May 2004
Volume 45, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2004
Analysis of chicken lens crystallins by mass spectrometry reveals an alternate start codon for beta A2 and a splice variant for beta B2
Author Affiliations & Notes
  • L.L. David
    Department of Integrative Biosciences, Oregon Health & Science Univ, Portland, OR
  • P.A. Wilmarth
    Department of Integrative Biosciences, Oregon Health & Science Univ, Portland, OR
  • J.R. Taube
    Department of Biological Sciences, University of Delaware, Newark, DE
  • M.A. Riviere
    Department of Integrative Biosciences, Oregon Health & Science Univ, Portland, OR
  • M.K. Duncan
    Department of Biological Sciences, University of Delaware, Newark, DE
  • Footnotes
    Commercial Relationships  L.L. David, None; P.A. Wilmarth, None; J.R. Taube, None; M.A. Riviere, None; M.K. Duncan, None.
  • Footnotes
    Support  NIH grants EY007755, EY010572, EY12221
Investigative Ophthalmology & Visual Science May 2004, Vol.45, 3969. doi:
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      L.L. David, P.A. Wilmarth, J.R. Taube, M.A. Riviere, M.K. Duncan; Analysis of chicken lens crystallins by mass spectrometry reveals an alternate start codon for beta A2 and a splice variant for beta B2 . Invest. Ophthalmol. Vis. Sci. 2004;45(13):3969.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Abstract: : Purpose: To characterize the crystallins of chicken lens by mass spectrometry to investigate several unusual properties of these proteins observed on 2D–electrophoresis gels. Methods: Soluble proteins from 10–week–old chicken lenses were separated by 2D–electrophoresis using pH 3–10 immobilized pH gradients in the first dimension and 22 cm wide SDS–PAGE gels in the second dimension. Spots containing crystallins were excised, proteins digested with trypsin, and digests analyzed by liquid chromatography/tandem mass spectrometry. Soluble proteins were also separated by gel filtration, proteins in major peaks resolved by reverse phase chromatography, and the masses of the undigested proteins determined. The sequences of chicken ßB3 cDNA, and an intron from the ßB2 gene were also determined. Results: Chicken lenses contained the same complement of α– and ß–crystallins found in mammalian lenses, except no ßA4 was detected, and two major spots were observed for both ßA2 and ßB2–crystallins. Tandem mass spectral analysis of the larger form of ßA2 indicated that it contained an acetylated N–terminus missing the N–terminal methionine, while analysis of the smaller form of ßA2 indicted the N–terminus began with the second methionine at residue 5 that was also acetylated. The shorter form of ßA2 comprised 60% of the total ßA2. Analysis of the two forms of ßB2 indicated that the basic higher molecular weight form contained 14 extra amino acids inserted after residue 101. Sequencing of the ßB2 gene indicated that the extra residues resulted from slippage of the 3' end of a 289 base pair intron. The larger basic form of ßB2 comprised 20% of the total ßB2. Measurement of the whole masses of the chicken ß–crystallins further confirmed these alternate forms of ßA2 and ßB2, since proteins of the predicted masses were found. Since the mass of ßB3 did not match the mass of the reported sequence, the cDNA of chicken ßB3 was reexamined. The additional sequence resulted in the substitution of 7 residues. The predicted mass of the new ßB3 sequence (24,361) then matched the measured mass. Conclusions: Similar to ßA3 and ßA1 in most species, chicken ßA2 is translated from two alternate in frame start codons. Two forms of ßB2 also exist in chicken, but result from slippage during excision of an intron, resulting in 14 extra amino acid residues within the chicken ßB2 connecting peptide. The additional sequences found in the alternate form of chicken ßB2 may significantly alter its oligomeric properties.

Keywords: crystallins • protein structure/function • proteomics 
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