Age-related changes in human lens crystallins identified by HPLC and mass spectrometry

Abstract

Analysis of water-soluble crystallins from human lenses, ages 32 week fetal to 55 years has led to identification of the major modifications of the proteins comprising the lens. These modifications were identified by the masses of the proteins determined by electrospray ionization mass spectrometry after the proteins were separated by gel filtration and reversed phase high performance liquid chromatography. Examination of all the proteins isolated from the water soluble portion demonstrated that the major age-related modifications causing significant alteration in the molecular weights of the lens crystallins include truncation of the N-termini of beta B1, beta A3 and beta A1, and partial phosphorylation and C-terminal degradation of alpha-crystallins. N-terminal degradation of beta B1, beta A3 and beta A1 was evident in human lenses less than one year old, and the proportion of these truncated proteins became greater with age. Phosphorylation of alpha A- and alpha B-crystallins increased from the fetal to the 3 year old lens, but did not change with further aging. Minor components indicating truncation of the C-termini of alpha-crystallins were found in older lenses. In contrast to beta B1, beta A3 and beta A1, the masses of the major species of alpha A, alpha B, beta B2, beta A4, gamma S, gamma C, and gamma D did not change with aging. This suggested that the major modifications to these crystallins are limited to deamidation and possibly intra-molecular disulfide bonds. These data, in conjunction with the data in the accompanying manuscript, established deamidation as a common modification, since deamidation, which causes only a one dalton change in mass, is the only modification that is consistent with the absence of a detectable change in molecular weight and the observed increased acidity demonstrated in the two-dimensional gels of the accompanying paper. Other age related changes included a decrease in beta B3 (M(r) 24224), a major component of the fetal lens, which was not detected in lenses older than 3 years, and increases in the ratios of alpha B:alpha A and gamma S:gamma C.