Studies on Filarial Proteinases and Their Inhibitors for Development of Filarial Drugs

1.从斯里兰卡采集的丝虫（Setcria digitata）粗提物中，经DEAE-cellulose、Sephacryl S-200、pepstatia-Sepharose和monoQ柱层析，分离纯化出两种天冬氨酸蛋白酶，并对其酶学性质和分子生物学性质进行了研究，测定了N端序列。此外，中性蛋白酶的部分纯化。2. cDNA文库制备的人丝虫寄生虫，包括马来丝虫的mRNA。扭转Ozwhocerca和潘氏丝虫，以及马来丝虫和班氏吴策线虫的基因组DNA文库。然后，两种天冬氨酸蛋白酶Asp 2和Asp 3的全长cDNA。从马来丝虫cDNA文库中克隆了天冬氨酸蛋白酶Asp 1的部分cDNA，测定了它们的碱基序列，并推导了氨基酸序列。构建了包括Asp 2和Asp 3在内的天冬氨酸蛋白酶系统进化树。指示。Asp 2在某种程度上 ...更多信息 Asp 3类似于哺乳动物组织蛋白酶D，而Asp 3是独特的，因为它与组织蛋白酶D和胃蛋白酶都显著不同。从马来丝虫的cDNA文库中分离出两种半胱氨酸蛋白酶Cys 1和Cys 2的全长cDNA，测定了它们的碱基序列，并推导了它们的氨基酸序列。Cys 1和Cys 2之间的序列非常相似，但与哺乳动物半胱氨酸蛋白酶的序列明显不同。4.另外，对一种新的膜结合的切割牛脑前胃泌素C端（88-101）肽的金属内肽酶进行了纯化和/或表征研究，该酶是一种特异性切割猪Ras及其相关蛋白C端CAAX基序的猪脑内肽酶。肠肽酶、胰胰蛋白酶和α-胰凝乳蛋白酶、大鼠肝微粒体膜结合丝氨酸蛋白酶（hepsin）、结肠蛋白酶（colopsin）（一种来自肠的新的膜结合丝氨酸蛋白酶）、人和小鼠组织蛋白酶E、腔肠胃蛋白酶原、结肠蛋白酶、霉菌酸性蛋白酶A和B、细菌金属内肽酶、sigaital肽酶和菠萝蛋白酶抑制剂。少

1.Two aspartic proteinases were purified from the crude extract of boviae filarial parasites (Setcria digitata) collected in Sri Lanka by sucessive steps of chromatography on columns of DEAE-cellulose, Sephacryl S-200, pepstatia-Sepharose, and monoQ, and their enzymat.ic and molecular properties were investigated and the N-terminal sequences were determined. In addition, neutral proteinases were partially purified.2.cDNA libraries were prepared from the mRNAs of human filarial parasites, including Brugia malayi. Ozwhocerca volvulus and Brugia phangi, and genomic DNA libraries from Brugia malayi and Wuchereria bancrofti. Then, full-length cDNAs of two aspartic proteinases, Asp2 and Asp3. and a partial length cDNA of one aspartic proteinase, Aspl, were isolated from the cDNA libraries of Brugia malayi and their base sequences were determined and amin acid sequences deduced. A phylogenetic tree of aspartic proteinases includingAsp2 and Asp3 was constructed. indicating. that Asp2 somewhat … More resembles mammalian cathepsin D, whereas Asp3 is unique in that it is significantly diverged from both cathepsin D and pepsin. AspI was shown to be fairly similar in sequence to Asp2.3, From the cDNA library of Brugia malayi, full-length cDNAs of two cysteine proteinases, Cysl and Cys2, were isolated, and their base sequences were determined and amino acid sequences deduced. The sequences are fairly similar between Cys1 and Cys2, but are markedly different from those of mammalian cysteine proteinases.4.In addition, purification and/or characterization studies were performed for a novel inembrane-bound inetalloendopeptidase cleaving progastrin C-terminal (88-101) peptide from bovine brain, a porcine brain endopeptidase specific for the C-terminal CAAX motif of Ras and related proteins, porcine . enteropeptidase, pancreatic trypsin and alpha-chymotrypsin, a rat liver microsomal membrane- bound serine proteinase(hepsin), colopsin (a novel membrane-bound serine proteinase from intestine), human and mouse cathepsin E, coelacant pepsinogen, nope nthesin, mold acid proteinases A and B, bacterial metalloendopeptidases, sigaital peptidase and brotnelain inhibitors. Less