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

1.采用DEAE-cellulose、Sephacryl S-200、pepstatia-Sepharose和monoQ色谱柱对斯里兰卡产牛丝虫（Setcria digitata）粗提物及其酶进行层层析，纯化出两种天冬氨酸蛋白酶。研究了其ic和分子性质，并确定了n端序列。此外，中性蛋白酶也得到了部分纯化。从包括马来布鲁氏菌在内的人丝虫寄生虫的mrna中制备cDNA文库。卷尾虾和棕腹虾，马来虾和斑腹虾基因组DNA文库。然后是两种天冬氨酸蛋白酶Asp2和Asp3的全长cdna。从马来Brugia malayi cDNA文库中分离到一个天冬氨酸蛋白酶Aspl的部分全长cDNA，并测定了其碱基序列和氨基酸序列。构建了包括asp2和Asp3在内的天冬氨酸蛋白酶的系统发育树。指示。Asp2在某种程度上更类似于哺乳动物的组织蛋白酶D，而Asp3的独特之处在于它与组织蛋白酶D和胃蛋白酶都有明显的差异。从马来Brugia malayi的cDNA文库中分离到两种半胱氨酸蛋白酶Cysl和Cys2的全长cDNA，并测定了它们的碱基序列和氨基酸序列。Cys1和Cys2的序列基本相似，但与哺乳动物半胱氨酸蛋白酶的序列有显著差异。此外，我们还从牛脑中分离了一种新型的膜结合inetalloendeptidase，用于分离原胃泌素c -末端（88-101）肽，这是一种猪脑内肽酶，专门用于猪Ras和相关蛋白的c -末端CAAX基序。肠肽酶、胰胰蛋白酶和α -凝乳胰蛋白酶、大鼠肝微粒体膜结合丝氨酸蛋白酶（hepsin）、结肠蛋白酶（一种来自肠道的新型膜结合丝氨酸蛋白酶）、人和小鼠组织蛋白酶E、凝乳胃蛋白酶原、不合成酶、霉菌酸性蛋白酶a和B、细菌金属内肽酶、信号肽酶和兄弟蛋白酶抑制剂。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