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Kirsten J. Lampi, Marjorie Shih, Yoji Ueda, Thomas R. Shearer, Larry L. David; Lens Proteomics: Analysis of Rat Crystallin Sequences and Two-Dimensional Electrophoresis Map. Invest. Ophthalmol. Vis. Sci. 2002;43(1):216-224.
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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
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.
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