Chen Y C, Reid G E, Simpson R J, Truscott R J
Australian Cataract Research Foundation, University of Wollongong, NSW, Australia.
Exp Eye Res. 1997 Dec;65(6):835-40. doi: 10.1006/exer.1997.0393.
The proteins of the lens which become insoluble, crosslinked and coloured as a result of the onset of human nuclear cataract have been studied using a combination of enzymatic digestion and HPLC/mass spectrometry (MS). The objective was to determine if such an approach could provide information on the identities of the polypeptide components involved in the colouration and crosslinking and to discover whether any crystallins predominate in this characteristic post-translational modification process. Initially, coloured high molecular weight peptides were isolated from a tryptic/chymotryptic digest of the 6 M guanidine hydrochloride-insoluble lens protein fraction. These tryptic/chymotryptic peptides were then incubated with pronase and the small peptides released, purified by gel filtration. All but one of the peptides analysed by HPLC/MS/MS were found to contain proline. Peptides derived from alpha-crystallin were found to comprise the great majority of the peptides characterised. No gamma-crystallin peptides were observed. Both alpha A-crystallin and alpha B-crystallin were represented. Further, all but one of these peptides were derived from the N-terminal region of the alpha-crystallin subunits: a region recently implicated in the chaperone activity of alpha-crystallin. This finding suggests that the putative N-terminal domain of alpha-crystallin may be involved at the molecular level in the process of crosslinking and colouration which is known to be characteristic of age-related nuclear cataract. It is, therefore, conceivable that an early stage of these cataractous modifications may involve alpha-crystallin acting as a molecular chaperone.
利用酶消化与高效液相色谱/质谱联用技术（HPLC/MS），对因人类核性白内障发作而变得不溶、交联且有色的晶状体蛋白质进行了研究。目的是确定这种方法是否能提供有关参与染色和交联的多肽成分的信息，并发现是否有任何晶状体蛋白在这种特征性的翻译后修饰过程中占主导地位。最初，从6M盐酸胍不溶性晶状体蛋白组分的胰蛋白酶/胰凝乳蛋白酶消化物中分离出有色高分子量肽。然后将这些胰蛋白酶/胰凝乳蛋白酶肽与链霉蛋白酶一起孵育，并通过凝胶过滤纯化释放出的小肽。通过HPLC/MS/MS分析的所有肽中，除一种外均发现含有脯氨酸。发现源自α-晶状体蛋白的肽占所鉴定肽的绝大多数。未观察到γ-晶状体蛋白肽。αA-晶状体蛋白和αB-晶状体蛋白均有代表。此外，这些肽中除一种外均源自α-晶状体蛋白亚基的N端区域：该区域最近与α-晶状体蛋白的伴侣活性有关。这一发现表明，α-晶状体蛋白假定的N端结构域可能在分子水平上参与交联和染色过程，而交联和染色是年龄相关性核性白内障的特征。因此，可以设想这些白内障修饰的早期阶段可能涉及α-晶状体蛋白作为分子伴侣发挥作用。
