Roles of protein deamidation in molecular and cellular aging.

Asn残基的脱酰胺是蛋白质的化学翻译后改变。现在认识到，在生理条件下，大多数蛋白质上的脱酰胺作用将以时间依赖性的方式发生。本研究的目的是阐明脱酰胺作为分子钟在细胞衰老中的作用和影响。我们分析了红细胞膜蛋白，蛋白4.1和锚蛋白，发生脱酰胺的氨基酸残基，并检查通过定点突变产生的脱酰胺蛋白的功能。质谱和氨基酸分析的蛋白质4.1衍生的蛋白水解肽显示，脱酰胺发生在Asn 502和Asn 478。我们证明，Asn 502的脱酰胺发生非常缓慢，在红细胞老化过程中的表观分子量的变化。通过诱变将蛋白4.1的同源物转化为脱酰胺形式证实了这一点。观察到多肽内胰蛋白酶敏感位点的变化，特别是在脱酰胺位点附近的结构域中，表明蛋白4.1分子通过脱酰胺而发生结构改变。然而，通过与已耗尽骨架蛋白的红细胞膜结合，在有或没有Asn 502脱酰胺的蛋白4.1的功能中观察到显著差异。我们还观察到锚蛋白N-末端89-kDa结构域的ANK重复结构内的第2和第8个Asn-Gly序列可被脱酰胺。

Deamidation of Asn residue is a chemical posttranslational alteration of the protein. it is now appreciated that deamidation would occur on most proteins in a time-dependent manner under physiological conditions. The purpose of this study was to elucidate roles and effects of deamidation in cellular aging as the molecular clock. We analyzed the amino acid residues of red cell membrane proteins, protein 4.1 and ankyrin, on which deamidation occurred and examined the functions of the deamidated proteins which was generated by site-directed mutagenesis. Mass spectrometry and amino acid analysis of the proteolytic peptides derived from protein 4.1 revealed that deamidation occurred at Asn502 and Asn478. We demonstrated that deamidation of Asn502 occurs very slowly during red cell aging with the change of apparent molecular mass. This was confirmed by converting a congenor of protein 4.1 to a deamidated form by mutagenesis. Changes of trypsin-sensitive sites within the polypeptide, particularly in the domains neighboring the deamidation sites, were observed, indicating a structural alteration of protein 4.1 molecule by deamidation. However, on significant differences was observed in the function of the protein 4.1 with or without Asn502 deamidation determined by binding to the red cell membranes which had been depleted of skeletal proteins. We also observed that the 2nd and 8th Asn-Gly sequence within the ANK repeat structure of the N-terminal 89-kDa domain of ankyrin were accessible to deamidation.