Department of Marine Life Sciences, School of Marine Biomedical Sciences, Jeju National University, Jeju Special Self-Governing Province 690-756, Republic of Korea.
Fish Shellfish Immunol. 2011 Jan;30(1):194-208. doi: 10.1016/j.fsi.2010.10.004. Epub 2010 Oct 15.
Heparin cofactor (HCII) is a serine protease inhibitor (SPI), and plays important physiological roles in various biological events including hemostasis. The gene encoding the HCII was isolated from GS-FLX™ genomic data of rock bream (Oplegnathus fasciatus), designated as RbHCII. The RbHCII (1950 bp) consists of a 1512 bp open reading frame (ORF) encoding 504 amino acids (aa), with a signal peptide of 19 aa residues. The predicted molecular mass and the estimated isoelectric point of RbHCII were 58 kDa and 5.9, respectively. The deduced aa sequence of RbHCII displayed a characteristic serpin domain and a serpin signature motif (FTVDQPFLFLI). RbHCII demonstrated homology with vertebrate HCIIs and the greatest degree of similarity (90.1%) was observed with Gasterosteus aculeatus HCII. Various functional domains including the reactive center loop (RCL), glycosaminoglycan (GAG) and thrombin binding sites and acidic repeats of human and RbHCII were found to be orthologs through the molecular modeling studies. Phylogenetic analysis revealed that RbHCII belongs to the clade D serpins, and is closely related to the clade A members. Constitutive expression of RbHCII mRNA was detected at different levels in various tissues in a tissue-specific manner. Interestingly, RbHCII transcription was significantly downregulated (p < 0.05) in liver after challenge with lipopolysaccharide (LPS), Edwardsiella tarda and rock bream iridovirus (RBIV). However, after the immune challenges, RbHCII showed a significant downregulation in blood tissue only at the late-phase of investigation. The recombinant RbHCII (rRbHCII) was overexpressed in Rosetta-gami (DE3) cells and purified using the pMAL™ system. The rRbHCII inhibited thrombin and chymotrypsin in a dose-dependent manner. Remarkably, heparin was found to be an enhancer of RbHCII's thrombin-inhibitory activity. Correlating the heparin-dependent thrombin-inhibition activity of RbHCII with its temporal downregulation against immune stimulants, it could be suggested that it is not only involved in the blood coagulation cascade, but also plays an incognito role in immune modulation.
肝素辅因子 II（HCII）是一种丝氨酸蛋白酶抑制剂（SPI），在包括止血在内的各种生物事件中发挥着重要的生理作用。编码 HCII 的基因是从石斑鱼（Oplegnathus fasciatus）的 GS-FLX™基因组数据中分离出来的，被命名为 RbHCII。RbHCII（1950 bp）由一个 1512 bp 的开放阅读框（ORF）编码 504 个氨基酸（aa）组成，带有 19 个残基的信号肽。RbHCII 的预测分子质量和估计等电点分别为 58 kDa 和 5.9。RbHCII 的推导 aa 序列显示出特征性的 serpin 结构域和 serpin 特征基序（FTVDQPFLFLI）。RbHCII 与脊椎动物的 HCIIs 具有同源性，与棘皮动物的 HCIIs 具有最大的相似性（90.1%）。通过分子建模研究发现，各种功能域，包括反应中心环（RCL）、糖胺聚糖（GAG）和凝血酶结合位点以及人和 RbHCII 的酸性重复，都是同源的。系统发育分析表明，RbHCII 属于 D 类 serpin，与 A 类成员密切相关。在组织特异性方式下，在不同组织中以不同水平检测到 RbHCII mRNA 的组成型表达。有趣的是，在用脂多糖（LPS）、迟钝爱德华氏菌和石斑鱼虹彩病毒（RBIV）刺激后，肝组织中 RbHCII 的转录显著下调（p<0.05）。然而，在免疫挑战后，仅在研究的后期阶段，RbHCII 在血液组织中表现出显著下调。重组 RbHCII（rRbHCII）在 Rosetta-gami（DE3）细胞中过表达，并使用 pMAL™系统纯化。rRbHCII 以剂量依赖性方式抑制凝血酶和糜蛋白酶。值得注意的是，肝素被发现是 RbHCII 凝血酶抑制活性的增强剂。将 RbHCII 对肝素依赖性凝血酶抑制活性与对免疫刺激物的时间下调相关联，可以表明它不仅参与了血液凝固级联反应，而且还在免疫调节中发挥了隐匿作用。
