Kondo S, Tokunaga F, Kario K, Matsuo T, Koide T
Department of Life Science, Faculty of Science, Himeji Institute of Technology, Harima Science Park City, Hyogo, Japan.
Blood. 1996 Feb 1;87(3):1006-12.
Heparin cofactor II (HCII) is a serine proteinase inhibitor in human plasma that rapidly inhibits thrombin in the presence of dermatan sulfate or heparin. To understand the molecular mechanism for HCII deficiency in a patient with reduced circulating HCII antigen, we studied a Japanese patient with type I HCII deficiency who suffered from angina pectoris and coronary artery disease. Polymerase chain reaction (PCR)-based sequence analysis showed that the propositus' gene for HCII (HCII Awaji gene) had a thymine insertion after codon (GAT) for Asp88 in exon II, resulting in a frameshift mutation. Consequently, the abnormal HCII Awaji protein was suggested to have an altered amino acid sequence from position 89 and terminate at 107, thus being composed of the NH2-terminal one fifth of normal HCII and dysfunctional for thrombin inhibition. The molecular weight and pI value of HCII Awaji were calculated to be 12,040 and 3.6, respectively, without posttranslational modification. Mutagenic PCR followed by the Tsp509I digestion showed that a half of the PCR products derived from the propositus and his sister was cleaved, suggesting that his sister also has the same mutant allele. Crossed-immunoelectrophoresis and Western blot analyses of plasma and urine from the the propositus and of plasma from his sister did not provide evidence for the existence of the abnormal HCII, suggesting that little truncated HCII was circulating in the patient's blood. However, stable expression assay using human kidney 293 cells transfected with the expression vector containing cDNA encoding wild-type or Awaji-type HCII showed that mutant as well as wild-type HCII was secreted into culture medium normally. These results suggest that the abnormal HCII Awaji protein is secreted normally, but rapidly degraded in the circulating blood.
肝素辅因子II（HCII）是人体血浆中的一种丝氨酸蛋白酶抑制剂，在硫酸皮肤素或肝素存在的情况下能快速抑制凝血酶。为了解一名循环中HCII抗原减少患者的HCII缺乏分子机制，我们研究了一名患有心绞痛和冠状动脉疾病的I型HCII缺乏日本患者。基于聚合酶链反应（PCR）的序列分析表明，先证者的HCII基因（HCII淡路岛基因）在外显子II中Asp88的密码子（GAT）后有一个胸腺嘧啶插入，导致移码突变。因此，异常的HCII淡路岛蛋白被认为从第89位开始氨基酸序列改变，并在第107位终止，从而由正常HCII的氨基末端五分之一组成，且对凝血酶抑制功能失调。未经翻译后修饰时，HCII淡路岛蛋白的分子量和pI值分别计算为12,040和3.6。诱变PCR后用Tsp509I酶切显示，先证者及其妹妹的一半PCR产物被切割，表明他的妹妹也有相同的突变等位基因。对先证者的血浆和尿液以及他妹妹的血浆进行交叉免疫电泳和蛋白质印迹分析，未提供异常HCII存在的证据，表明患者血液中循环的截短HCII很少。然而，使用转染了含有编码野生型或淡路岛型HCII cDNA的表达载体的人肾293细胞进行的稳定表达试验表明，突变型和野生型HCII均正常分泌到培养基中。这些结果表明，异常的HCII淡路岛蛋白正常分泌，但在循环血液中迅速降解。
