Gene duplication and separation of functions in αB‐crystallin from zebrafish (Danio rerio)

Abstract

The a-crystallins are evolutionarily related members of the small heat shock protein (sHSP) superfamily which are taxonomically ubiquitous components of the vertebrate eye lens [1]. The aA-crystallin and aB-crystallin genes arose through a gene duplication event that occurred early in vertebrate history and are most closely related to sHsp20 [2]. Mammalian aA-crystallin is primarily lens specific and has lost the stress induction response that characterizes most sHsps, although some metals induce its expression [3]. In contrast, multiple cellular stresses induce mammalian aB-crystallin expression in a variety of tissues [4]. The mammalian a-crystallins act as chaperone-like molecules by binding to and preventing the aggregation of non-native We previously reported that zebrafish aB-crystallin is not constitutively expressed in nervous or muscular tissue and has reduced chaperone-like activity compared with its human ortholog. Here we characterize the tissue expression pattern and chaperone-like activity of a second zebrafish aBcrystallin. Expressed sequence tag analysis of adult zebrafish lens revealed the presence of a novel a-crystallin transcript designated cryab2 and the resulting protein aB2-crystallin. The deduced protein sequence was 58.2% and 50.3% identical with human aB-crystallin and zebrafish aB1-crystallin, respectively. RT-PCR showed that aB2-crystallin is expressed predominantly in lens but, reminiscent of mammalian aB-crystallin, also has lower constitutive expression in heart, brain, skeletal muscle and liver. The chaperone-like activity of purified recombinant aB2 protein was assayed by measuring its ability to prevent the chemically induced aggregation of a-lactalbumin and lysozyme. At 25°C and 30°C, zebrafish aB2 showed greater chaperone-like activity than human aB-crystallin, and at 35°C and 40°C, the human protein provided greater protection against aggregation. 2D gel electrophoresis indicated that aB2-crystallin makes up 0.16% of total zebrafish lens protein. Zebrafish is the first species known to express two different aB-crystallins. Differences in primary structure, expression and chaperone-like activity suggest that the two zebrafish aB-crystallins perform divergent physiological roles. After gene duplication, zebrafish aB2 maintained the widespread protective role also found in mammalian aB-crystallin, while zebrafish aB1 adopted a more restricted, nonchaperone role in the lens. Gene duplication may have allowed these functions to separate, providing a unique model for studying structure-function relationships and the regulation of tissue-specific expression patterns.Abbreviations sHSP, small heat shock protein.

αA-crystallin expression prevents γ-crystallin insolubility and cataract formation in the zebrafishclochemutant lens

“…We also isolated a second ␣B-crystallin gene, which we called ␣Bb-crystallin (cryabb; GenBank Accession Number, AY939876). This gene is identical to ␣B2-cystallin (GenBank Accession Number; DQ113417) (Smith et al, 2006) and similar to a previously reported ␣B1-crystallin (GenBank Accession Number, NM 131157) (Posner et al, 1999).…”

“…The membranes were incubated with antimouse ␣A-crystallin antisera (1/2000 dilution) or anti-newt ␥-crystallin antisera (1:2000 dilution) followed by HRP-conjugated anti-guinea pig IgG (Jackson ImmunoResearch Laboratories, 1:5000) or anti-rabbit IgG (Amersham Pharmacia Biotech; 1: 10,000), and visualization with enhanced chemiluminescence (Perkin Elmer Life Science). The specificity of each antibody was confirmed by western blotting of lysates prepared by transiently transfecting Chinese Hamster Ovarian cells (American Type Culture Collection) with expression constructs of zebrafish ␣A- (Runkle et al, 2002) (GenBank Accession Number, NM 152950), ␣B1- (Posner et al, 1999;Smith et al, 2006) (GenBank Accession Number, NM 131157), ␤B1- (Chen et al, 2001) (GenBank Accession Number, NM 173231) and ␥-crystallin genes. Cell maintenance and gene transfection procedures were as described previously (Goishi et al, 2003).…”

“…␣ ␣A-crystallin gene expression is down-regulated in cloche There are several classes of crystallins, including ␣-, ␤-and ␥-crystallin, and each class has several different genes (Chen et al, 2001;Posner et al, 1999;Runkle et al, 2002;Vihtelic et al, 2005a;Wistow et al, 2005;Smith et al, 2006). The expression levels of ␣A-, ␣Bb-, ␤B1-and ␥-crystallin were analyzed by semi-quantitative RT-PCR ( Fig.…”

Lengsin expression and function during zebrafish lens formation

“…In adults, αB1-crystallin transcripts were largely restricted to the lens tissue, while αB2-crystallin transcription was identified in brain, heart, liver and skeletal muscle, in addition to the lens (Posner et al, 1999;Smith et al, 2006). This suggests the expression patterns of the zebrafish αB-crystallins may change between development and the adult, although more detailed analysis of the gene and protein expression patterns will be necessary to fully examine this possibility.…”

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“…sHsps have been identified in the zebrafish, ten of which are likely orthologs of human sHsps, and each of which corresponds to one of the thirteen teleost sHSPs (Franck et al, 2004). Through searching all available expressed gene and genomic sequence databases, seven additional zebrafish sHsps exist (Hspb1, Hspb2, Hspb3, Hspb4, Hspb5a, Hspb5b, and Hspb12) (Posner et al, 1999;Franck et al, 2004;Mao et al, 2005;Smith et al, 2006). The zebrafish protein is 57% similar to human Hsp27, 56% similar to mouse Hsp25 and 64% similar to an Hsp27 protein cloned from the desert topminnow, Poeciliopsis lucida (Norris et al, 1997).…”