Qin S F, Tan W D, Chen Y N, Lu Y R, Lu X F, Li Y P, Li H X, Wang L, Cheng J Q
Key Laboratory of Transplant Engineering and Immunology, Ministry of Health, West China Hospital, Sichuan University, P R China.
Transplant Proc. 2008 Mar;40(2):603-6. doi: 10.1016/j.transproceed.2008.01.030.
Hemorrhagic diseases have been considered to be one of the main causes of xenotransplantation failure. To explore the role of the rhesus monkey (Macaca mulatta) coagulation system in a pig-to-human xenotransplantation model, we primarily studied the full-length cDNA and the three-dimensional (3-D) structures of the important coagulation factor-prothrombin of the rhesus.
The full-length cDNA of rhesus prothrombin was obtained by rhesus monkey liver cDNA library screening and a 5'RACE technique. The 3-D protein structure was modeled using the SWISS-MODEL program. The important macromolecular interaction sites were compared with human and porcine prothrombin.
At first, the full-length rhesus prothrombin cDNA was cloned; the sequence was submitted to the Genebank (accession number: EF057490). The full-length cDNA is 2029 bp, with a complete open reading frame of 1884 bp, coding 626 amino acids. The deduced protein sequence contains a signal peptide, propeptide, Gla domain, two Kringle domains, and a Trypsin domain. The nucleotide similarities of rhesus-human and rhesus-porcine genes are 95.64% and 86.14%, and those of the amino acids, 94.51% and 82.82%, respectively. Some important functional sites, such as the catalytic triad DHS, RGD, Na+ -binding, and the carboxylase-binding site, are highly conserved. However, among the three species some variations are observed in potential N-glycosylation, O-glycosylation, phosphorylation, cleavage site, FXa-binding sites. Especially, the autolysis loop shows great differences in both amino acid sequence and 3-D model.
The great similarity of prothrombin among rhesus, human, and porcine confirmed the great value of the pig-to-rhesus xenotransplantation model. The variation especially in some important recognition sites between rhesus and pig would vary the binding affinity of enzymes to xenosubstrates and their reaction velocities in postoperative coagulation processes, which would be one possible reason for the disordered regulation of clotting seen during the xenorejection in animal models.
出血性疾病一直被认为是异种移植失败的主要原因之一。为了探讨恒河猴凝血系统在猪 - 人异种移植模型中的作用，我们主要研究了恒河猴重要凝血因子 - 凝血酶原的全长cDNA及其三维（3 - D）结构。
通过恒河猴肝脏cDNA文库筛选和5'RACE技术获得恒河猴凝血酶原的全长cDNA。使用SWISS - MODEL程序构建3 - D蛋白质结构模型。将重要的大分子相互作用位点与人及猪的凝血酶原进行比较。
首先，克隆了恒河猴凝血酶原全长cDNA；该序列已提交至Genebank（登录号：EF057490）。全长cDNA为2029 bp，具有1884 bp的完整开放阅读框，编码626个氨基酸。推导的蛋白质序列包含信号肽、前肽、Gla结构域、两个kringle结构域和一个胰蛋白酶结构域。恒河猴 - 人基因和恒河猴 - 猪基因的核苷酸相似性分别为95.64%和86.14%，氨基酸相似性分别为94.51%和82.82%。一些重要的功能位点，如催化三联体DHS、RGD、Na + 结合位点和羧化酶结合位点高度保守。然而，在这三个物种中，在潜在的N - 糖基化、O - 糖基化、磷酸化、切割位点、FXa结合位点存在一些差异。特别是，自溶环在氨基酸序列和3 - D模型中都显示出很大差异。
恒河猴、人和猪的凝血酶原高度相似，证实了猪 - 恒河猴异种移植模型的巨大价值。恒河猴和猪之间特别是一些重要识别位点的差异会改变酶与异种底物的结合亲和力及其在术后凝血过程中的反应速度，这可能是动物模型异种排斥期间凝血调节紊乱的一个原因。
