Analysis of genes of C-type lectin domain-containing proteins with useful biological activities.

许多含有C型凝集素样结构域的蛋白质都是从蛇毒中分离出来的。它们表现出各种各样的生物活性。IX/X-BP是从哈布蛇毒中分离到的一种异二聚体蛋白，由两个C型凝集素样结构域组成。两个C型凝集素样结构域互换。在钙离子存在的情况下，该蛋白与凝血因子IX和X结合，并抑制凝血级联反应。IX/X-BP的互补DNA克隆表明，IX/X-BP的每一条链都由各自的基因编码。蛇毒C型凝集素样蛋白的基因结构尚未见报道。在本研究中，我们克隆了编码IX/X-BP A链的基因。从黄绿色竹叶青的血液中提取总基因组DNA，用Sau3Al对其进行部分消化。用BamH1简化的Lambda EMBL3克隆载体构建基因组文库。对含有IX/X-BP A链基因的大肠杆菌克隆进行了培养，制备了重组表达载体。用Sall和Not1双酶切质粒中插入的DNA，用地高辛标记，作为探针筛选基因组文库。从基因组文库中分离到一个编码IX/X-BP A链的基因克隆，并用双脱氧链终止法测定了其核苷酸序列。IX/X-BP A链的基因结构不同于许多脊椎动物的C型凝集素结构域，如去唾液酸糖蛋白受体和LGE Fcε受体，它们的蛋白质结构与IX/X-BP非常相似，但Drickamer将其归入除VII类以外的其他类群。在铰链区插入了一个内含子，表明内含子插入对结构域交换机制的贡献。

Many proteins that contain C-type lectin-like domains are isolated from snake venom. They express a great variety of biological activities. IX/X-bp is one of the heterodimeric proteins isolated from the venom of habu snake (Trimeresurus flavoviridis) consisting of two C-type lectin-like domains. Two C-type lectin-like domains are swapped. This protein binds to blood coagulation factors IX and X in the presence of Ca ions and inhibits the blood coagulation cascade. Complementary DNA cloning of IX/X-bp indicated that each chain of IX/X-bp is encoded by respective gene.. Gene structure of snake venom C-type lectin-like protein is not yet reported. In the present study, we cloned the gene encoding IX/X-bp A chain. Total genomic DNA was prepared from the blood of Trimeresurus flavoviridis, and partially digested with Sau3Al. A genomic library was constructed using a Lambda EMBL3 cloning vector predigested by BamHl according to the manufacture's protocol. E. coli clone containing IX/X-bp A chain cDNA plasmid was cultured and the plasmid was prepared. Inserted DNA was cleaved off from the plasmid by the digestion with Sall and Notl and labeled with DIG to use as probe to screen the genomic library. One gene clone encoding the IX/X-bp A chain was isolated from the genomic library and the nucleotide sequence was determined by the dideoxynucleotide chain-termination method. The gene structure of IX/X-bp A chain is different from that of many vertebrate C-type lectin domains such as asialoglycoprotein receptors and lgE Fc ε receptor whose protein structures are very similar to that of IX/X-bp but are classified in the groups other than group VII by Drickamer. One of introns is inserted in the hinge region suggesting the contribution of the intron insertion to the domain swapping mechanism.