Srivastava O P, Srivastava K, Harrington V
School of Optometry, University of Alabama at Birmingham, Birmingham, Alabama, 35294, USA.
Biochem Biophys Res Commun. 1999 May 19;258(3):632-8. doi: 10.1006/bbrc.1999.0506.
The aim of this study was to determine age-related degradation of betaA3/A1-crystallin in human lenses. The betaA3/A1-crystallin fragments were identified by Western blot analysis using two site-specific anti-betaA3/A1-crystallin antibodies. The first antibody was raised against a N-terminal region (residues 35-66), and the second to the C-terminal (residues 203-214) region of the crystallin. During the analyses, either preparative SDS-PAGE-separated fragments from betaH-crystallin fraction or water-soluble (WS) protein fractions from lenses of different aged donors were used. In lenses from 27- to 30-year-old donors, four major crystallin fragments of about 5, 16, 17, and 18 kDa immunoreacted with the anti-betaA3/A1-N-terminal antibody, suggesting their intact N-terminus but cleaved C-terminus. A similar analysis with the anti-betaA3/A1-C-terminal antibody identified 15-, 18-, 19-, and 20-kDa species and also five species between 4 and 11 kDa that had intact C-terminus but cleaved N-terminus. In lenses from a 5-year-old donor only two crystallin species, a major 15-kDa and a minor 18-kDa species, showed an intact N-terminus and cleaved C-terminus, whereas, eight species with Mr's between 4 and 19 kDa exhibited intact C-terminus but cleaved N-terminus. Upon two-dimensional gel electrophoresis of a betaH-crystallin fraction from the lenses of a 70-year-old donor, a degradation profile almost similar to the crystallin mentioned above was observed. However, the existence of multiple spots with identical Mr's of truncated betaA3/A1-crystallin species on the 2D-gel suggests their existence as isoforms (identical size species with different charges) because of post-translational modifications. Five species of 4, 6, 11, 15, and 18 kDa showed an identical partial N-terminal sequence of N-F-Q-G, suggesting cleavage at the E39-N40 bond during their production. Together, the data suggest that the majority of age-related cleavages in betaA3/A1-crystallin occur at the N-terminal region, with a major cleavage site at the E39-N40 bond generating some of these fragments.
本研究的目的是确定人晶状体中βA3/A1-晶状体蛋白与年龄相关的降解情况。使用两种位点特异性抗βA3/A1-晶状体蛋白抗体，通过蛋白质免疫印迹分析鉴定βA3/A1-晶状体蛋白片段。第一种抗体是针对晶状体蛋白的N端区域（第35 - 66位氨基酸残基）制备的，第二种是针对其C端区域（第203 - 214位氨基酸残基）制备的。在分析过程中，使用了从βH-晶状体蛋白组分经制备性SDS-PAGE分离得到的片段，或来自不同年龄供体晶状体的水溶性（WS）蛋白组分。在27至30岁供体的晶状体中，约5、16、17和18 kDa的四个主要晶状体蛋白片段与抗βA3/A1-N端抗体发生免疫反应，表明它们的N端完整但C端被切割。用抗βA3/A1-C端抗体进行的类似分析鉴定出了15、18、19和20 kDa的蛋白条带，以及4至11 kDa之间的五个蛋白条带，这些蛋白条带的C端完整但N端被切割。在一名5岁供体捐献的晶状体中，只有两种晶状体蛋白，一种主要的15 kDa蛋白和一种次要的18 kDa蛋白，显示出完整的N端和切割的C端，而8种分子量在4至19 kDa之间的蛋白显示出完整的C端但切割的N端。对一名70岁供体晶状体的βH-晶状体蛋白组分进行二维凝胶电泳后，观察到了一个与上述晶状体蛋白几乎相似的降解图谱。然而，二维凝胶上存在多个具有相同分子量的截短βA3/A1-晶状体蛋白异构体（相同大小但电荷不同的蛋白），这表明它们是由于翻译后修饰而存在的。4、6、11、15和18 kDa的五种蛋白显示出相同的部分N端序列N-F-Q-G，表明它们在产生过程中在E39 - N40键处发生了切割。总之，这些数据表明，βA3/A1-晶状体蛋白中大多数与年龄相关的切割发生在N端区域，E39 - N40键是主要切割位点，产生了其中一些片段。
