BIOCHEMICAL AND MOLECULAR BIOLOGICAL ANALYSIS OF THE FUCOSYLTRANSFERASES IN C.ELEGANS

2001 Fiscal Year Final Research Report Summary

• Project/Area Number: 12680679
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Molecular biology
• Research Institution: SOKA UNIVERSITY
• Principal Investigator: KONDO Kazunori SOKA UNIVERSITY, DEPARTMENT OF BIOENGINEERING, LECTURER, 工学部, 講師 (10211913)
• Co-Investigator(Kenkyū-buntansha): NISHIHARA Shoko SOKA UNIVERSITY, INSTITUTE OF LIFE SCIENCE, PROFESSOR, 生命科学研究所, 教授 (00164575)
• Project Period (FY): 2000 – 2001
• Keywords: GLYCOSYLTRANSFERASE / FUCOSYLTRANSFERASE / C.ELEGANS / NEMATODE / MOLECULAR BIOLOGY / MOLECULAR GENETICS

Research Abstract

Biological functions of the carbohydrate on glycoconjugates have been recognized in a variety of cell activities. The nematode, Coenorhabditis elegans should be a suitable model organism for the study of the functions of carbohydrates, since in this animal not only molecular biological techniques but also classical genetics are available. Carbohydrates are synthesized by sugar transferases. We have been studying fucosyl-transferases in C. elegans, because these enzymes are well characterized in mammalian system and their activities synthesizing Lewis X antigen are evolutionary conserved from bacteria (ex : Helicobacter pylori) to mammals (Homo sapience). Five genes which are homologous to mammalian α-1,3(4)-fucosyltransferase turned out to exist on C. elegans genome through the genome-sequencing project. All of them are coded on the linkage group II. We cloned cDNAs of them by RT-PCR method (CEFT1-CEFT5). The CEFT1 cDNA was subcloned into the expression vector pCDM8 and transfected to the COS1 cells. Homogenates of the cells are used as an enzyme source, and transfer activities of ^<14>C-labeled GDP-fucose were assayed for several acceptor substrates. An incorporation of ^<14>C-fucose was observed when C. elegans total proteins were used as acceptor substrates, while no activity was shown when lipids or mammlian Le^x precursors were used as acceptor substrates. For the remaining four putative fucosyltransferase genes, we used pAMof2 vector for secretory expression of the recombinant enzymes, but they did not express successfully. Then we are trying baculovirus expression system, pBacPAK9-BacPAK6 (Clontech). Also we are planning to analyze the gene-knock out phenotype of thse five fucosyltransferases by RNAi method.