Sanchez-Lopez R, Nicholson R, Gesnel M C, Matrisian L M, Breathnach R
Laboratoire de Génétique Moléculaire des Eucaryotes, Institut National de la Santé et de la Recherche Médicale, Faculté de Médecine, Strasbourg, France.
J Biol Chem. 1988 Aug 25;263(24):11892-9.
We have developed a system for studying the proteinase activity of a collagenase family member, transin. Cos cells transfected with a vector designed to direct synthesis of a secretable fusion protein between staphylococcal protein A and transin secrete a latent proteinase, activable by 4-aminophenylmercuric acetate, which binds to IgG-Sepharose. Treatment with 4-aminophenylmercuric acetate leads to cleavage of the fusion protein and elution of the active proteinase transin. Based on results obtained with this system we propose that transin comprises an N-terminal proteinase domain and an independent C-terminal hemopexin-like domain. The latter domain is not required for binding of inhibitors or for maintenance of transin in its inactive form. The sequence PRCGVPDV is present in the proenzyme forms of collagenase family proteinases just upstream from the N termini of the active enzymes. We show that mutations within this sequence lead to transin variants with a much increased tendency to undergo spontaneous activation. Finally, we show that mutations within a region of transin having sequence similarity to the zinc-binding site of bacterial metalloproteinases inactivate the proteinase activity of transin, lending support to the notion that this region represents part of transin's active site.
我们开发了一种用于研究胶原酶家族成员——转化生长因子β诱导蛋白（transin）蛋白酶活性的系统。用一种载体转染的Cos细胞，该载体设计用于指导合成葡萄球菌蛋白A与transin之间的可分泌融合蛋白，可分泌一种潜在的蛋白酶，该蛋白酶可被乙酸4-氨基苯汞激活，乙酸4-氨基苯汞可与IgG-琼脂糖结合。用乙酸4-氨基苯汞处理会导致融合蛋白的裂解以及活性蛋白酶transin的洗脱。基于该系统获得的结果，我们提出transin包含一个N端蛋白酶结构域和一个独立的C端血红素结合蛋白样结构域。后一个结构域对于抑制剂的结合或维持transin的无活性形式并非必需。序列PRCGVPDV存在于胶原酶家族蛋白酶的酶原形式中，就在活性酶N端的上游。我们表明，该序列内的突变会导致transin变体自发激活的倾向大大增加。最后，我们表明，transin中与细菌金属蛋白酶锌结合位点具有序列相似性的区域内的突变会使transin的蛋白酶活性失活，这支持了该区域代表transin活性位点一部分的观点。
