Post-genome study on filarial proteases for development of inhibitors.

2000 Fiscal Year Final Research Report Summary

• Project/Area Number: 11672218
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: 医薬分子機能学
• Research Institution: Tokyo University of Pharmacy and Life Science
• Principal Investigator: HIDESHI Inoue TOKYO UNIVERSITY OF PHARMACY AND LIFE SCIENCE, SCHOOL OF LIFE SCIENCE, ASSOCIATE PROFESSOR, 生命科学部, 助教授 (20184765)
• Co-Investigator(Kenkyū-buntansha): KOJIMA Masaki TOKYO UNIVERSITY OF PHARMACY AND LIFE SCIENCE, SCHOOL OF LIFE SCIENCE, ASSISTANT PROFESSOR, 生命科学部, 助手 (90277252) ; TAKAHASHI Kenji TOKYO UNIVERSITY OF PHARMACY AND LIFE SCIENCE, SCHOOL OF LIFE SCIENCE, PROFESSOR, 生命科学部, 教授 (70011533)
• Project Period (FY): 1999 – 2000
• Keywords: filaria / nematode / Caenorhabditis elegans / protease / post genome / parasite / Brugia malayi / RNAi

Research Abstract

1) We performed molecular cloning of various proteases from a parasitic nematode Brugia malayi, whiich causes human filariasis, and obtained six distinct cDNA clones containing complete open reading frames coding for two aspartic protease, three cysteine protease, and one metalloprotease-like sequences. Additionally, we obtained fourteen partial cDNA sequences.
2) Among the cDNA clones, two aspartic protease-like proteins, BmAsp2 and BmAsp3, were expressed by Escherichia coli to form inclusion bodies. The expressed proteins were extracted from the inclusion bodies with a buffer containing 8 M urea and were purified through DE52 column chromatography under denaturing conditions. The purified proteins were refolded by removing the denaturant. After refolding, protease activity toward hemoglobin could be detected under acidic conditions. Using antibodies raised against the BmAsp2 and BmAsp3 proteins, immunohistochemical analyses of adult male and female B.malayi were performed. BmAsp2 was detected in reproduction system and body wall muscle, while BmAsp3 was detected in intestine.
3) In order to investigate the functions of nematode proteases, we performed reverse-genetic analyses using RNAi by feeding worms with bacteria that express double-stranded RNA to target 130 distinct genes coding for protease-like sequences. RNAi phenotypes of most of the genes were normal, but 23 genes were found to have lethal RNAi phenotypes, and some other genes showed RNAi phenotypes with abnormal gonadogenesis.