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

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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