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1 From the Departments of Oral Molecular Biology and 4 Ophthalmology, Schools of Dentistry and Medicine, Oregon Health and Science University, Portland, Oregon; and the 2 Department of Animal Sciences, Oregon State University, Corvallis, Oregon.
PURPOSE. To determine the sequence of four rat ß-crystallins, confirm the sequences by mass spectrometry, and produce a two-dimensional electrophoresis (2-DE) map of soluble crystallins in young rat lens.
METHODS. New or additional sequences were determined for ßB1, ßB3, ßA3, and ßA4-crystallin cDNAs from Sprague-Dawley rats, and the deduced protein sequences confirmed by mass spectrometry. The identity and relative abundance of each crystallin was then determined by 2-DE of soluble protein from whole lenses of 12-day-old rats, image analysis, and tandem mass spectrometry (MS/MS) spectra of peptides from in-gel digests.
RESULTS. The previously unreported sequence of rat ßA4 cDNA encoded a 195-amino-acid protein. Additional cDNA sequencing provided the previously unknown N-terminal sequence of rat ßA3, found two differences from the previous amino acid sequences of both rat ßB1 and ßB3, and detected a polymorphism at residue 54 in rat ßB3. These new sequences were then confirmed by whole protein masses and MS/MS spectra of proteolytic digests. 2-DE analysis provided a more detailed map of rat crystallins than previously available and allowed the composition of crystallins in young rat lens to be compared with that in young human lens.
CONCLUSIONS. This report provides baseline data that will facilitate the analysis of posttranslational modifications in rat crystallins during cataract. Detection of a polymorphism in the sequence of rat ßB3 suggests that crystallins in humans could also exhibit polymorphisms. The unusual abundance of rat ßB3 and low abundance of ßB2 may account for the increased susceptibility of rat crystallins to insolubilization during aging and cataract.
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