Devlopment of MACIF as a new medicine for regulation of membrane attack complex of complement

当这项研究在两年前开始时，我们关于Macif的证据有限。我们最初知道的Macif的性质是，Macif是一种糖基磷脂酰肌醇(GPL)锚定蛋白，能特异性地抑制同源互补的膜攻击复合体的形成。(1)利用现有的技术几乎不可能制备大量的膜形式的MACIF。(2)尝试用三种二聚体调味汁制备可溶形态的Macif，而不是膜形态的Macif。第一种来自人尿，含有约0.5 mg/L的可溶性形式。第二个和第三个分别来自大肠杆菌和CHO细胞，其中通过基因工程生产重组的可溶性形式的Macif。(3)Macif由77个氨基酸组成，一个N-糖苷寡糖单元和一个GPL-寡糖单元。尿液中含有两个单位，而来自大肠杆菌的重组形式没有单位，而来自CHO细胞的重组形式只有一个N-糖苷低聚糖单位。(4)用豚鼠红细胞和人补体的溶血抑制法测定，三种可溶性形式的活性仅为膜形式的0.1%。这一结果表明，MACIF的膜结合对活性是重要的，而碳水化合物部分对活性不是必需的。(5)尿Macif对恒河猴肾功能低下再灌流所致的细胞毒性有明显的保护作用，但其作用机制尚不清楚。

We had only a limited line of evidence on MACIF when this research had started two years ago. The properties of MACIF we knew at the beginning were that MACIF was a glycosylphosphatidylinositol(GPl)-anchored protein which specifically inhibited the formation of membrane attack complex of homologous complement.New findings obtained from this reseach project are as follows. (1) it was almost impossible to prepare a large amount of the membrane form of MACIFby using presently available techniques. (2) we tried to prepare the soluble forms of MACIF, instead of the membrane form by three dimerent sauces. The first one was from human urine which contained about 0.5 mg/L of the soluble form. The second and third ones were from Eschericia coli and CHO cells, respectively, in which recombinant soluble forms of MACIF were produced by gene engineering. (3) MACIF consists of 77 amino acids, a N-glycosidic oligosaccharide unit and a GPl-oligosaccharide unit. The urine form had both units, and the recombinant form from E. coli had no units while that from CHO cells had only a N-glycosidic oligosaccharide unit. (4) activities of the three soluble forms were only 0.1% as much as that of the membrane form wheer those activities were assayed by the inhibition of hemolysis using guinea pig erythrocytes and human complement. This result indicated that the membrane binding of MACIF was important for the activity, and the carbohydrate moieties were not essential for the activity. (5) the urine MACIF significantly protected the cytotoxicity caused by recirculation after kidney hypaemia of marmosets, although the reaction mechanism was not clear.