Proteomic and sequence analysis of chicken lens crystallins reveals alternate splicing and translational forms of βB2 and βA2 crystallins

Phillip A. Wilmarth, Jennifer R. Taube, Michael A. Riviere, Melinda K. Duncan, Larry L. David

Research output: Contribution to journalArticle

32 Scopus citations

Abstract

PURPOSE. To characterize the adult chicken lens proteome using mass spectrometry and two-dimensional gel electrophoresis (2-DE). METHODS. Lens proteins from 10-week old chickens were separated by gel filtration and reversed-phase chromatography, and whole protein masses were measured with electrospray mass spectrometry. Water-soluble lens proteins were separated by 2-DE and identified by tandem mass spectrometry of in-gel digests. RESULTS. Whole protein masses were consistent with all major chicken lens crystallin sequences, except for βB2 and βB3. Subsequent cDNA sequencing revealed errors in published sequences translating into 2- and 7-amino-acid differences, respectively, for βB2 and βB3, which were in better agreement with the measured masses. Previously uncharacterized forms of βA2 and βB2 were observed. The novel form of βA2 had four fewer amino acids, was more abundant, and resulted from translation at a second start codon. The novel form of βB2 contained 14 additional amino acids in the interdomain linker and resulted from alternate splicing within intron 4 of the transcript. All examined crystallins, except βA3, for which data could not be obtained, were N-terminally acetylated, and all β-crystallins lacked an initial methionine, except for the smaller βA2 form. In-gel digests identified 29 proteins on the 2-DE map and indicated that truncation occurs within N-terminal extensions of β-crystallins during lens maturation. CONCLUSIONS. The complementary techniques 2-DE, mass spectrometry, and DNA sequencing were used to provide the most complete description of the adult chicken lens proteome to date and identified alternate forms of βA2 and βB2.

Original languageEnglish (US)
Pages (from-to)2705-2715
Number of pages11
JournalInvestigative Ophthalmology and Visual Science
Volume45
Issue number8
DOIs
StatePublished - Aug 1 2004

ASJC Scopus subject areas

  • Ophthalmology
  • Sensory Systems
  • Cellular and Molecular Neuroscience

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