|Molecular Vision 2002;
Received 23 January 2002 | Accepted 7 March 2002 | Published 11 March 2002
Sequence and spatial expression of zebrafish (Danio rerio) aA-crystallin
Stephanie Runkle,1 Julie Hill,1
1Department of Biology, Ashland University, Ashland, OH; 2Department of Biology, West Virginia University, Morgantown, WV; 3Jules Stein Eye Institute, UCLA School of Medicine, Los Angeles, CA
Correspondence to: Mason Posner, Department of Biology, Ashland University, 401 College Avenue, Ashland, OH, 44805; Phone: (419) 289-5691; FAX: (419) 289-5283; email: firstname.lastname@example.org
Purpose: To determine the nucleotide sequence, amino acid sequence and tissue specificity of zebrafish aA-crystallin.
Methods: RACE, both 3' and 5', was used to clone the zebrafish aA-crystallin gene. The peptide sequence of the encoded protein was deduced and compared to cavefish, shark, amphibian, bird and human orthologues using the CLUSTAL W algorithm. aA-crystallin transcript was evaluated in brain, heart, lens, liver, skeletal muscle/skin, and spleen by semi-quantitative RT-PCR.
Results: The 173 amino acid sequence of zebrafish aA-crystallin was determined to be 73% and 86% similar to its human and cavefish orthologues, respectively. We detected high expression of zebrafish aA-crystallin in the lens and very low expression in liver and spleen.
Conclusions: Few amino acids identified as being functionally important to chaperone function differ between zebrafish and mammalian aA-crystallin. The expression of aA-crystallin is mainly confined to the lens in both taxa. These data suggest that zebrafish aA-crystallin plays a physiologically limited role outside of the zebrafish lens, similar to its mammalian orthologues.
a-Crystallin is a member of the small heat shock protein family that can account for 30% of the protein content of the vertebrate ocular lens [1,2]. While this high protein concentration contributes to the optical properties of the lens, a-crystallin also plays a non-refractive role as a chaperone. The protein's two subunits, aA- and aB-crystallin, both act as molecular chaperones by preventing the stress induced aggregation of denatured proteins in vitro [3-5]. By protecting lens crystallins, metabolic enzymes and cytoskeletal proteins, a-crystallin may play a role in the prevention of cataracts. In the lens, aA- and aB-crystallin combine to form large heterogeneous multimers of approximately 800 kDa . Outside of the lens, the two subunits are found as separate homogenous aggregates. In mammals, extralenticular expression of aA-crystallin is restricted to low levels in the spleen and thymus, while aB-crystallin is expressed in numerous non-lenticular tissues including skeletal and heart muscle, nervous tissue, skin and lung [7,8]. Only aB-crystallin is stress inducible in mammals and accumulates in association with several neurological diseases such as Parkinson's, Alexander's, Creuztfeld-Jacob's and Multiple Sclerosis [9-11]. aB-crystallin expression is also upregulated in diseased human hearts .
Studies indicating that aA- and aB-crystallin have different biochemical and biophysical properties support the hypothesis that these proteins perform different physiological roles. Datta and Rao  showed that bovine aB-crystallin has greater chaperone activity at physiological temperatures than aA-crystallin, but that aA-crystallin chaperone function increases with increasing temperature. Reddy et al.  found that as temperature increases, structural changes expose more hydrophobic residues on aA-crystallin than are found on aB-crystallin. Additionally, Liang et al.  found that human recombinant aA-crystallin is more resistant than aB-crystallin to heat induced conformational changes, and Wang et al.  demonstrated that each subunit shows a different phosphorylation pattern under stress conditions. Collectively, these studies support the conclusion that aA- and aB-crystallin have different biophysical properties and physiological functions. To understand the physiological role of a-crystallin it is important to examine the structure and function of both subunits independently.
Many investigators have attempted to identify the functionally important regions of mammalian a-crystallin using point-mutations, NMR spectroscopy and naturally occurring mutations in human populations. Smulders et al.  showed that the flexible C-terminal region of a-crystallin plays an important role in chaperone function. They used NMR spectroscopy to demonstrate that immobilization of this region in bovine aA-crystallin reduced chaperone activity. Other studies have indicated that a-crystallin is resilient to point mutations . Three substitution mutants in a highly conserved region of aB-crystallin produced no significant changes in chaperone function . However, a naturally occurring mutation at residue R120 of human aB-crystallin destroys chaperone activity, leads to cataracts and produces a desmin related myopathy . This last study demonstrates the benefits of using naturally occurring variations to study structure/function relationships in a-crystallin. Few studies, however, have taken advantage of the naturally occurring variations between different vertebrate taxa to analyze a-crystallin function. de Jong et al.  compared a-crystallin and small heat shock protein sequences from a large number of taxa to identify conserved, functionally important regions in these proteins. Yet few others have used this comparative approach. A more focused analysis of a-crystallin variation between different vertebrate taxa can help to identify functionally important residues.
Comparison of differential expression patterns between vertebrate taxa can shed light on the physiological roles of both a-crystallin subunits. However, virtually no data exist on the tissue specific expression of a-crystallin in non-mammalian vertebrates. We recently used a comparative approach to find putatively important amino acid differences between mammalian and zebrafish aB-crystallin . The zebrafish protein lacks two phosphorylation sites shown to be functionally important in other vertebrates, and it lacks four residues at the C-terminal end known to play a role in protein-protein interactions. Zebrafish aB-crystallin expression is more restricted to the lens compared to mammals. These data suggest that zebrafish aB-crystallin plays a more limited physiological role outside of the lens than its mammalian orthologues.
These observed differences between zebrafish and mammalian aB-crystallin structure and expression led us to investigate the zebrafish aA-crystallin subunit. Amino acid sequences for aA-crystallin have been reported from mammals , birds , amphibians , sharks  and one bony fish (Astyanax fasciatus) . Here we report the nucleotide and amino acid sequence and expression pattern for a second bony fish, the zebrafish, Danio rerio. We found that the zebrafish protein contains many of the amino acid residues thought to be functionally important in the mammalian protein. Furthermore, its expression is similar to that in mammals, except for possibly higher expression in the liver. These data suggest that zebrafish aA-crystallin plays a physiologically limited role outside of the zebrafish lens, similar to its mammalian orthologues.
Cloning and sequencing
Total RNA was collected from pet-store purchased adult zebrafish using the RNEasy kit (Qiagen, Valencia, CA) and used as template in 3' and 5' rapid amplification of cDNA ends (RACE) procedures . A partial zebrafish aA-crystallin sequence from Genbank (AI626170) containing 423 basepairs was used to design gene specific primers for 3' and 5' RACE. The 3'-RACE system for Rapid Amplification of cDNA ends (Gibco-BRL, Gaithersburg, MD) was used with 2 mg of total lens RNA and gene specific primer DR 12 (5'-CCAGGGCAAGCATGGAGAAA-3'). The 5' RACE system for Rapid Amplification of cDNA ends version 2.0 (Gibco-BRL) was used with 2 mg of total lens RNA and gene specific primer DR 18 (5'-GAAAGAATAAAGGGCCAGAT-3'). Two nested amplifications were then performed with primers DR 15 (5'-GCCCCACTCACACCTCCATA-3') and DR 11 (5'-ACGGGAGATGTAGCCATGAT-3'). All RACE amplifications were for 35 cycles with the following parameters: 94 °C for 1 min, 55 °C for 1 min and 74 °C for 2 min. Products from both RACE procedures were gel purified and inserted into the TOPO TA vector (Invitrogen, Carlsbad, CA), which was used to transform TOP10 bacteria (Invitrogen). At least five clones spanning the coding region were sequenced using a commercial nucleotide sequencing service (Cleveland Genomics, Cleveland, OH). The amino acid sequence for the complete zebrafish aA-crystallin cDNA was deduced from the overlapping RACE products using Biology Workbench and aligned with orthologues from other vertebrate groups using the CLUSTAL W algorithm .
Spatial expression of zebrafish aA-crystallin
Total RNA was collected from adult zebrafish brain, heart, lens, liver, skeletal muscle/skin and spleen as described above. Total RNA (150 ng) from each tissue was subjected to reverse-transcription polymerase chain reaction (RT-PCR) using the Access kit (Promega, Madison, WI). Each sample was reverse transcribed for 45 min at 48 °C, and then amplified for 25 cycles with primers DR 10 (5'-AGGAGTTACCAGGTCTGACA-3') and DR 11 (5'-ACGGGAGATGTAGCCATGAT-3') using the following parameters: 94 °C for 1 min, 55 °C for 1 min and 74 °C for 2 min. To avoid amplification of contaminating genomic DNA, primers were designed to cross intron/exon boundaries as inferred from the genomic aA-crystallin sequence of Astayanax fasciatus (Y11301). Control reactions without reverse-transcriptase were also performed and lens RNA was amplified for a range of cycles to ensure that these experiments where conducted within the linear range of amplification. Parallel reactions using tubulin specific primers were performed to confirm that mRNA from each tissue was not degraded. The tubulin reactions were performed for 35 cycles using the same parameters as above.
To detect possible low levels of aA-crystallin expression, 450 ng of total zebrafish RNA from brain, heart, lens, liver, skeletal muscle/skin and spleen was reverse transcribed and amplified for 40 cycles using the same cycling parameters as above using primers DR 12 and DR 15 (sequences described above). This primer pair spanned intron/exon boundaries. Negative control reactions were also performed without reverse transcriptase to ensure that any products resulted from amplification of mRNA.
Cloning and sequencing
RACE (3' and 5') was used to obtain the aA-crystallin mRNA sequence as described in the Methods. An amplification product obtained by 3' RACE was sequenced and found to contain regions identical to the partial zebrafish aA-crystallin sequence in Genbank (AI626170). An amplification product produced by 5' RACE was sequenced and found to be identical to the partial zebrafish aA-crystallin sequence. The 5' end of this product allowed identification of the beginning of the 5' untranslated region. The 3' and 5' RACE products overlapped and were used to reconstruct a mRNA sequence of 730 basepairs (Figure 1). Only one position (171) varied between the nine clones analyzed. Six clones and the partial Genbank sequence contained a guanine, while three clones contained an adenine at this position. This variation would change the resulting amino acid from a glycine to a glutamic acid. Glycine is likely to be encoded since it is found in six of the nine clones, the previously reported Genbank sequence and because it is conserved at this position in other known vertebrate taxa.
Translation of the nucleotide sequence indicated a protein of 173 amino acids. A BLASTP search (National Library of Medicine, Bethesda, MD) found this zebrafish protein to be most like aA-crystallin orthologues from other vertebrate taxa. Sequence alignment identified high similarity between the zebrafish protein and orthologues from several vertebrate groups (Figure 2). The zebrafish protein was 86% identical to that from the bony fish Astyanax fasciatus and 73% identical to the human protein. Several comparisons between the vertebrate aA-crystallins are worth noting. First, the two bony fish sequences contain a two amino acid deletion after position 15, a three amino acid insertion at positions 58-60, and a one amino acid deletion after position 147 compared to all other vertebrate sequences. Second, the zebrafish protein contains an arginine at position 117 (116 in humans) that has been shown to be important to chaperone function in human aA-crystallin . Third, the C-terminal extension in zebrafish is similar to other vertebrate taxa except the dogfish shark (Squalus acanthias), which has an unusually elongated C-terminal end. Fourth, the zebrafish protein contains a serine at position 123 (122 in mammals) that is phosphorylated in the bovine and human lens , but lacks a threonine at position 4 involved in stress induced phosphorylation in the rat . And fifth, one of the few residues found to reduce chaperone function when substituted in human a-crystallin, the aspartic acid at position 69 , is substituted by glutamic acid at position 70 of the zebrafish protein. Aspartic acid is found in the only other known bony fish sequence, and in all other vertebrate taxa.
Spatial expression of zebrafish aA-crystallin
Levels of aA-crystallin expression were examined in total RNA isolated from zebrafish brain, heart, lens, liver, skeletal muscle/skin and spleen using RT-PCR as described in the Methods. aA-crystallin was only detected in the lens after 25 amplification cycles (Figure 3A). Control reactions indicated that 25 cycles was within the linear range of amplification for lens RNA (data not shown). Parallel reactions using tubulin specific primers as an internal control showed that the mRNA from each tissue was not degraded (Figure 3B).
To detect possible extralenticular expression of aA-crystallin, higher amounts of total RNA (3 times the amount used for the initial quantitation) from the same tissues was subjected to RT-PCR for 40 cycles. These reactions produced a strong aA-crystallin band from lens RNA, fainter bands for liver and spleen, and a very faint band for heart (Figure 4A). A different total RNA preparation produced an additional very faint band from brain (Figure 4B). aA-crystallin products from lens, liver and spleen were sequenced to confirm their identity. The brain, heart, liver and spleen reactions also produced non-specific bands. The non-specific brain and heart bands were consistently stronger than the aA-crystallin product from the same tissues, while in liver and spleen the non-specific band was sometimes stronger than, and sometimes equal to the aA-crystallin product. Two amplification products were isolated from the non-specific liver band and sequenced. Neither showed similarity to either aA-crystallin or any other identified gene.
Comparison of the zebrafish aA-crystallin protein sequence with its mammalian orthologues indicates few variations in functionally important amino acid residues. While there are several insertions and deletions that distinguish the zebrafish and cavefish sequences from those of other vertebrate classes, none of these differences involve residues implicated in chaperone function. One residue that does differ is the aspartic acid that is found at position 69 in the human protein, which Smulders et al.  identified as functionally important using site directed mutagenesis. In the zebrafish this amino acid has been replaced by glutamic acid at position 70. It is interesting to note that the only other known bony fish aA-crystallin sequence, that for the cavefish, contains aspartic acid at this site, as do all other vertebrate taxa. It is questionable whether the substitution of aspartic acid with another charged amino acid in the zebrafish would cause a change in chaperone function. In fact, substitutions between aspartic acid and glutamic acid are common in vertebrate aA-crystallins (see zebrafish positions 31, 84, 156 and 165 in Figure 2). The only other known functionally important residue that differs in the zebrafish sequence is the threonine at position 4 that undergoes stress-induced phosphorylation in the rat . Because the role of phosphorylation in a-crystallin chaperone ability is unclear, it is difficult to conclude whether this substitution has an effect on function.
The minimal variation in functionally important residues between zebrafish and mammalian aA-crystallin may reflect similarities in their chaperone function. Alternatively, this minimal variation in functionally important residues could be due to our inadequate knowledge of the amino acids important to chaperone function. This latter explanation is more likely considering the vast evolutionary distance between bony fishes and mammals. The differing physiological demands of these two groups, and the differing biophysical environments of their lenses, makes it unlikely that their aA-crystallins would exhibit identical chaperone function. For example, it is well established that aA-crystallin chaperone function changes with temperature [13,14]. The difference in physiological temperature between the ectothermic zebrafish (25-31 °C) and endothermic mammals (37 °C) is likely to produce different selective pressures on the thermal sensitivity of aA-crystallin chaperone function. This selective pressure would be predicted to promote evolutionary changes in both the structure and function of the protein. Therefore, there may be functionally important structural differences between the zebrafish and mammalian proteins yet to be identified.
The alternatively spliced exon 2 that is expressed to form the aAins-crystallin of some mammalian species is not expressed in the zebrafish. van Dijk et al.  recently showed that this alternative exon is present in the genomes of many mammals that do not express the aAins-crystallin form. Because we cloned zebrafish aA-crystallin from its mRNA, we are not able to comment on the presence or absence of this alternate exon in the zebrafish genome. However, Behrens et al.  did not identify the alternate exon 2 in their genomic sequence for the bony fish Astyanax fasciatus.
The very low extralenticular expression of zebrafish aA-crystallin reported here is similar to the pattern seen for mammalian aA-crystallin. As with the mammalian protein, this limited expression suggests that zebrafish aA-crystallin does not play a widespread physiological role throughout the body. RT-PCR analysis did identify extralenticular aA-crystallin expression in brain, heart, liver and spleen. However, when separated on an agarose gel the aA-crystallin products from brain and heart were less intense than a non-specific band produced with the same amplification primers. Zebrafish liver and spleen samples produced aA-crystallin products equal to or lesser in intensity than a non-specific band. The amplification products from the non-specific liver band showed no similarity to aA-crystallin, indicating that at least in liver these non-specific products were not amplified from aA-crystallin itself.
Considering the large number of amplification cycles used (40) and the intensity of co-occurring non-specific products, expression appears to be extremely low in these extralenticular tissues. The appearance of these light bands could be an artifact related to the background expression of aA-crystallin that could be expected in all tissues. It is important to note that we have only examined the constitutive expression of zebrafish aA-crystallin. While mammalian aA-crystallin expression is not stress induced, no study has investigated the effect of stress on expression in the zebrafish. Furthermore, possible changes in aA-crystallin expression during development have not been examined.
We previously found similarly low extralenticular expression for zebrafish aB-crystallin . Our current data support the conclusion that a-crystallin plays a limited physiological role outside of the lens in the adult zebrafish. Variation in primary structure and possible differences in function between the zebrafish and mammalian proteins can be used in future studies of a-crystallin structure/function relationship. Natural variations between vertebrate a-crystallins will be a useful tool for understanding the function and physiological role of this important stress protein.
We would like to thank Scott Shors for helpful comments during this research. This research has been funded by a grant from the National Eye Institute to M.P. (EY13535-01).
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