Roles of protein deamidation in molecular and cellular aging.

ASN残基的脱酰胺是蛋白质的一种化学翻译后改变。现在人们意识到，在生理条件下，大多数蛋白质都会以一种依赖于时间的方式发生脱酰胺。这项研究的目的是阐明作为分子时钟的脱酰胺在细胞衰老中的作用和效果。我们分析了发生脱酰胺作用的红细胞膜蛋白、蛋白4.1和锚蛋白的氨基酸残基，并检测了通过定点突变产生的脱胺蛋白的功能。蛋白质4.1的蛋白质水解肽的质谱分析和氨基酸分析表明，脱酰胺发生在Asn502和Asn478处。我们证明，随着表观分子质量的变化，Asn502的脱酰胺在红细胞老化过程中发生得非常缓慢。通过突变将蛋白质4.1的同源转化为去胺化形式，证实了这一点。在多肽中观察到胰酶敏感部位的变化，特别是在邻近脱酰胺部位的区域，这表明蛋白质4.1分子的结构在脱酰胺作用后发生了变化。然而，有或没有Asn502去酰胺化的蛋白质4.1的功能有显著的差异，通过与已经耗尽骨骼蛋白的红细胞膜的结合来确定。我们还观察到，锚蛋白N端89 kDa结构域ANK重复结构中的第二和第八个ASN-Gly序列可以被脱酰胺。

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.