Lens proteomics: analysis of rat crystallin sequences and two-dimensional electrophoresis map.
OBJECTIVE
To determine the sequence of four rat beta-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 betaB1, betaB3, betaA3, and betaA4-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 betaA4 cDNA encoded a 195-amino-acid protein. Additional cDNA sequencing provided the previously unknown N-terminal sequence of rat betaA3, found two differences from the previous amino acid sequences of both rat betaB1 and betaB3, and detected a polymorphism at residue 54 in rat betaB3. 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 betaB3 suggests that crystallins in humans could also exhibit polymorphisms. The unusual abundance of rat betaB3 and low abundance of betaB2 may account for the increased susceptibility of rat crystallins to insolubilization during aging and cataract.
Lampi
Kirsten J
KJ
Department of Oral Molecular Biology, Schools of Dentistry and Medicine, Oregon Health and Science University, Portland, Oregon 97201, USA.
Shih
Marjorie
M
Ueda
Yoji
Y
Shearer
Thomas R
TR
David
Larry L
LL
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003459:Crystallins / Q000032:analysis* / Q000302:isolation & purification; D015180:Electrophoresis, Gel, Two-Dimensional; D008401:Gas Chromatography-Mass Spectrometry; D007908:Lens, Crystalline / Q000737:chemistry*; D008969:Molecular Sequence Data; D020543:Proteome / Q000032:analysis*; D051381:Rats; D017207:Rats, Sprague-Dawley; D017422:Sequence Analysis, DNA; D020539:Sequence Analysis, Protein
D003459:Crystallins; D020543:Proteome