CHARACTERIZATION OF TRYPANOSOMA BRUCEI GP63 PROTEIN

布鲁氏锥虫是非洲锥虫病的病原体，在其哺乳动物宿主的血液中细胞外繁殖，但对补体介导的裂解(CML)具有抵抗力。本研究的目的是确定布氏毛滴虫GP63(TB-GP63)基因在慢性粒细胞白血病抗性中的作用。TB-GP63是利什曼原虫GP63(L-GP63)的同源物，是一种细胞表面锌金属蛋白酶，在利什曼原虫对慢性粒细胞白血病的抵抗中起关键作用。已鉴定出两组TB-GP63基因A和B，它们与L-GP63的同源性约为40%，与彼此的同源性约为60%。L-GP63中所有的二硫键半胱氨酸和TB-GP63-A和-B中的9个脯氨酸都是保守的，这表明TB-GP63蛋白在结构上将与L-GP63相似。为了研究TB-GP63在布鲁氏毛滴虫对慢性粒细胞白血病耐药中的作用，将TB-GP63在杆状病毒表达系统中进行表达，产生针对Th-GP63的纯化抗体，并建立过表达或缺失TB-GP63的细胞系。用这些试剂。将讨论以下问题：(I)TB-Gp63-A是一种功能性细胞表面蛋白水解酶吗？(Ii)TB-GP63-B也是功能性的GP63同系物吗？(3)TB-GP63是否增加了慢性粒细胞白血病耐药细胞？(4)布氏毛滴虫在小鼠体内的生长是否需要TB-GP63？以及(V)TB-GP63基因3‘端非编码区是否调控TB-GP63的阶段特异性表达？这些研究可能会确定抗锥虫药物的新靶点，并将为布鲁氏锥虫作为寄生虫成功的分子基础提供洞察。

Trypanosoma brucei, the causative agent of African trypanosomiasis, multiplies extracellularly in the bloodstream of its mammalian hosts but is resistant to complement mediated lysis (CML). The objective of this proposed research is to determine the role of T. brucei GP63 (Tb-GP63) genes in resistance to CML. Tb-GP63 is a homolog of leishmania GP63 (L- GP63), a cell surface zinc metalloprotease that is critical for the resistance of leishmania to CML. Two groups of Tb-GP63 genes have been identified A and B. which are approximately 40 % identical and 60 % similar to L- GP63 and to each other. All disulfide bonded cysteines in L-GP63 and nine prolines are conserved in Tb-GP63-A and -B, suggesting that the Tb-GP63 proteins will be structurally similar to L-GP63. To study the role of Tb- GP63 in the resistance of T. brucei to CML Tb-GP63 will expressed in a baculovirus expression system and purified antibodies will be generated against Th-GP63 and cell lines that over-express or that lack Tb-GP63 will be created. With these reagents. the following questions will be addressed: (i) is Tb-Gp63-A is a functional cell surface protease? (ii) is Tb-GP63-B also a functional GP63 homolog? (iii) does Tb-GP63 increase the resistance cells to CML? (iv) is Tb-GP63 is required for growth of T. brucei in mice? and (v) do the Tb-GP63 gene 3' UTRs regulate stage specific expression of Tb-GP63? These studies may identify a new target for anti-trypanosome drugs and will provide insight into the molecular basis for the success of T. brucei as a parasite.