| Co-Investigator(Kenkyū-buntansha) |
SOMAMOTO Tomonori Kyushu University, Department of Bioscience and Biotechnology, Assistant Professor, 大学院・農学研究院, 助手 (40403993)
KATO-UNOKI Yoko Kyushu University, Department of Bioscience and Biotechnology, Technical Manager, 農学部, 技術専門職員 (10380560)
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| Budget Amount *help |
¥15,700,000 (Direct Cost: ¥15,700,000)
Fiscal Year 2005: ¥6,800,000 (Direct Cost: ¥6,800,000)
Fiscal Year 2004: ¥8,900,000 (Direct Cost: ¥8,900,000)
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| Research Abstract |
The present study was aimed at clarifying functional differentiation among isotypes of bony fish complement component including C1r/s, C3,C4,C5,factor B and factor I, at both DNA and protein levels. Tissue distribution of mRNA encoding the isotypes was analyzed by RT-PCR. It was noted that isotypes of C4,C5,factors B and I showed different tissue distribution of their mRNA. Furthermore, C5 and factor B isotypes differed each other not only in expression sites but also in their expression response against bacterial infection. In particular, because two factor B isotypes, A1 and A3,showed marked difference in expression sites and response, their transcriptional elements were analyzed at the genomic DNA level. For this purpose, a carp genomic DNA library was constructed using lambda-FIX vector, from which factor A1 and A3 genes were isolated and sequenced over the 5'-upstream region. As a result, transcriptional elements C/EBP-alpha and HNF-1,which can account for liver-specific and constitutive expression of factor B-A1 isotype were identified in the A1 gene. At the protein level, on the other hand, factor B-A3 isotype, produced as a recombinant protein using a baculovirus/insect cell system, showed a novel reaction with C3-S isotype, which lacks the catalytic histidine residue, in a factor D-independent manner. This suggest the existence of a novel activation pathway in the alternative complement pathway of carp, forming a C3-convertase composed of C3-S and uncleaved factor B-A3. Two homologous but distinct collectins, designated MBL and GalBL, were purified from carp serum and demonstrated to be complexed with MASP2-like serine protease, which is responsible for C4 activation. The two lectins differ in carbohydrate-binding specificity. The data obtained by the present study strongly suggest that duplication of complement component genes contribute to expand and enhance innate immune defense relevant to complement system in bony fish.
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