Contribution of D-Aspartic Acit to formation of indigestible aggregate of protein

近年来，研究发现蛋白质的难消化性聚集与朊病毒、阿尔茨海默病等“折叠病”密切相关，但蛋白质聚集的机制尚不清楚。从L-天冬氨酸（L-Asp）到D-天冬氨酸（D-Asp）的转化被认为是这种聚集体形成的原因之一。本研究通过合成10 ~ 30个氨基酸残基的多肽，研究了D-Asp对模型肽或蛋白质构象的影响，以及D-Asp对模型肽或蛋白质聚集的影响。我们使用CD和FT-IR光谱比较了仅包含L-氨基酸的肽和其中L-Asp被D-Asp置换的队列肽的构象。淀粉样纤维的形成也通过荧光染料的结合来探测。对大多数多肽而言，D-Asp取代促进了分子间β折叠的形成。在本研究中，我们还研究了在不同的食品加工条件下，人tau蛋白在蛋白质聚集体中D-Asp的存在情况，以及D-Asp在蛋白质聚集体中解离成单体形式的方法。我们发现，高于玻璃化转变温度的加热可以有效地使二级结构从分子间的β-折叠折叠转变为α-螺旋结构，从而使喷雾干燥诱导的聚集体解离。

Recently, indigestible aggregate of protein is found to be closely relating to the "folding disease" such as prion, Alzheimer disease, but the mechanism of protein aggregation remains unclear. Transformation from L-aspartic acid(L-Asp) to D-aspartic acid(D-Asp) is suggested to be one reason for such aggregate formation. In this study, the following experiments are carried out in order to understand the effects of the transformation to D-Asp on the conformation of model peptides or proteins, and the following aggregation of peptides or proteins.Several kinds of peptides with amino acid residues varying from 10 to 30 were synthesized. We compared the conformation of peptides including only L-amino acids and the cohort peptides in which L-Asp was displaced by D-Asp using CD and FT-IR spectroscopy. The formation of amyloid fibril was also probed by the binding of fluorescent dye. For most of peptides, the substitution to D-Asp enhanced the formation of inter-molecular β-sheet. Human tau protein was found to form the amyloid fibril by the substitution to D-Asp, suggesting that D-Asp is closely related to the formation of indigestible aggregates of proteins.In this study, we also investigated the occurrence of D-Asp in the protein aggregate in food proteins by several food processing conditions, and the way to dissociate such aggregates to monomeric forms. We found that the heating above glass transition temperature is effective to cause the refolding the secondary structure from inter-molecular β-sheet to α-helix structure, thereby dissociating the aggregates induced by spray-drying.