Engineering D-Amino Acid Containing Collagen Like Peptide at the Cleavage Site of Clostridium histolyticum Collagenase for Its Inhibition

Collagenase is an important enzyme which plays an important role in degradation of collagen in wound healing, cancer metastasis and even in embryonic development. However, the mechanism of this degradation has not yet been completely understood. In the field of biomedical and protein engineering, the design and development of new peptide based materials is of main concern. In the present work an attempt has been made to study the effect of DAla in collagen like peptide (imino-poor region of type I collagen) on the structure and stability of peptide against enzyme hydrolysis. Effect of replacement of DAla in the collagen like peptide has been studied using circular dichroic spectroscopy (CD). Our findings suggest that, DAla substitution leads to conformational changes in the secondary structure and favours the formation of polyproline II conformation than its L-counterpart in the imino-poor region of collagen like peptides. Change in the chirality of alanine at the cleavage site of collagenase in the imino-poor region inhibits collagenolytic activity. This may find application in design of peptides and peptidomimics for enzyme-substrate interaction, specifically with reference to collagen and other extra cellular matrix proteins.

胶原酶（Collagenase）是一类重要的酶，在伤口愈合、癌症转移乃至胚胎发育过程中的胶原蛋白降解环节发挥关键作用。然而，该降解过程的完整分子机制至今尚未完全阐明。在生物医学与蛋白质工程领域，新型肽基材料的设计与开发是核心研究方向之一。本研究以I型胶原蛋白的贫亚氨基酸区域为模板构建胶原样肽（collagen-like peptide），旨在探究其中D-丙氨酸（D-Ala）对肽段结构及其抗酶解稳定性的影响；采用圆二色光谱（circular dichroic spectroscopy, CD）技术分析了D-丙氨酸替换的作用效果。研究结果表明，相较于L-丙氨酸对应体，在该类胶原样肽的贫亚氨基酸区域引入D-丙氨酸替换会引发二级结构构象变化，且更利于聚脯氨酸II型构象（polyproline II conformation）的形成。在贫亚氨基酸区域的胶原酶切割位点处，丙氨酸手性的改变会抑制胶原酶解活性。该研究成果可应用于酶-底物相互作用相关的肽与肽模拟物设计，尤其适用于胶原蛋白及其他细胞外基质蛋白相关的研究场景。