| Budget Amount *help |
¥3,300,000 (Direct Cost: ¥3,300,000)
Fiscal Year 1998: ¥1,800,000 (Direct Cost: ¥1,800,000)
Fiscal Year 1997: ¥1,500,000 (Direct Cost: ¥1,500,000)
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| Research Abstract |
We have previously isolated three distinct full-length cDNA clones and five RT-PCR fragments, all of which encode C3, from a single individual of carp. Interestingly, three of the fragments (C3-S, C3-Q1, and C3-Q2) predicted that thioester-associating His residue, which is found in all other C3 reported so far, is replaced by Ser, GIn, and GIn, respectively. Since recombinant human C4B carrying Ser and rat a-macroglobulin carrying Asn at the corresponding position have been reported to show a human C4A-like binding specificity of the thioester, these amino acid substitution may account for the difference in binding specificity to various target surfaces, which has recently been described for teleost C3. In this study, we have isolated cDNA clones corresponding to the C3-S, C3-Q1, and C3-Q2 from carp hepatopancreas library to further characterize their molecular entity.
C3-S (5.0 kbp) contained a single long open reading frame which encodes pre-pro form of C3 and the deduced amino acid sequence showed about 80% identity with other types of carp C3. On the other hand, C3-Q1 contained several stop codons in the region corresponding to B-chain, suggesting that C3-Q 1 would represent the transcript from a pseudogene. Although C3-Q2 (3.8 kbp) had an open reading frame, it lacked the region corresponding to the 330 amino acids in the middle of B-chain and the C3a region. Moreover, C3-Q2 had a GSGEQ sequence at the thioester site instead of GCGEQ, indicating that C3-Q2 unlikely functions as C3. RT-PCR analysis detected C3-Q2 mRNA in hepatopancreas from seven of twelve carp individuals tested. These results suggest that the genes of C3-Q1 and C3-Q2 would be essentially non-functional, but C3-S seems to exist as a functional protein. Isolation of C3-S protein and analyses of its binding specificity are in progress.
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