Sajid Mohammed, McKerrow James H, Hansell Elizabeth, Mathieu Mary A, Lucas Kimberley D, Hsieh Ivy, Greenbaum Doron, Bogyo Matthew, Salter Jason P, Lim Kee C, Franklin Christopher, Kim Jea-Hyoun, Caffrey Conor R
Department of Pathology, Tropical Disease Research Unit and Sandler Centre for Basic Research in Parasitic Diseases, University of California-San Francisco, Box 0511, San Francisco, CA 94143, USA.
Mol Biochem Parasitol. 2003 Sep;131(1):65-75. doi: 10.1016/s0166-6851(03)00194-4.
Peptidases are essential for the establishment and survival of the medically important parasite, Schistosoma mansoni. This helminth expresses a number of gut-associated peptidases that degrade host blood proteins, including hemoglobin, as a means of nutrition. Using irreversible affinity probes, we demonstrate that S. mansoni cathepsin B-like endopeptidase 1 (SmCB1) is the most abundant papain family cysteine peptidase in both the parasite gut and somatic extracts. SmCB1 zymogen (SmCB1pm) was functionally expressed in Pichia pastoris (4-11mgl(-1)). Monospecific and immunoselected antibodies raised against SmCB1pm localized the enzyme exclusively to the gut lumen and surrounding gastrodermis of adult worms. Recombinant SmCB1pm was unable to catalyze its activation, even at low pH. However, recombinant S. mansoni asparaginyl endopeptidase (SmAE), another gut-associated cysteine peptidase, processed and activated SmCB1pm in trans. Consistent with the known specificity of AEs, processing occurred on the carboxyl side of an asparagine residue, two residues upstream of the start of the mature SmCB1 sequence. The remaining pro-region dipeptide was removed by rat cathepsin C (dipeptidyl-peptidase I)-an action conceivably performed by an endogenous cathepsin C in vivo. The activated recombinant SmCB1 is biochemically identical to the native enzyme with respect to dipeptidyl substrate kinetics and pH profiles. Also, the serum proteins, hemoglobin, serum albumin, IgG, and alpha-2 macroglobulin were efficiently degraded. Further, a novel application of an assay to measure the peptidyl carboxypeptidase activity of SmCB1 and other cathepsins B was developed using the synthetic substrate benzoyl-glycinyl-histidinyl-leucine (Bz-Gly-His-Leu). This study characterizes the major digestive cysteine peptidase in schistosomes and defines novel trans-processing events required to activate the SmCB1 zymogen in vitro which may facilitate the digestive process in vivo.
肽酶对于医学上重要的寄生虫曼氏血吸虫的生存和繁衍至关重要。这种蠕虫表达多种与肠道相关的肽酶，这些肽酶可降解宿主血液中的蛋白质，包括血红蛋白，以此作为营养来源。我们使用不可逆亲和探针证明，曼氏血吸虫组织蛋白酶B样内肽酶1（SmCB1）是寄生虫肠道和体细胞提取物中最丰富的木瓜蛋白酶家族半胱氨酸肽酶。SmCB1酶原（SmCB1pm）在毕赤酵母中实现了功能性表达（4 - 11mg l⁻¹）。针对SmCB1pm产生的单特异性抗体和免疫选择抗体将该酶仅定位在成虫的肠腔和周围的胃皮层。重组SmCB1pm即使在低pH值下也无法催化自身激活。然而，另一种与肠道相关的半胱氨酸肽酶——重组曼氏血吸虫天冬酰胺内肽酶（SmAE），可反式加工并激活SmCB1pm。与天冬酰胺内肽酶已知的特异性一致，加工过程发生在天冬酰胺残基的羧基侧，该残基位于成熟SmCB1序列起始位置上游两个残基处。剩余的前肽区域二肽由大鼠组织蛋白酶C（二肽基肽酶I）去除——这一作用在体内可能由内源性组织蛋白酶C完成。就二肽基底物动力学和pH曲线而言，活化的重组SmCB1在生化性质上与天然酶相同。此外，血清蛋白、血红蛋白、血清白蛋白、IgG和α-2巨球蛋白均被有效降解。此外，利用合成底物苯甲酰-甘氨酰-组氨酰-亮氨酸（Bz-Gly-His-Leu）开发了一种新的测定方法，用于测量SmCB1和其他组织蛋白酶B的肽基羧肽酶活性。本研究对血吸虫中的主要消化性半胱氨酸肽酶进行了表征，并确定了在体外激活SmCB1酶原所需的新的反式加工事件，这可能有助于体内的消化过程。
