Expression mechanism of subunit-specific low tolerance to protease digestion of collagen in bivalve molluscs

已知的几种双壳类软体动物的胶原蛋白的SDS-PAGE图谱被胃酶消化后发生了显著的变化，在SDS-PAGE上特定的a链的相对染色强度降低。在本研究中，我们试图建立一种从双壳类组织中制备一级结构不被酶消化改变的完整胶原蛋白的方法，并对其性质进行检测，以收集胃酶消化后胶原蛋白结构变化的信息。明确了用盐酸胍溶液从碱提残渣中提取胶原蛋白(RS-AL)的方法，提取的胶原蛋白称为盐酸胍可溶性胶原蛋白(GSC)。此外，用解聚液(0.1MTris-HCl缓冲液，含有0.05M EDTA，0.5M氯化钠和0.2M2-硫醇)对RS-AL进行预处理，可以提高S的…胃酶溶解的胶原蛋白制剂的主要胶原蛋白的亚基组成，观察到该物种的胃酶消化的凝胶电泳性变化相对较小。从主要的胶原蛋白中分离出两条遗传上截然不同的α链。氨基酸分析结果表明，主胶原蛋白可能是一个杂三聚体，其亚基组成为(α1)_2GSC2。每个组分的N端氨基酸序列都是几个双壳类的α链。所有被检测的α链的N-末端都不被吡谷氨酸封闭。特别令人感兴趣的是，所研究的许多α链具有独特的天冬氨酸-谷氨酸-N末端氨基酸序列。此外，日本扇贝外套膜GSC的一条特异的a链(暂时命名为α2)与[Gly-X-Y]三联体具有不同的内部序列，这表明α2链的三螺旋结构域中存在胃蛋白酶敏感区。在变性条件下，阳离子交换柱层析证实日本扇贝外套膜的GSC至少含有3条a链。目前正在进行进一步的研究，以提纯每个a链并澄清它们的结构特征。较少

SDS-PAGE pattern of collagen is known to be significantly changed by pepsin digestion for several species of bivalve molluscs, relative staining intensity of a specific a chain being decreased on SDS-PAGE. In the present study, we tried to establish a method to prepare intact collagen, of which primary structure is not modified by protease digestion, from the tissues of bivalves and to examine the properties of the intact collagen and its constituent a chains for the purpose of collecting information on the structural changes of collagen by pepsin digestion.It was clarified that collagen could be extracted in intact form from the residue after alkali extraction (RS-AL) by guanidine hydrochloride (GuHCl) solution, and the extracted collagen was referred to as guanidine hydrochloride-soluble collagen (GSC). Moreover, a pretreatment of the RS-AL by the disaggregating solution (0.1 M Tris-HC1 buffer containing 0.05 M EDTA,0.5 M NaCl and 0.2 M 2-mercapoethanol) was revealed to enhance the s … More olubility of collagen in the GuHCl extraction.Subunit composition of the major collagen of giant Pacific oyster was examined for the pepsin-solubilized collagen preparation on the basis of the observation that the electrophoretic change by pepsin digestion was relatively small for this species. Two genetically distinct a chains were isolated from the major collagen. The results of amino acid analysis for these a chains suggested that the major collagen may be a heterotrimer of which subunit composition was (α1)_2α2.N-terminal amino acid sequence was examined for each constituent a chain of GSC from several bivalves. N-termini of all of the α chains examined were revealed to be not closed by piroglutamate. It was of special interest that many of the α chains examined had a distinct N-terminal amino acid sequence of Asp-Glu-. Moreover, a specific a chain (temporarily named α2) of the GSC from Japanese scallop mantle had distinct internal sequences from [Gly-X-Y] triplet, suggesting the existence of pepsin-sensitive region in the triple helical domain of the chain α2. The GSC from the Japanese scallop mantle was elucidated to contain at least three a chains by cation-exchage column chromatography under denaturing conditions. Further studies are now in progress to purify each a chains and to clarify the structural characteristics of them. Less