Divergent genes encoding the third complement component (C3) in carp.

我们之前已经从单个鲤鱼个体中分离出三个不同的全长cDNA克隆和五个RT-PCR片段，它们都编码C3。有趣的是，其中三个片段（C3- s， C3- q1和C3- q2）预测了硫酯相关的His残基，这是迄今为止报道的所有其他C3中发现的，分别被Ser， GIn和GIn取代。由于在相应位置携带Ser的重组人C4B和携带Asn的大鼠a-巨球蛋白显示出类似人c4a的硫酯结合特异性，这些氨基酸替换可能解释了对不同靶表面结合特异性的差异，最近对硬骨鱼C3进行了描述。本研究从鲤鱼肝胰脏文库中分离出C3-S、C3-Q1和C3-Q2对应的cDNA克隆，进一步表征其分子实体。C3- s （5.0 kbp）包含一个长开放阅读框，该阅读框编码C3的前体，推断出的氨基酸序列与其他类型鲤鱼C3的同源性约为80%。另一方面，C3-Q1在b链对应的区域含有多个终止密码子，表明C3-Q1可能代表假基因的转录本。C3-Q2 （3.8 kbp）虽然有一个开放的阅读框，但缺少b链中间330个氨基酸对应的区域和C3a区。此外，C3- q2在硫酯位点有一个GSGEQ序列，而不是GCGEQ序列，这表明C3- q2不太可能具有C3的功能。RT-PCR分析检测到12条鲤鱼中有7条的肝胰腺中含有C3-Q2 mRNA。这些结果表明C3-Q1和C3-Q2基因本质上是无功能的，但C3-S似乎是作为功能蛋白存在的。C3-S蛋白的分离及其结合特异性分析正在进行中。

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.