Lens proteomics: Analysis of rat crystallin sequences and two-dimensional electrophoresis map

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Abstract

Purpose. To determine the sequence of four rat β-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 βB1, βB3, βA3, and βA4-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 βA4 cDNA encoded a 195-amino-acid protein. Additional cDNA sequencing provided the previously unknown N-terminal sequence of rat βA3, found two differences from the previous amino acid sequences of both rat βB1 and βB3, and detected a polymorphism at residue 54 in rat βB3. 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 βB3 suggests that crystallins in humans could also exhibit polymorphisms. The unusual abundance of rat βB3 and low abundance of βB2 may account for the increased susceptibility of rat crystallins to insolubilization during aging and cataract.

Original languageEnglish (US)
Pages (from-to)216-224
Number of pages9
JournalInvestigative Ophthalmology and Visual Science
Volume43
Issue number1
StatePublished - Jan 15 2002

ASJC Scopus subject areas

  • Ophthalmology
  • Sensory Systems
  • Cellular and Molecular Neuroscience

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