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

近年来，人们发现蛋白质的不可消化聚集与朊病毒、阿尔茨海默病等“折叠疾病”密切相关，但蛋白质聚集的机制尚不清楚。l -天冬氨酸（L-Asp）向d -天冬氨酸（D-Asp）的转化被认为是这种聚集形成的原因之一。在本研究中，为了了解D-Asp转化对模型肽或蛋白质构象的影响，以及随后肽或蛋白质聚集的影响，我们进行了以下实验。合成了几种氨基酸残基从10到30不等的多肽。我们使用CD和FT-IR光谱比较了仅含l -氨基酸的肽和L-Asp被D-Asp取代的肽群的构象。通过荧光染料的结合对淀粉样纤维的形成进行了探讨。对于大多数肽，D-Asp的取代促进了分子间β-sheet的形成。发现人tau蛋白通过取代D-Asp形成淀粉样蛋白纤维，提示D-Asp与蛋白质不可消化聚集体的形成密切相关。在本研究中，我们还研究了几种食品加工条件下食品蛋白中蛋白质聚集体中D-Asp的发生，以及将这些聚集体解离成单体形式的方法。我们发现在玻璃化转变温度以上的加热可以有效地使二级结构从分子间的β-片结构重新折叠为α-螺旋结构，从而解离喷雾干燥引起的聚集体。

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.