Brockton Virginia, Hammond John A, Smith Valerie J
Comparative Immunology Group, Gatty Marine Laboratory, University of St. Andrews, Scotland, UK.
Mol Immunol. 2007 Feb;44(5):943-9. doi: 10.1016/j.molimm.2006.03.017. Epub 2006 May 18.
Carcinin is a whey acidic protein (WAP) domain-containing antimicrobial protein produced by the circulating haemocytes of the shore crab, Carcinus maenas. Cloning of its full coding cDNA reveals that it shows some similarity to invertebrate defensins, has a valine-rich signal sequence followed by a defined cleavage site but no obvious acidic anionic 'pro' sequence. The C-terminus exhibits a unique cysteine array that is predicted to form six disulphide bonds in the tertiary structure. This 12 cysteine array arrangement is conserved in expressed sequence tags (ESTs) from related genera and seems to represent a novel tertiary structure amongst antimicrobial proteins (AMPs), unique to the Crustacea. There are at least five putative isoforms that arise through the transcription of a multi-exon gene. These isoforms do not arise as a result of alternate splicing of the exons, but by either the transcription of different alleles and/or single point mutation of the transcript at up to four loci in the gene. Several of the same transcripts have been found in different animals. The most commonly expressed transcript of the protein was recombinantly expressed in bacterial fusion system to a yield of ca. 2-3 microg ml(-1) of culture. In vitro expression with or without the leader sequence confirms the bioinformatic prediction that the stability of the mature protein is reduced when the leader sequence is removed. Carcinin is one of very few invertebrate AMPs characterised at the gene, transcript and protein level and to be recombinantly expressed in vitro in a bacterial system.
癌抑素是一种含乳清酸性蛋白（WAP）结构域的抗菌蛋白，由海滨蟹（Carcinus maenas）的循环血细胞产生。其完整编码cDNA的克隆表明，它与无脊椎动物防御素具有一定相似性，有一个富含缬氨酸的信号序列，其后是一个确定的切割位点，但没有明显的酸性阴离子“前体”序列。C端表现出独特的半胱氨酸阵列，预计在三级结构中形成六个二硫键。这种12个半胱氨酸的阵列排列在相关属的表达序列标签（EST）中是保守的，似乎代表了抗菌蛋白（AMP）中的一种新型三级结构，这是甲壳纲动物特有的。通过一个多外显子基因的转录产生至少五种推定的异构体。这些异构体不是由于外显子的可变剪接产生的，而是通过不同等位基因的转录和/或基因中多达四个位点的转录本的单点突变产生的。在不同动物中发现了几种相同的转录本。该蛋白最常表达的转录本在细菌融合系统中进行了重组表达，培养物产量约为2 - 3微克/毫升。有或没有前导序列的体外表达证实了生物信息学预测，即去除前导序列时成熟蛋白的稳定性会降低。癌抑素是极少数在基因、转录本和蛋白质水平上得到表征并在细菌系统中进行体外重组表达的无脊椎动物AMP之一。
