Gaussem P, Gandrille S, Duchemin J, Emmerich J, Alhenc-Gelas M, Aillaud M F, Aiach M
Groupe de Recherche sur la Thrombose, INSERM CJF 91-01, UFR des Sciences Pharmaceutiques et Biologiques, Université Paris V, France.
Thromb Haemost. 1994 Jun;71(6):748-54.
The protein C Gla domain was studied in six families presenting a type II hereditary deficiency characterized by low activity in a coagulation assay and normal activity in an amidolytic assay. Five of these mutations, previously described by our group, affected Arg-5, Arg-1, Arg 229 and Ser 252. We report here the first natural Glu 7 to Asp mutation in a sixth family. We evaluated the binding of the mutated protein C to H11, a monoclonal antibody (mAb) known to recognize the sequence Phe4 to Arg9 of the Gla domain; the presence of calcium ions suppresses the recognition of this epitope by H11. Mutation of Arg229 to Gln and Ser252 to Asn did not modify the inhibition of protein C binding, whereas the Arg-1 to His mutation resulted in a loss of inhibition in the presence of CaCl2. This suggests that the protein C of this patient shows impaired carboxylation. The protein C from patients bearing the mutations Arg-5 to Trp, Arg-1 to Cys and Glu 7 to Asp bound poorly to H11 mAb, even in the absence of calcium ions. The calcium affinity of the Gla domain was studied by pseudo-affinity chromatography, in which protein C was successively eluted from a Mono Q column by CaCl2 10 mM and NaCl 0.6 M. Protein C from the patient bearing the Arg-5 to Asp mutation had a normal elution profile, suggesting that a modification of the propeptide cleavage site impairs the conformation of the Gla domain but not carboxylation.(ABSTRACT TRUNCATED AT 250 WORDS)
对六个家族中的蛋白C Gla结构域进行了研究，这些家族呈现II型遗传性缺陷，其特征为凝血检测中活性低，而酰胺水解检测中活性正常。我们团队先前已描述过其中五个突变，这些突变影响了精氨酸-5、精氨酸-1、精氨酸229和丝氨酸252。我们在此报告第六个家族中首次发现的天然谷氨酸7突变为天冬氨酸的情况。我们评估了突变型蛋白C与H11的结合，H11是一种已知可识别Gla结构域中苯丙氨酸4至精氨酸9序列的单克隆抗体（mAb）；钙离子的存在会抑制H11对该表位的识别。精氨酸229突变为谷氨酰胺以及丝氨酸252突变为天冬酰胺并未改变对蛋白C结合的抑制作用，而精氨酸-1突变为组氨酸则导致在氯化钙存在时抑制作用丧失。这表明该患者的蛋白C羧化受损。携带精氨酸-5突变为色氨酸、精氨酸-1突变为半胱氨酸以及谷氨酸7突变为天冬氨酸突变的患者的蛋白C与H11 mAb的结合很差，即使在没有钙离子的情况下也是如此。通过假亲和色谱法研究了Gla结构域的钙亲和力，其中蛋白C先后用10 mM氯化钙和0.6 M氯化钠从Mono Q柱上洗脱。携带精氨酸-5突变为天冬氨酸突变的患者的蛋白C具有正常的洗脱图谱，这表明前肽切割位点的修饰会损害Gla结构域的构象，但不会影响羧化。（摘要截短为250字）
