Zhang Xi, Azuma Noriko, Hagihara Seishi, Adachi Shinji, Ura Kazuhiro, Takagi Yasuaki
Graduate School of Fisheries Sciences, Hokkaido University, Hakodate, Japan.
Graduate School of Fisheries Sciences, Hokkaido University, Hakodate, Japan.
Gene. 2016 Mar 15;579(1):8-16. doi: 10.1016/j.gene.2015.12.038. Epub 2016 Jan 6.
To characterize type I and II collagen in the Amur sturgeon at the molecular level, mRNAs encoding the proα chain of both types of collagen were cloned and sequenced. Full sequences of both were obtained, and the molecular phylogeny based on the deduced amino acid sequence indicated that the correct sequences of the target genes were obtained. Analyses of primary structure of the proα chains revealed that type I and II collagen share the basic structure of the proα chain of fibril collagen, but have different characteristics, especially in residues related to thermal stability. In the triple helical domain, Gly-Pro-Pro sequence stabilizing the tripeptide unit was more frequent in type II than in type I, and Gly-Gly, which likely decline in thermal stability, was more frequent in type I than in type II. These results suggested that the denaturation temperature of type II would be remarkably higher than type I. The spatial pattern of gene expression was analyzed by quantitative real-time PCR, which showed that relatively ubiquitous type I gene and strongly skewed distribution of type II gene, which highly expressed only in vertebra, snout cartilage, and notochord. This pattern was similar to the distribution pattern of each collagen protein detected by previous biochemical analyses using Amur and Bester sturgeons. The present study is the first report of the cloning of the full-length cDNAs for both of type I and type II collagen in the Amur sturgeon, and is the first comparative analysis of type I and II collagens in a sturgeon species at the molecular level. The results provide basic and general information on collagens in sturgeons.
为了在分子水平上表征鲟鱼中的I型和II型胶原蛋白，克隆并测序了编码这两种胶原蛋白原α链的mRNA。获得了两者的完整序列，基于推导的氨基酸序列的分子系统发育分析表明获得了目标基因的正确序列。对原α链一级结构的分析表明，I型和II型胶原蛋白共享原纤维胶原蛋白原α链的基本结构，但具有不同的特征，特别是在与热稳定性相关的残基方面。在三螺旋结构域中，稳定三肽单元的甘氨酸-脯氨酸-脯氨酸序列在II型中比在I型中更频繁，而可能导致热稳定性下降的甘氨酸-甘氨酸在I型中比在II型中更频繁。这些结果表明，II型的变性温度将明显高于I型。通过定量实时PCR分析基因表达的空间模式，结果显示I型基因相对普遍存在，而II型基因分布强烈偏斜，仅在椎骨、吻软骨和脊索中高表达。这种模式与之前使用鲟鱼和贝斯特鲟鱼进行的生化分析检测到的每种胶原蛋白的分布模式相似。本研究是首次报道鲟鱼中I型和II型胶原蛋白全长cDNA的克隆，也是首次在分子水平上对鲟鱼物种中的I型和II型胶原蛋白进行比较分析。这些结果提供了关于鲟鱼胶原蛋白的基本和一般信息。
