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A Comparative View of Alpha Crystallins: The contribution of comparative studies to understanding function1
1 Department of Biology, Ashland University, Ashland, Ohio 44805
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Integration between comparative biology and cellular/molecular biology has helped advance understanding of the structure, function and physiology of the vertebrate small heat shock proteins
A- and B-crystallin. These proteins are expressed at high concentration in the eye lens where they contribute to transparency and refractive power. But they also function similarly to molecular chaperones by preventing the aggregation of denatured proteins that can cause opacities, or cataracts. -crystallins also serve a number of other roles in and out of the lens that are still not completely understood. Comparative examination of -crystallins and closely related small heat shock proteins from diverse taxa has helped provide insights into the proteins' three-dimensional shape and structure/function relationships. Until recently, no studies had examined the tissue specific expression or chaperone-like activity of -crystallins from a non-mammalian vertebrate. I have been investigating the -crystallins of the zebrafish, Danio rerio, as a first step towards utilizing the bony fishes as a model group for understanding the evolution of -crystallin function. Zebrafish A-crystallin displays similar structure and expression and increased chaperone-like activity compared to its human orthologue. Zebrafish B-crystallin, however, has a truncated C-terminal extension, more limited expression and lower chaperone-like activity than its human orthologue. These data suggest that A-crystallin physiological function may be conserved between zebrafish and mammals, while B-crystallin physiological function has diverged. Understanding zebrafish -crystallin physiology is necessary before this species can be used for developmental and genetic studies, and provides a foundation for further comparative studies. |
INTRODUCTION |
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Comparative biology and cellular/molecular biology have often been cast as mutually exclusive fields. By concentrating on a small number of model organisms, the cellular/molecular field has often not taken advantage of the insights available from comparative studies. Likewise, comparative biologists have not taken advantage of the powerful techniques and expertise developed by cellular/molecular labs. Fortunately, this situation is changing. Over the past decade the comparative community has widely adopted the molecular techniques that at first seemed inappropriate for their field. And the cellular/molecular community has realized the power of examining questions in an evolutionary context. Continuing integration of ideas and techniques will benefit both communities.
Investigations into the small heat shock proteins A- and B-crystallin highlight the benefits of this integrative approach. These proteins have received intense study because of their possible role in preventing cataracts, one of the leading causes of human blindness. The majority of this research has involved the usual group of mostly mammalian model species. However, a smaller number of comparative studies has contributed greatly to our understanding of the -crystallins' structure, function and physiology. In this review I hope to detail an example of how comparative studies can beneficially augment typical cellular/molecular approaches. I will also show how molecular tools can allow comparative biologists to ask novel questions about the evolution of physiology.
Before the 1980s the -crystallins appeared to be simply structural proteins of the vertebrate ocular lens. But then Ingolia and Craig (1982)
found that the -crystallins shared a highly conserved stretch of 76 amino acids with four small heat shock proteins from Drosophila melanogaster. This conserved region, known as the -crystallin domain, is the signature feature of the small heat shock protein family, which has members in all domains of life. A decade later, Horwitz (1992) demonstrated that -crystallins function like a molecular chaperone by protecting denatured proteins from aggregation. Unfolded proteins expose hydrophobic regions that can lead to irreversible aggregation that disrupts cellular homeostasis. Molecular chaperones bind to these unfolded proteins and either aid in folding or release them to another chaperone that aids in folding. This chaperone function is vital when proteins are first made or when they denature under stressful conditions. Because -crystallins do not refold or release denatured proteins, they are usually referred to as being chaperone-like. The realization that -crystallins had chaperone-like functions opened the door to the past decade of studies into their functional role both in and outside the lens (see for review: Horwitz, 2000, 2003; Van Montfort et al., 2001). -crystallins are no longer considered passive proteins simply refracting light. They are now known to play a dynamic role in vertebrate physiology. The evolutionary perspective provided by comparative studies has played an important role in detailing this role.
Why is there a molecular chaperone in the lens?
The vertebrate lens refracts light by accumulating densely packed proteins called crystallins, which can make up to 90% of the dry weight of the lens (Bloemendal, 1981). A number of diverse crystallin families are found in the vertebrate lens. Like the -crystallins, some are found in almost all vertebrate taxa and are termed the ubiquitous crystallins. Others are more restricted in their distribution and are termed taxon-specific crystallins (see for review: Piatigorsky, 1992). Many of the lens crystallins serve other roles in addition to contributing to the refraction of light. For example, 
-crystallin of the bird lens is encoded by the same gene for lactate dehydrogenase and retains its enzymatic function (Wistow and Piatigorsky, 1987; Hendriks et al., 1988). Most of the other taxon-specific crystallins are also identical to or closely related to functioning enzymes. Crystallins are a good example of gene sharing, in which a single gene product is used for two or more functions. While not a new concept, the idea of gene sharing has recently gained more attention with the realization from the human genome project that many genes can produce a number of proteins with varied roles. The discovery that -crystallin was a small heat shock protein with chaperone-like activity provided another interesting example of gene sharing.
The possible need for a molecular chaperone in the lens can be explained by considering lens development. The lens continues to increase in diameter throughout life, even in vertebrates with determinate growth. As it grows, the lens must maintain its transparency and refractive properties, while also countering optical problems such as spherical and chromatic aberration. Increase in lens size results from the production of new cells within an epithelial layer lining the anterior edge of the lens in mammals, or along the entire outer surface in fishes. As these cells move to the edges of the epithelium they express large amounts of crystallins and differentiate into elongated lens fiber cells, which form a series of concentric layers within the lens. During fiber cell differentiation the organelles and nuclei degrade and eventually disappear. Fiber cells, therefore, lose the ability to replace both aging crystallins and the many housekeeping enzymes normally needed to maintain cellular metabolism. The result is lens fiber cells filled with aging proteins, with the center of the lens containing proteins produced before birth. As these proteins grow older, they are likely to denature, expose hydrophobic residues, and aggregate. These aggregates disrupt cellular homeostasis, and interfere with the orderly arrangement of proteins needed for transparency and the proper refraction of light. The end result is opacity in the lens, or cataract.
The co-option of a protein with chaperone-like activity into the lens makes sense in light of this lack of protein turnover. The -crystallins apparently perform at least two functions in the lens. First, they are structural proteins that contribute to the refractive powers of the lens. But they also serve as a trashcan for aging proteins, preventing their aggregation. This chaperone-like activity is apparent in vitro, but studies also suggest that -crystallins perform this role in vivo as well (Boyle and Takemoto, 1994; Rao et al., 1995).
The two alpha crystallins
A- and B-crystallin occur in the vertebrate lens as a heteroaggregate of variable size, ranging from 300,000 to over 1,000,000 daltons. Each monomer is approximately 20,000 daltons in size, meaning that the aggregates are composed of 15 to over 50 subunits. The proportion of A- and B- varies in each aggregate, but the molar ratio of the entire mammalian lens is typically 3:1 A- to B-crystallin. The two genes that encode for the -crystallins are the product of a gene duplication event that must have occurred very early in vertebrate evolution (de Jong et al., 1998). A recent search of the human genome found a total of ten small heat shock protein genes that have apparently evolved from a single common ancestral gene (Kappe et al., 2003). While not all of these genes have been identified in other vertebrate classes, many of them can be expected to occur. The great age of the duplication events that led to this cluster of genes make it difficult to develop a well-supported gene phylogeny for the entire family. However, there is good support for a close relationship between the two -crystallins and Hsp20 (Kappe et al., 2003).
Mammalian A-crystallin is expressed constitutively in significant amounts only in the lens. Much smaller amounts have been found in the rat spleen and thymus (Kato et al., 1991). But mammalian B-crystallin is expressed outside of the lens in many tissues including heart, spinal cord, brain, skeletal muscle and kidney (Bhat and Nagineni, 1989; Dubin et al., 1989). In mammals, B-crystallin expression is upregulated by various stresses (Klemenz et al., 1991), and its levels increase in numerous neurological diseases such as Alzheimer's (Renkawek et al., 1994) and Creutzfeldt-Jacob (Renkawek et al., 1992). A-crystallin expression was not thought to be stress induced, but Hawse et al. (2003) recently showed that human lens cell lines upregulate A-crystallin when exposed to metals. The difference in transcriptional control and subsequent expression of the two -crystallins suggests that they play divergent roles in mammalian physiology. Lack of studies on -crystallin expression in non-mammalian vertebrates makes generalizations about all vertebrates difficult.
The expanding roles of -crystallins
The -crystallins' role within the lens may go beyond protecting aging proteins from aggregation. Boyle and Takemoto (2000) have shown that -crystallins are involved in lens fiber cell differentiation. They induced cultured primary epithelial cells from adult bovine lenses to differentiate into fiber cells by loading them with -crystallins. Bhat et al. (1999) found -crystallins localized in the nucleus of cultured cells, suggesting that they may play some role in nuclear signaling. Cobb and Petrash (2000; 2002) have investigated the interaction of -crystallins with plasma membranes. The function, if any, for this interaction is unclear. And numerous studies have found that -crystallins associate with cytoskeletal proteins, possibly playing a role in cellular reorganization (Muchowski et al., 1999; Quinlan, 2002).
The importance of the -crystallins' chaperone-like activity is not restricted to the lens. As mentioned above, numerous neurological disorders involve overexpression of B-crystallin. Mutations in human B-crystallin can also lead to abnormal aggregates of the cytoskeletal protein desmin in cardiac tissue (Der Perng et al., 1999). Abnormal muscle development occurs in B-crystallin knockout mice (Brady et al., 2001), and B-crystallin has been shown to prevent apoptosis of retinal pigment epithelial cells during heat shock (Alge et al., 2002). We are clearly just beginning to understand the roles of -crystallins both within the lens and throughout the vertebrate body.
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COMPARATIVE STUDIES OF ALPHA CRYSTALLIN |
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Sequence comparisons
Early comparative studies by de Jong were instrumental in demonstrating the homology between
-crystallins of diverse vertebrate taxa and characterizing the structural and functional domains of the proteins. The work of de Jong et al. (1976) was the first to compare the amino acid sequences of vertebrate -crystallins. In it they detailed partial primary structures from six classes of vertebrates, including mammals, birds, reptiles, bony and cartilaginous fishes and a lamprey. Later de Jong et al. (1984) reported complete A-crystallin primary structures from 28 mammalian species and from chicken and frog. This was the first effort to determine the structural changes that have occurred during -crystallin evolution. These primary structures were also used to develop early molecular phylogenies for mammals (de Jong et al., 1981). Over the years -crystallin sequences have played an important role in vertebrate systematics (Van Dijk et al., 1999; Poux et al., 2002).
The advent of the polymerase chain reaction in the 1990s allowed de Jong and his colleagues to more readily accumulate diverse -crystallin sequences and compare them to each other and to their closely related small heat shock protein paralogs (see for review: de Jong et al., 1998). These sequence comparisons helped to identify three functionally important regions in the small heat shock protein family. Foremost is the highly conserved -crystallin domain, a region composed of 80–100 residues towards the protein's C-terminal end. This region is thought to directly interact with denaturing proteins to prevent aggregation. Small heat shock proteins also contain a highly variable N-terminal region and a short, flexible C-terminal extension. The C-terminal extension usually ends in two charged or polar amino acids, and is thought to maintain the solubility of -crystallin complexes after they are bound to denatured protein. Truncation of the C-terminal extension, or replacement of the charged amino acids with hydrophobic residues, reduces chaperone activity (Smulders et al., 1996).
Insights into structure from other small heat shock proteins
The high degree of evolutionary conservation of the -crystallin domain suggests that this region plays an important role in the structure and function of small heat shock proteins. Kokke et al. (2001) have taken advantage of the particularly small Hsp12.2 from the roundworm Caenorhabditis elegans to investigate the role of this domain in chaperone function. Caenorhabditis elegans Hsp12.2 is reduced to an -crystallin domain, with an extremely small N-terminal region and C-terminal extension. Like most other small heat shock proteins, Hsp12.2 forms oligomers, but it does not have chaperone-like activity. Kokke et al. (2001) found that adding the N-terminus and C-terminal extension from human B-crystallin did not restore chaperone-like activity to Hsp12.2. They suggested two explanations for this result. First, the -crystallin domain of Hsp12.2 may be inherently unfit for chaperone activity. But secondly, interactions between the -crystallin domain and the other two regions may be important for chaperone-like activity. In this more holistic view, small heat shock proteins are not modular in function. Although the -crystallin domain may interact with denaturing proteins, proper synergism between the different regions is also necessary for chaperone-like activity.
A small heat shock protein from the thermophilic archaebacterium Methanococcus janaschii has provided important information on -crystallin structure. Determining the three-dimensional structure of any small heat shock protein has been elusive. Although many labs have tried, it has not been possible to perform x-ray crystallography on -crystallins. Alternate approaches such as circular dichroism, spin-labeling and cryo-electron microscopy have provided clues to the proteins' structure (Horwitz, 2003). But Hsp16.5 from M. janaschii has been crystallized and provides some of the best insights into small heat shock protein tertiary and quaternary structure. Unlike the -crystallins, which form aggregates of various sizes, Hsp16.5 from M. janaschii always forms a complex of 24 monomers. Kim et al. (1998) found that the -crystallin domain of each of these monomers is composed of eight ß sheets that fold onto each other to form a ß sandwich fold. This tertiary structural conformation seems to be conserved throughout the small heat shock protein family. Crystallographic data and cryo-electron microscopy both indicate that the 24 monomers of M. janaschii Hsp16.5 come together to form a symmetrical, hollow sphere (Kim et al., 1998; Haley et al., 2000). -crystallins also form a hollow sphere, but unlike Hsp16.5, these spheres are not symmetrical (Haley et al., 1998, 2000). The varied size and asymmetrical nature of -crystallin aggregates may be important to their chaperone-like function. A better understanding of -crystallin quaternary structure will have to await more refined cryo-electron microscopy and other techniques. A second crystal structure, from wheat Hsp16.9, showed that this protein forms dodecamers composed of 2 disks each containing 6 monomeric units (van Montfort et al., 2001). Although this quaternary structure differs from both the -crystallins and M. janaschii Hsp16.5, the presence of exposed hydrophobic regions along the -crystallin domain does suggest a common mechanism for the chaperone-like activity of all of these proteins.
Studies on blind species
Studies on species with regressed eyes have been used to suggest that A-crystallin may play a functional role outside of the differentiated adult lens. The blind mole rat, Spalax ehrenbergi, has been a focus of several such studies. This species lives in a subterranean habitat and terminates lens development at an early embryonic stage. If A-crystallin only functions in the adult lens, the reduced selection pressures on the protein should have led to divergence in structure. Hendriks et al. (1987) found that the mole rat A-crystallin amino acid sequence has indeed diverged from closely related species. However, the rate of divergence is less than would be expected for a pseudogene with no function, suggesting that there is still some selective pressure on protein structure. Smulders et al. (2002) showed that the tertiary structure of mole rat A-crystallin is similar to that of other mammals. The mole rat protein also retains chaperone-like activity, but at a slightly lower effectiveness than rat A-crystallin. The authors attribute this reduced chaperone-like activity to a less flexible C-terminal extension, which is known to be important in maintaining solubility of chaperone/protein aggregations. The conservation of normal structure and maintenance of some chaperone-like activity suggest that mole rat A-crystallin still plays some functional role, even though adults of this species lack a transparent lens. The authors suggest that as yet unidentified functions in the spleen and retina may provide selection pressure that maintains certain structural aspects of the protein. Alternatively, A-crystallin may play a role as a morphogen during early eye development, which still occurs in the mole rat. Other studies on the blind cave fish, Astyanax mexicanus, seem to confirm a role for A-crystallin outside of the adult lens. The subterranean populations of this species have regressed eyes and never express A-crystallin in the lens. However, Behrens et al. (1998) found that only one amino acid differs between the eyed and blind populations. The promoter region of the blind form is also highly conserved. These data suggest that the protein's structure and the gene's promoter region are under selective pressure for some function outside of the lens. This function remains unclear.
Insights from natural variations in human -crystallin
Correlating functional changes in proteins with modifications to structure is a powerful approach for understanding structure/function relationships. Many studies have produced artificial changes in the structure of -crystallins using site directed mutagenesis only to find that the protein's function remains relatively stable (Hepburne-Scott and Crabbe, 1999; Derham et al., 2001). Some of the best breakthroughs in discovering functionally important residues have come from studies of natural mutations. Litt et al. (1998) identified a natural mutant in a human population that causes cataract due to a single amino acid substitution in A-crystallin. Vicart et al. (1998) reported that a mutation in the homologous amino acid of B-crystallin also causes early onset of cataract and leads to the abnormal deposition of the cytoskeletal protein desmin in cardiac muscle. Chaperone assays of recombinant proteins containing these mutations showed that these point mutations alter protein structure and reduce chaperone-like activity (Bova et al., 1999; Shroff et al., 2000). Additional natural mutations in human -crystallins have since been found (Pras et al., 2000; Berry et al., 2001).
Examination of natural variations in human -crystallins has been a very successful approach for understanding structure/function relationship. However, this comparative approach has not been extended beyond humans. Although the primary structures of many vertebrate -crystallins have been reported, little work has been done on the expression pattern and function of these proteins. A large resource of evolutionary variation has been left unexamined. A broader study of vertebrate taxa can shed light not only on structure/function relationships, but also on the range of physiological roles for -crystallins. Examining the physiological function of -crystallins in an evolutionary context can help make sense of the data obtained from human and other mammalian studies. Work with non-mammalian model organisms can also provide new tools for developmental and genetic studies.
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BONY FISHES AS A MODEL GROUP FOR COMPARATIVE STUDIES |
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Vertebrate
-crystallins contain sufficient variation in chaperone function, expression and possible physiological roles to make them a valuable subject for comparative studies. The existence and utility of this variation was suggested by early studies on vertebrate crystallins, and has been confirmed by recent work with the zebrafish. McFall-Ngai and Horwitz (1990) showed that ectothermic vertebrates from wide-ranging thermal environments have lens crystallins with different thermal tolerances. The lens crystallins from the Antarctic ice fish denature at lower temperatures than those of a desert lizard. Species from intermediate temperatures have crystallins that denature at intermediate temperatures. While these results are not surprising, this study was the first to show that lens crystallins adapt to different physiological conditions. With current molecular techniques it is now possible to conduct similar experiments on isolated lens crystallins, and attempt to correlate functional differences with structure.
Among vertebrates, bony fishes are a great model group for comparative studies of -crystallin because of the large number of species and their physiological diversity. A couple of factors that could exert selective pressures on -crystallin chaperone-like activity vary in bony fishes. First, they occur in a wide range of environmental temperatures from the Antarctic to freshwater desert environments. Several studies have shown that the chaperone-like function of -crystallins is temperature dependent (Datta and Rao, 1999; Reddy et al., 2000). As temperature increases, both A- and B-crystallin expose more hydrophobic residues, increasing their ability to bind to denatured protein and enhancing their protective abilities. Differences in physiological temperature may lead to evolutionary variations in chaperone-like function. The lower body temperatures of many fishes compared to mammals could also lead to differences in chaperone-like function because of the decreased thermal stress on proteins. A second factor that could affect -crystallin chaperone-like function in fishes is lifespan. Species with decreased longevities may require less of a protective role from their -crystallins since they would face reduced protein denaturation. A reduced need for protection from -crystallins could be reflected in decreased expression and/or reduced chaperone activity. The lower body temperatures and shorter lifespans may explain the decreased levels of -crystallin in the bony fish lens compared to mammals. While mammal lenses may contain one-third -crystallin by weight, a wide diversity of bony fishes all contain far lower levels (Joe Horwitz, personal communication).
Bony fishes are also an attractive group for comparative studies because of their growing use in genetic and developmental studies. A complete genome is known for the puffer, Takifugu rubripes, with several other genomes nearing completion. Diverse protocols have been developed for genetic manipulations of species such as Danio rerio and Oryzias latipes that allow for testing of transgenes and knockouts. And the ease with which many species are bred makes studies of the developmental role of -crystallins very convenient.
Initial attempts to test the chaperone-like function of bony fish -crystallins were hampered by an inability to purify these proteins from fish lenses. Mammalian lens crystallins are easily separated by gel filtration chromatography. However, I consistently found that fish -crystallins remained bound to the ß-crystallins, another family of vertebrate lens crystallins (unpublished data). Several other techniques, such as immunoprecipitation and ion-exchange chromatography also failed to purify the -crystallins. These results suggested that the interaction between -crystallins and ß-crystallins differed between the bony fish and mammalian lens. It was therefore necessary to produce recombinant fish -crystallins to study chaperone-like activity. Using recombinant techniques would also allow the separate production of purified A- and B-crystallin. Because these two -crystallins occur as a coaggregate within the lens, it is difficult to assay their function individually using native protein. The use of recombinant proteins has also become the standard technique for studying human -crystallins.
I chose to start a comparative study of vertebrate -crystallins with the zebrafish, Danio rerio, for several reasons. First, its widespread use as a vertebrate model species provides potential for future studies in -crystallin genetics and development. Second, basic information on the -crystallins of this species would be of benefit to a large number of researchers. For example, several labs are beginning to investigate the potential role of -crystallins in zebrafish mutants with abnormal lens development. And finally, initial work with zebrafish provides a starting point for more in depth comparisons of bony fish species.
Cloning, sequencing and spatial expression of zebrafish alpha crystallins
PCR techniques were used to clone both zebrafish A- and B-crystallin. Behrens et al. (1998) had previously reported the genomic sequence for A-crystallin from the bony fish Astyanax mexicanus, the blind cave fish. I designed PCR primers based on the A. mexicanus sequence that annealed to conserved regions of the A-crystallin coding region. Total RNA from zebrafish lens was reverse transcribed and subjected to PCR using these primers. The amplified coding region was sequenced and inserted into an expression plasmid so that recombinant protein could be produced in Escherichia coli. The same procedure was used to clone zebrafish B-crystallin, except that no bony fish sequence had yet been published. PCR primers for this amplification were based instead on published frog (Rana catesbeiana) and chicken (Gallus gallus) B-crystallin and amplifications were performed at low temperature because of the lower specificity in primer design. Full-length coding sequences were obtained for both zebrafish -crystallins and used to deduce the amino acid sequence of the proteins. These protein sequences were aligned with their vertebrate orthologues to identify structural differences that could potentially lead to variations in function.
Zebrafish A-crystallin does not differ greatly from its vertebrate orthologues (Runkle et al., 2002). It is 73% and 86% identical to human and cavefish A-crystallin, respectively. More importantly, few residues that have been found to be functionally important in mammalian A-crystallins differ in the zebrafish protein (Fig. 1). For example, the zebrafish protein contains an arginine at position 117 that is homologous to an arginine in the human protein that, when mutated, drastically reduces chaperone activity (Shroff et al., 2000). Also, the C-terminal extension of the zebrafish protein is similar to those from other vertebrate taxa. Only the C-terminal extension from Squalus acanthias (the dogfish shark) differs significantly from the other taxa. Modification of this extension has been shown to reduce chaperone activity (Smulders et al., 1996). Based on the known functionally important residues in A-crystallin there are no obvious modifications in the zebrafish protein that would affect chaperone-like activity.
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Comparison of zebrafish
B-crystallin to other vertebrate orthologues yields a different story. Percent identities are lower, with only 58% of residues identical between the zebrafish and human proteins. There are also some significant differences in putatively functional residues. Two of the serines known to be phosphorylated in mammals are substituted in the zebrafish protein (Fig. 2; positions 19 and 43). Since the impact of phosphorylation on B-crystallin function is unclear, it is difficult to predict the effect of these substitutions. The most obvious difference in zebrafish B-crystallin is the loss of four amino acids in the C-terminal extension (Posner et al., 1999).
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The differing levels of variation between the two zebrafish
-crystallins and their respective vertebrate orthologues extends to spatial expression as well. Reverse-transciptase polymerase chain reaction (RT-PCR) was used to identify tissues in adult zebrafish that expressed the two -crystallins. Zebrafish A-crystallin expression is mostly restricted to the lens, with only minimal expression in the spleen indicated after a large number of amplification cycles (Fig. 3A). This is similar to the tissue specific expression found in mammals. The spatial expression of zebrafish B-crystallin, however, differs markedly from mammals. Mammalian B-crystallin is expressed outside of the lens in numerous tissues, such as heart, skeletal muscle, nervous tissue and skin (Bhat and Nagineni, 1989; Dubin et al., 1989). But we could not find zebrafish B-crystallin in heart, skeletal muscle or brain (Fig. 3B). Even when amplifying for a large number of cycles, we see non-specific products in several of these tissues, but no B-crystallin product. Of the tissues we have examined, we only find B-crystallin expressed in the lens. This limited expression of zebrafish B-crystallin is interesting considering the apparent widespread importance of the protein in mammalian physiology. It appears that B-crystallin plays a different and more limited physiological role in the zebrafish. This difference may be reflected in the protein's chaperone-like activity, as described below.
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Chaperone-like activity of zebrafish alpha crystallins
Although zebrafish and mammalian
A-crystallins share similar amino acid sequences and expression patterns, it seemed likely that there would be differences in chaperone-like activity due to the large evolutionary distance between these taxa and variations in lens biochemistry between fish and mammals. Differences in the chaperone-like activity of zebrafish and mammalian B-crystallins seemed even more likely considering the greater divergence in structure and expression pattern. To test these hypotheses, I have compared the chaperone-like activities of zebrafish and human -crystallins by measuring their ability to prevent the aggregation of denatured target proteins. Aggregation can be induced by breaking disulfide bonds with the chemical DTT and quantified by measuring increase in light scattering with a spectrophotometer (Horwitz et al., 1998). -crystallins are not affected by DTT because they lack disulfide bonds. By denaturing the target protein lactalbumin in the presence of either recombinant zebrafish or human A-crystallin, I found that the chaperone-like activity of the zebrafish protein was greater than that of its human orthologue (Fig. 4A; unpublished data). This difference occurred at each temperature tested between 25°C and 40°C (Fig. 5; unpublished data). It should be noted, however, that at their relevant physiological temperatures the human and zebrafish proteins exhibit similar chaperone-like activity. The percent protection provided by the zebrafish protein at approximately 28°C would be roughly equivalent to the human protein at 37°C (Fig. 5; unpublished data). Together these data suggest that A-crystallin plays an important role as a molecular chaperone in the zebrafish lens, as it does in the human lens. Despite potentially important differences in lens physiology and biochemistry between the zebrafish and humans, the two A-crystallins may have the same fundamental role as both a structural protein and as a molecular chaperone. If this is true, the zebrafish may make a good model for studying human A-crystallin function.
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Zebrafish
B-crystallin chaperone-like activity also differed from its human orthologue. But in this case the difference was a reduced activity (Fig. 4B; unpublished data). Zebrafish B-crystallin showed lower chaperone-like activity compared to human B-crystallin at all temperatures tested (Fig. 5; unpublished data). It required over ten times more zebrafish protein to provide the same protection as human B-crystallin at 35°C. And at their relevant physiological temperatures the protection provided by the zebrafish protein would be much lower than that of the human protein (Fig. 5; unpublished data). This low chaperone-like activity and the restricted expression described above both support the hypothesis that zebrafish B-crystallin plays a more limited physiological role compared to its mammalian orthologues. Because we currently have a limited understanding of this protein's role in mammals, it is difficult to make any conclusions from these limited data about the physiological role of zebrafish B-crystallin. The lack of studies on B-crystallin expression and chaperone-like function from other vertebrate taxa also makes it difficult to determine whether zebrafish or mammals represent the ancestral condition for B-crystallin. Clearly, we need more comparative studies to understand the interesting story behind B-crystallin evolution.
It is tempting to hypothesize that the truncated C-terminal extension of the zebrafish protein leads to this reduced activity. To test this hypothesis I produced a PCR construct that combined the zebrafish sequence with a human-like C-terminal extension (Table 1). As described above, Kokke et al. (2001) found that adding a human C-terminal extension to the -crystallin domain of C. elegans Hsp16.5 did not restore chaperone activity. But in the case of this zebrafish/human hybrid regions are being swapped between more closely related proteins. Nevertheless, the addition of the human C-terminal extension had a minimal effect on chaperone-like activity (Fig. 5; unpublished data). Protection was increased slightly over the wildtype zebrafish B-crystallin, but did not come close to the protection of the human orthologue.
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There are two possible explanations for the continued reduction in chaperone-like activity even with the addition of the human C-terminal extension. First, this small region may not be important for preventing protein aggregation. However, this conclusion contradicts several studies that have shown that removal of the C-terminal extension in the human protein reduces chaperone-like activity (Smulders et al., 1996
). Second, the C-terminal extension may be important for chaperone-like activity, but requires as yet unidentified structural components somewhere else in the protein. This second conclusion takes a more global view of -crystallin function. Under this view the protein is not modular, but rather the different regions interact with each other to produce chaperone-like function. This view is in accord with Kokke et al.'s (2001) work with C. elegans. Additional mutation experiments with zebrafish B-crystallin can be used to help detail these interactions by providing a model for determining how the human sequence produces enhanced chaperone-like activity. Instead of using site directed mutagenesis to disable a strong molecular chaperone like human B-crystallin, the zebrafish protein provides a model for restoring function. Simply stated, it is easier to break something than to fix it. Restoring chaperone-like activity to zebrafish B-crystallin can provide a more stringent test of the functional importance of different amino acids. Likewise, zebrafish A-crystallin can help to identify structural components that enhance chaperone-like activity over its human orthologue.
How can the zebrafish help us to understand alpha crystallin?
The structural and functional studies on zebrafish -crystallins described above provide a foundation for two future research pursuits. First, these data can be used as a baseline for future comparative studies. While comparison of zebrafish and human -crystallins can help us to understand structure/function relationship, it is not a suitable comparison for determining the physiological selection pressures that have impacted -crystallin evolution. The large evolutionary distance between bony fishes and humans makes such conclusions difficult. Furthermore, limiting comparative studies to two species does not allow you to assess the polarity of any evolutionary changes (Garland and Adolph, 1994). Study of additional -crystallins from groups of closely related bony fishes can identify the effects of different environmental conditions and physiological change on -crystallin evolution. Well-developed phylogenies for many bony fish taxa are available for these comparative studies. Furthermore, a comparative study examining different vertebrate classes can help describe the changes in -crystallin physiology that have occurred during vertebrate evolution. For example, is the restriction of B-crystallin to the lens the ancestral condition for vertebrates, with expression becoming widespread later in evolution? Or did expression narrow during the evolution of bony fishes, with the ancestral condition being maintained in mammals. Further examination of other vertebrate taxa can help to answer this question and provide insights into the physiological role of -crystallins in all vertebrates.
A second future research direction that can build on this initial zebrafish work is to utilize the zebrafish as a model for genetic and developmental studies of -crystallins. Zebrafish are well suited for knockout and transgenic experiments that can be used to examine the in vivo role of these proteins. Similar studies with mice have been valuable (Boyle et al., 2003), and a bony fish model can provide a good source of variation for comparison. The accumulation of zebrafish lens and eye mutants also provides a valuable resource for understanding the role of -crystallins in normal lens development and physiology.
The integration of comparative techniques into -crystallin research has provided novel information not available from traditional cellular/molecular approaches alone. Comparisons of diverse taxa have added greatly to our understanding of structure/function relationships and the physiological role of -crystallins both within and outside of the lens. A greater research emphasis on -crystallins from diverse vertebrate species will strengthen this valuable comparative perspective on the function of these important stress proteins.
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ACKNOWLEDGMENTS |
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I would like to thank Joseph Horwitz and Marc Kantorow for their valuable collaboration on the work with zebrafish
-crystallin. Thanks also to the students at Ashland University who helped with many of the experiments involved in these studies: Jason Dahlman, Julie Hill, Sara Low, Kelli Margot and Stephanie Runkle. Much of this research was funded by the National Eye Institute of the National Institutes of Health (EY 13535-01). The symposium to which this paper is a contribution would not have been possible without the support of the National Science Foundation and the Society for Integrative and Comparative Biology. |
FOOTNOTES |
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1 From the Symposium Comparative and Integrative Vision Research presented at the Annual Meeting of the Society for Integrative and Comparative Biology, 4–8 January 2003, at Toronto, Canada.
2 E-mail: mposner{at}ashland.edu
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