Burleigh B A, Andrews N W
Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06510.
J Biol Chem. 1995 Mar 10;270(10):5172-80. doi: 10.1074/jbc.270.10.5172.
Trypomastigotes, the infective stages of the intracellular parasite Trypanosoma cruzi, induce rapid and repetitive cytosolic free Ca2+ transients in fibroblasts. Buffering or depletion of intracellular free Ca2+ inhibits cell entry by trypomastigotes, indicating a role for this signaling event in invasion. We show here that the majority of the Ca(2+)-signaling activity is associated with the soluble fraction of parasites disrupted by sonication. Distinct cell types from different species are responsive to this soluble factor, and intracellular free Ca2+ transients occur rapidly and reach concentrations comparable to responses induced by thrombin and bombesin. The Ca(2+)-signaling activity does not bind concanavalin A and is strongly inhibited by a specific subset of protease inhibitors. The only detectable protease in the fractions with Ca(2+)-signaling activity is an unusual alkaline peptidase of 120 kDa, to which no function had been previously assigned. The activity of the protease and cell invasion by trypomastigotes are blocked by the same specific inhibitors that impair Ca(2+)-signaling, suggesting that the enzyme is required for generating the response leading to infection. We demonstrate that the 120-kDa peptidase is not sufficient for triggering Ca(2+)-signaling, possibly being involved in the processing of precursors present only in infective trypomastigotes. These findings indicate a biological function for a previously identified unusual protozoan protease and provide the first example of a proteolytically generated parasite factor with characteristics of a mammalian hormone.
无鞭毛体是细胞内寄生虫克氏锥虫的感染阶段，可在成纤维细胞中诱导快速且重复的胞质游离钙离子瞬变。缓冲或耗尽细胞内游离钙离子会抑制无鞭毛体进入细胞，表明这一信号事件在入侵过程中发挥作用。我们在此表明，大部分钙离子信号活性与经超声处理破坏的寄生虫可溶性部分相关。来自不同物种的不同细胞类型对这种可溶性因子有反应，细胞内游离钙离子瞬变迅速发生，其浓度与凝血酶和蛙皮素诱导的反应相当。钙离子信号活性不与伴刀豆球蛋白A结合，且受到特定子集的蛋白酶抑制剂的强烈抑制。具有钙离子信号活性的组分中唯一可检测到的蛋白酶是一种分子量为120 kDa的异常碱性肽酶，此前未赋予其任何功能。蛋白酶的活性和无鞭毛体对细胞的入侵被损害钙离子信号的相同特异性抑制剂所阻断，这表明该酶是产生导致感染的反应所必需的。我们证明120 kDa的肽酶不足以触发钙离子信号，可能参与仅存在于感染性无鞭毛体中的前体的加工。这些发现表明一种先前鉴定的异常原生动物蛋白酶具有生物学功能，并提供了第一个具有哺乳动物激素特征的蛋白水解产生的寄生虫因子的例子。
