Macdonald M H, Morrison C J, McMaster W R
Department of Medical Genetics, University of British Columbia, Vancouver, Canada.
Biochim Biophys Acta. 1995 Dec 6;1253(2):199-207. doi: 10.1016/0167-4838(95)00155-5.
The major surface glycoprotein of Leishmania promastigotes, referred to as GP63, is a zinc metalloproteinase of 63,000 M(r) containing a glycosylphosphatidylinositol (GPI) membrane anchor. Recent studies demonstrated that recombinant GP63 (rGP63) expressed by the baculovirus insect cell system was secreted as a glycosylated latent proteinase that required activation for full proteinase activity (Button et al. (1993) Gene 134, 75-81). To extend these studies, the active site of L. major GP63 was characterized by site-directed mutagenesis and the activation mechanism of latent rGP63 was studied using both secreted and cell surface expression systems. To determine whether the proposed active site of L. major GP63 conforms to other well characterized zinc metalloproteinases, the proposed GP63 catalytic Glu-265, corresponding to catalytic Glu-147 of thermolysin, was changed to Asp-265. Using a transient expression system in COS-7 cells, expression of the Asp-265 mutant GP63 gene resulted in rGP63 with no detectable proteinase activity, whereas expression of the wild-type GP63 gene resulted in rGP63 with a level of proteinase activity similar to native GP63. Thus, the mechanism of GP63 proteinase activity is predicted to be homologous to that of other well characterized zinc metalloproteinases. NH2-Terminal sequence analysis revealed that activation with HgCl2 resulted in removal of the pro region, ultimately generating the mature NH2-terminus. This processing included the removal of a conserved Cys residue (Cys-48) and occurred by a cis mechanism, since the addition of previously activated rGP63 did not lead to an enhancement of latent rGP63 proteinase activation. The mechanism of activation of GP63 is consistent with the cysteine switch mechanism proposed for matrix metalloproteinases and thus has been conserved from protozoa to mammals.
利什曼原虫前鞭毛体的主要表面糖蛋白，称为GP63，是一种分子量为63,000的锌金属蛋白酶，含有糖基磷脂酰肌醇（GPI）膜锚定结构。最近的研究表明，杆状病毒昆虫细胞系统表达的重组GP63（rGP63）作为一种糖基化的潜伏蛋白酶分泌，其完全蛋白酶活性需要激活（Button等人，（1993年）《基因》134卷，75 - 81页）。为了扩展这些研究，通过定点诱变对大利什曼原虫GP63的活性位点进行了表征，并使用分泌型和细胞表面表达系统研究了潜伏rGP63的激活机制。为了确定大利什曼原虫GP63的假定活性位点是否与其他特征明确的锌金属蛋白酶一致，将对应于嗜热菌蛋白酶催化性Glu - 147的假定GP63催化性Glu - 265替换为Asp - 265。在COS - 7细胞中使用瞬时表达系统，Asp - 265突变型GP63基因的表达产生了无明显蛋白酶活性的rGP63，而野生型GP63基因的表达产生了蛋白酶活性水平与天然GP63相似的rGP63。因此，预计GP63蛋白酶活性的机制与其他特征明确的锌金属蛋白酶的机制同源。氨基末端序列分析表明，用HgCl₂激活导致前区去除，最终产生成熟的氨基末端。这种加工包括去除一个保守的半胱氨酸残基（Cys - 48），并且通过顺式机制发生，因为添加先前激活的rGP63不会导致潜伏rGP63蛋白酶激活的增强。GP63的激活机制与为基质金属蛋白酶提出的半胱氨酸开关机制一致，因此从原生动物到哺乳动物都得以保留。
