结构完整性是必需的。其中四个残基（Arg(2074)、Asp(2098)、Arg(2171)和Arg(2174)）对应于凝血因子VIII C型结构域中的位置，这些位置已被确定为A型血友病患者中的点突变位点。抑制性单克隆抗体HV - 1的表位已定位到凝血因子V C2结构域中从Lys(2060)到Glu(2069)的区域。抑制性单克隆抗体6A5的表位由从His(2128)到Lys(2137)的氨基酸组成。凝血因子V C2结构域中的PS结合位点包括氨基酸残基Trp(2063)和Trp(2064)。该位点与单克隆抗体HV - 1的表位重叠。这些凝血因子V C2结构域突变体应为进一步确定负责凝血因子V与磷脂膜结合的分子相互作用提供有价值的工具。
