基于蛋白质组学的章鱼肽-钙螯合物跨膜转运促钙吸收机制研究

Calcium is an essential element of human body with important physiological functions. Calcium deficiency is a global health problem. There are many varieties of calcium supplements on the market. However, low bioavailability is the bottleneck of effective calcium supplementation of human body. The preparation of calcium-peptide organic chelate can significantly improve the absorption and utilization of calcium ions. Elucidation of the peptide-calcium chelation pattern and the mechanism of calcium transmembrane transport and absorption are the keys to revealing its high bioavailability.The project aims to purify the specific calcium-chelating peptides from the octopus scraps protein hydrolysate with high calcium chelating activity. The structural characteristics, peptide-calcium binding sites and chelation thermodynamics /kinetics will be determined through MS, sequence determination, ITC, FTIR and NMR.. Caco-2 cell monolayer model will be constructed to evaluate the calcium transmembrane transport efficiency of peptide-calcium chelate. The iTRAQ proteomics technology will be employed to analyze the effects of peptide-calcium chelate on Caco-2 cell metabolism. The Western blot and qPCR techniques will be used to elucidate the transmembrane transport and absorption mechanism of octopus scraps peptide-calcium chelate. This study will provide the theoretical basis for the development of peptide-calcium supplements.

钙是人体必不可少且具重要生理功能的元素，钙营养不足已成为全球性的公共健康问题，市场上补钙产品花样繁多，而生物利用率不高是制约人体有效补钙的瓶颈。利用蛋白肽制备钙-肽有机螯合物能显著提高钙离子的吸收利用率，而如何阐明肽-钙的螯合作用模式及跨膜转运促钙吸收机制是揭示其吸收利用率高的关键。本项目拟在已优化制备具有钙螯合力的章鱼蛋白肽的基础上，利用生化分离手段纯化特异性钙螯合肽，结合质谱、全序列测定、等温滴定量热法、红外光谱和核磁共振等技术确定其结构特征、钙离子结合位点和螯合热/动力学过程，揭示螯合作用的分子机制。构建Caco-2细胞模型，解析章鱼肽-钙螯合物的促钙跨膜转运吸收效率；应用iTRAQ蛋白质组学技术系统解析肽-钙螯合物对Caco-2细胞代谢的影响，结合Western blot和qPCR技术，阐明章鱼肽-钙螯合物的跨膜转运促钙吸收机制。研究结果将为开发蛋白肽-钙营养补充剂提供理论依据。

蛋白肽螯合钙离子制备肽钙螯合物是近年来开发补钙制剂的一个研究热点，其能显著提高钙离子的吸收利用率，可同时发挥补钙和蛋白肽的双重营养功效。本项目在前期已优化定向酶解条件制备的具有高效钙螯合力的章鱼蛋白酶解产物的基础上，利用凝胶过滤色谱技术分离具有高效钙螯合活力的章鱼肽组分，通过光谱技术解析肽与钙离子结合位点。制备章鱼肽钙螯合物，通过磷酸钙结晶抑制试验和体外模拟胃肠道消化试验，证明章鱼肽钙螯合物具有良好的稳定性。章鱼肽钙螯合物可提高Caco-2细胞对钙离子的吸收、抵抗膳食中钙离子吸收抑制剂的作用，提高钙离子跨膜转运吸收效率；结合qPCR和抑制剂试验结果表明，肽钙螯合物通过促进钙离子吸收通道相关因子的表达实现促钙吸收作用，也可通过肽的吸收途径PepT1转运体和胞吞作用进入细胞中，从而促进细胞的钙吸收，是一个多通道协同作用的过程。利用肽组学技术从章鱼肽中筛选特异性钙螯合肽序列，应用蛋白质组学技术分析肽钙螯合物在Caco-2细胞中的代谢涉及细胞旁路途径、转运蛋白和囊泡转运等。