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Invest Ophthalmol Vis Sci 2005;46: E-Abstract 3870.
© 2005 ARVO


3870—B228

A Second Alpha B Crystallin From the Zebrafish (Danio rerio)

M. Posner1, A. Smith1, T.S. Vihtelic2 and G.J. Wistow3

1 Dept Biology, Ashland University, Ashland, OH
2 Biological Sciences, University of Notre Dame, Notre Dame, IN
3 Section on Molecular Structure and Functional Genomics, National Eye Institute, Bethesda, MD

Commercial Relationships: M. Posner, None; A. Smith, None; T.S. Vihtelic, None; G.J. Wistow, None.

Support: NIH Grant EY13535

Abstract

Purpose: We previously reported that zebrafish {alpha}B–crystallin is not constitutively expressed in nervous or muscular tissue. It also has reduced chaperone–like activity compared to its human ortholog. Here we report that zebrafish contains a second {alpha}B–crystallin that differs from the first in both expression pattern and chaperone activity.

Methods: Messenger RNA from zebrafish lens was used to construct a cDNA library for the NEIBank project. Almost 4000 clones were sequenced and analyzed. A novel {alpha}B–crystallin (z{alpha}B2) was identified and the complete sequence was obtained. Reverse–transcriptase polymerase chain reaction (RT–PCR) using primers specific to z{alpha}B2 was used to determine its tissue specific expression. Recombinant z{alpha}B2 was expressed in E. coli and purified using ion exchange and size exclusion chromatography. The chaperone–like activity of the purified protein was assayed by measuring its ability to prevent the chemically induced aggregation of lactalbumin at temperatures between 25 and 40 degrees Celsius.

Results: The complete z{alpha}B2 cDNA is 1,953bp in length and encodes a protein sequence of 165 amino acids that is 58.2% and 50.3% identical to human {alpha}B–crystallin and zebrafish {alpha}B1–crystallin, respectively. Semi–quantitative RT–PCR indicated expression in lens, heart, brain, skeletal muscle and liver. Expression was highest in lens and lowest in the liver. At 25 and 30 degrees Celsius z{alpha}B2 demonstrated greater chaperone–like activity than human {alpha}B–crystallin. At 35 and 40 degrees Celsius the human protein provided greater protection against aggregation.

Conclusions: The zebrafish is now the first species known to express two different copies of {alpha}B–crystallin. The low level of identity between the two zebrafish {alpha}B–crystallins (50.3%) suggests that these two proteins are under different selection pressures. Divergent physiological roles for these two proteins would explain the differences in both expression and chaperone–like activity. It is possible that gene duplication has allowed a separation of functions, with z{alpha}B2 maintaining the widespread chaperone–like role of mammalian {alpha}B–crystallin, while zebrafish {alpha}B1–crystallin has acquired a more specialized function in the lens. The presence of two zebrafish {alpha}B–crystallins differing in expression and chaperone–like activity provides a unique model for studying the mechanisms behind {alpha}–crystallin function.

Keywords: crystallins • chaperones

 © 2005, The Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any part of this abstract, contact the ARVO Office at arvo{at}arvo.org.





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