厚壳贻贝一种新型贝壳胶原蛋白的重组表达与功能分析

Recombinant Expression and Functional Analysis of a Novel Type of Shell Collagen from Mytilus coruscus

贝壳是一种具有优异力学性能的生物硬组织,贝壳基质蛋白质对贝壳的形成具有重要意义。厚壳贻贝(Mytilus coruscus)贝壳中发现一种类似胶原蛋白质的新型贝壳基质蛋白质,命名为collagen-like protein 2(CLP-2)。然而,该蛋白质的结构与功能以及对贝壳形成的影响机制尚不清楚。为此,本研究对CLP-2开展了序列分析;进一步采取密码子优化结合原核重组表达策略,开展了CLP-2的重组表达;在此基础上分析了重组CLP-2对酸钙结晶的诱导、结晶速率抑制以及碳酸钙结合能力。对CLP-2的序列分析结果表明,该蛋白质序列中含有信号肽及两个Von Willebrand factor A(VWA)结构域。CLP-2在数据库中尚无高同源性蛋白质存在,表明这是一种较为新颖的贝壳基质蛋白。所获得的重组CLP-2对碳酸钙体外结晶表现出明显的诱导作用,扫描电镜以及傅里叶红外光谱结果表明,重组CLP-2可诱导碳酸钙晶体的形貌由立方体形转化为球形,并在高浓度下进一步转化为哑铃形;同时,重组CLP-2可促使碳酸钙晶体的晶型由方解石型向文石型转化;重组CLP-2在体外具有碳酸钙晶体结合作用;此外,重组CLP-2能显著抑制碳酸钙晶体的结晶速度(P<0.01),并具有浓度依赖性。上述结果表明,厚壳贻贝贝壳CLP-2蛋白质在贝壳,特别是文石型肌棱柱层的生物矿化过程中具有重要作用。上述研究为深入了解贻贝贝壳的形成机制,以及胶原类蛋白质对生物矿化过程的影响奠定了基础。

Mollusk shell is the hard tissue with excellent mechanical properties.A novel shell matrix protein named collagen-like protein 2(CLP-2)was previously identified from the shell of Mytilus coruscus.However,the structure and the function of CLP-2 are unknown for now.For exploring the possible functions of CLP-2 in the formation of the shell of Mytilus coruscus,the sequence of the CLP-2 protein has been analyzed and recombinant expressed using bioinformatics methods and prokaryotic recombinant systems with codon optimization,respectively.The effects of recombinant CLP-2 in calcium carbonate crystallization were analyzed using scanning electron microscope and Fourier transform infrared spectroscopy,respectively.Sequence analysis showed that the protein sequence of CLP-2 contained signal peptide and two Von Willebrand factor A(VWA)domains.In addition,CLP-2 has no high homologous proteins in the current database.The recombinant CLP-2 could induce the crystallization of calcium carbonate in vitro,including the morphology changing from the cubic shape to dumb-bell shape,and the transformation from calcite to aragonite,of the calcium carbonate crystal.Furthermore,the recombinant CLP-2 could bind with calcium carbonate crystals and inhibit the crystallization rate significantly(P<0.01).These results indicated that CLP-2 plays an important role in the biomineralization of the mussel shell,especially in the myostraum layer with aragonite.These studies provided a foundation for further understanding the formation mechanism of mussel shells and the effect of collagen on the process of biomineralization.