Lonsdale-Eccles J D, Mpimbaza G W, Nkhungulu Z R, Olobo J, Smith L, Tosomba O M, Grab D J
Department of Biochemistry, University of Natal, Pietermaritzburg, South Africa.
Biochem J. 1995 Jan 15;305 ( Pt 2)(Pt 2):549-56. doi: 10.1042/bj3050549.
African trypanosomes contain cysteine proteases (trypanopains) the activity of which can be measured by in vitro digestion of fibrinogen, after electrophoresis in fibrinogen-containing SDS/polyacrylamide gels. When assessed by this procedure, trypanopain from Trypanosoma brucei (trypanopain-Tb) is estimated to have a molecular mass of 28 kDa. However, two additional bands of trypanopain activity (87 kDa and 105 kDa) are observed if serum is added to the trypanopain before electrophoresis. Formation of the 87 and 105 kDa bands is frequently accompanied by a reduction in the intensity of the 28 kDa activity which suggests that the extra bands are complexes of the 28 kDa trypanopain-Tb and a molecule from rat serum called rat trypanopain moledulator (rTM). The rTM-induced activation of cysteine proteases is not restricted to T. brucei as it is also observed with proteases from other protozoan parasites such as bloodstream forms of Trypanosoma congolense and the mammalian-infective in vitro-derived promastigote forms of Leishmania donovani and Leishmania major. The physical properties of rTM resemble those of the kininogen family of cysteine protease inhibitors. rTM is an acidic (pI 4.7) heat-stable 68 kDa glycoprotein with 15 kDa protease-susceptible domains. This resemblance between rTM and kininogens was confirmed by the positive, albeit weak, immunoreactivity between anti-(human low-molecular-mass kininogen) antibody and rTM as well as anti-rTM antibody and human low-molecular-mass kininogen. Furthermore, commercial preparations of human-low-molecular-mass kininogen and chicken egg white cystatin mimicked rTM by forming extra bands of proteolytic activity in the presence of trypanopain-Tb. In some instances, low-molecular-mass kininogen was also observed to increase the rate of hydrolysis of 7-(benzyloxycarbonyl-phenylalanyl-arginyl-amido)-4- methylcoumarin by live T. brucei. Although this effect was rather erratic, in no instance was significant inhibition observed when this putative cysteine protease inhibitor was used under these conditions. The activation of parasite cysteine proteases by commonly accepted cysteine protease inhibitors is unexpected and may have important pathological repercussions.
非洲锥虫含有半胱氨酸蛋白酶（锥虫蛋白酶），其活性可通过在含纤维蛋白原的SDS/聚丙烯酰胺凝胶中电泳后，对纤维蛋白原进行体外消化来测定。用此方法评估时，布氏锥虫的锥虫蛋白酶（锥虫蛋白酶-Tb）估计分子量为28 kDa。然而，如果在电泳前向锥虫蛋白酶中加入血清，则会观察到另外两条锥虫蛋白酶活性带（87 kDa和105 kDa）。87 kDa和105 kDa条带的形成常常伴随着28 kDa活性强度的降低，这表明额外的条带是28 kDa锥虫蛋白酶-Tb与大鼠血清中一种名为大鼠锥虫蛋白酶调节剂（rTM）的分子形成的复合物。rTM诱导的半胱氨酸蛋白酶激活并不局限于布氏锥虫，在其他原生动物寄生虫的蛋白酶中也观察到这种现象，如刚果锥虫的血流型以及杜氏利什曼原虫和硕大利什曼原虫的哺乳动物感染性体外衍生前鞭毛体形式。rTM的物理性质类似于半胱氨酸蛋白酶抑制剂的激肽原家族。rTM是一种酸性（pI 4.7）、热稳定的68 kDa糖蛋白，具有15 kDa的蛋白酶敏感结构域。抗（人低分子量激肽原）抗体与rTM以及抗rTM抗体与人类低分子量激肽原之间存在阳性（尽管较弱）免疫反应，这证实了rTM与激肽原之间的这种相似性。此外，人低分子量激肽原和鸡卵清半胱氨酸蛋白酶抑制剂的商业制剂在锥虫蛋白酶-Tb存在的情况下通过形成额外的蛋白水解活性带来模拟rTM。在某些情况下，还观察到低分子量激肽原会增加活布氏锥虫对7-（苄氧羰基-苯丙氨酰-精氨酰胺基）-4-甲基香豆素的水解速率。尽管这种效应相当不稳定，但在这些条件下使用这种假定的半胱氨酸蛋白酶抑制剂时，在任何情况下都未观察到明显的抑制作用。公认的半胱氨酸蛋白酶抑制剂对寄生虫半胱氨酸蛋白酶的激活是出乎意料的，可能具有重要的病理影响。
