S ummary and I mplications We have described a correlation between the ratio of synthesis of the higher to the lower molecular weight polypeptides of δ‐crystallin and the intracellular concentrations of Na + and K + in the cultured embryonic chick lens. The only structural differences we have found between the larger and smaller δ‐crystallin polypeptides are two, acidic, methionine‐containing tryptic peptides which are absent from the lower molecular weight polypeptides. We do not yet know whether the alteration in the ratio of synthesis of the polypeptides is regulated at the post‐transcriptional or post‐translational level. We are continuing to investigate this problem by studies on the cell‐free synthesis of δ‐crystallin and by analysis of the sequences and organization of δ‐crystallin DNA. One implication of these experiments concerns protein synthesis in cataractous lenses, as has been reviewed elsewhere. 15 Numerous cataracts are associated with appreciable changes in ion concentrations, particularly with an increase in Na + and a decrease in K + . Differential reduction in crystallin synthesis has now been correlated with an increase in the Na + /K + ratio in three types of cataracts, namely, in hereditary cataracts in mice (Nakano mouse), 29 in galactose cataracts in rats, 30 and in drug‐induced experimental cataracts in rats. 31 It seem reasonable to propose that changes in ion concentrations will be correlated with alterations in protein synthesis in other cataracts and. possibly, with other diseases which result in electrolyte imbalances within cells.
Here we examine the molecular basis for the known preferential expression of rabbit aldehyde dehydrogenase class 1 (ALDH1A1) in the cornea. The rabbit Aldh1a1 promoter-firefly luciferase reporter transgene (-3519 to +43) was expressed preferentially in corneal cells in transfection tests and in transgenic mice, with an expression pattern resembling that of rabbit Aldh1a1. The 5' flanking region of the rabbit Aldh1a1 gene resembled that in the human gene (60.2%) more closely than that in the mouse (46%) or rat (51.5%) genes. We detected three xenobiotic response elements (XREs) and one E-box consensus sequence in the rabbit Aldh1a1 upstream region; these elements are prevalent in other highly expressed corneal genes and can mediate stimulation by dioxin and repression by CoCl(2), which simulates hypoxia. The rabbit Aldh1a1 promoter was stimulated fourfold by dioxin in human hepatoma cells and repressed threefold by CoCl(2) treatment in rabbit corneal stromal and epithelial cells. Cotransfection, mutagenesis, and gel retardation experiments implicated the hypoxia-inducible factor 3alpha/aryl hydrocarbon nuclear translocator heterodimer for Aldh1a1 promoter activation via the XREs and stimulated by retinoic acid protein 13 for promoter repression via the E-box. These experiments suggest that XREs, E-boxes, and PAS domain/basic helix-loop-helix transcription factors (bHLH-PAS) contribute to preferential rabbit Aldh1a1 promoter activity in the cornea, implicating hypoxia-related pathways.
It is accepted that the taxon-specific, multifunctional crystallins (small heat-shock proteins and enzymes) serve structural roles contributing to the transparent and refractive properties of the lens. The transparent cornea also accumulates unexpectedly high proportions of taxon-specific, multifunctional proteins particularly, but not only, in the epithelium. For example, aldehyde dehydrogenase 3 (ALDH3) is the main water-soluble protein in corneal epithelial cells of most mammals (but ALDH1 predominates in the rabbit), whereas gelsolin predominates in the zebrafish corneal epithelium. Moreover, some invertebrates (e.g., squid and scallop) accumulate proteins in their corneas that are similar to their lens crystallins. Pax-6, among other transcription factors, is implicated in development and tissue-specific gene expression of the lens and cornea. Environmental factors appear to influence gene expression in the cornea, but not the lens. Although no direct proof exists, the diverse, abundant corneal proteins may have evolved a crystallinlike role, in addition to their enzymatic or cytoskeletal functions, by a gene sharing mechanism similar to the lens crystallins. Consequently, it is proposed that the cornea and lens be considered as a single refractive unit, called here the “refracton,” to emphasize their similarities and common function.
The major proteins (crystallins) of the transparent, refractive eye lens of vertebrates are a surprisingly diverse group of multifunctional proteins. A number of lens crystallins display taxon‐specificity. In general, vertebrate crystallins have been recruited from stress‐protective proteins (i.e the small heat‐shock proteins) and a number of metabolic enzymes by a gene‐sharing mechanism. Despite the existence of refractive lenses in the complex and compound eyes of many invertebrates, relatively little is known about their crystallins. Here we review for the first time the state of knowledge of inverteorate crystallins. The major cephalopod (squid, octopus, and cuttlefish) crystallins ( S ‐crystallins) have, like vertebrate crystallins, been recruited from a stress protective metabolic enzyme, glutathione S ‐transferase. The presence of overlapping AP‐1 and antioxidant responsive‐like sequences that appear functional in transfected vertebrate cells suggests that the recruitment of glutathione S ‐transferase to S ‐crystallins involved response to oxidative stress. Cephalopods also have at least two taxon‐specific crystallins: Ω‐crystallin, related to aldehyde dehydrogenase, and O‐crystallin, related to a superfamily of lipid‐binding proteins. L‐crystallin (probably identical to Ω‐crystallin) is the major protein of the lens of the squid photophore, a specialized structure for emitting light. The use of L/Ω‐crystallin in the ectodermal lens of the eye and the, mesodermal lens of the photophore of the squid contrasts with the recruitment of different crystallins in the ectodermal lenses of the eye and photophore of fish. S – and Ω‐crystallins appear to be lens‐specific (some S ‐crystallins are also expressed in cornea) and, except for one S ‐crystallin polypeptide (SL11/Lops4; possibly a molecular fossil), lack enzymatic activity. The S ‐crystallins (except SL11/Lops4) contain a variable peptide that has been inserted by exon shuffling. The only other invertebrate crystallins that have been examined are in one marine gastropod ( Aplysia , a sea hare), in jellyfish and in the compound eyes of some arthropods; all are different and novel proteins. Drosocrystallin is one of three calcium binding taxon‐specific crystallins found selectively in the acellular corneal lens of Drosophila , while antigen 3G6 is a highly conserved protein present in the ommatidial crystallin cone and central nervous system of numerous arthropods. Cubomedusan jellyfish have three novel crystallin familes (the J‐crystallins); the J1‐crystallins are encoded in three very similar intronless genes with markedly different 5′ flanking sequences despite their almost identical encoded proteins and high lens expression. The numerous refractive structures that have evolved in the eyes of invertebrates contrast markedly with the limited information on their protein composition, making this field as exciting as it is underdeveloped. The similar requirement of Pax‐6 (and possibly other common transcription factors) for eye development as well as the diversity, taxon‐specificity and recruitment of stress‐protective enzymes as crystallins Suggest that borrowing multifunctional proteins for refraction by a gene sharing strategy may have occurred in invertebrates as it did in vertebrates.
While many of the diverse crystallins of the transparent lens of vertebrates are related or identical to metabolic enzymes, much less is known about the lens crystallins of invertebrates. Here we investigate the complex eye of scallops. Electron microscopic inspection revealed that the anterior, single layered corneal epithelium overlying the cellular lens contains a regular array of microvilli that we propose might contribute to its optical properties. The sole crystallin of the scallop eye lens was found to be homologous to Ω-crystallin, a minor crystallin in cephalopods related to aldehyde dehydrogenase (ALDH) class 1/2. Scallop Ω-crystallin (officially designated ALDH1A9) is 55–56% identical to its cephalopod homologues, while it is 67 and 64% identical to human ALDH 2 and 1, respectively, and 61% identical to retinaldehyde dehydrogenase/η-crystallin of elephant shrews. Like other enzyme-crystallins, scallop Ω-crystallin appears to be present in low amounts in non-ocular tissues. Within the scallop eye, immunofluorescence tests indicated that Ω-crystallin expression is confined to the lens and cornea. Although it has conserved the critical residues required for activity in other ALDHs and appears by homology modeling to have a structure very similar to human ALDH2, scallop Ω-crystallin was enzymatically inactive with diverse substrates and did not bind NAD or NADP. In contrast to mammalian ALDH1 and -2 and other cephalopod Ω-crystallins, which are tetrameric proteins, scallop Ω-crystallin is a dimeric protein. Thus, ALDH is the most diverse lens enzyme-crystallin identified so far, having been used as a lens crystallin in at least two classes of molluscs as well as elephant shrews.
To identify proteins that physically interact with Pax-6, a paired domain- and homeodomain (HD)-containing transcription factor that is a key regulator of eye development.Protein-protein interactions involving Pax-6, TATA-box-binding protein (TPB), and retinoblastoma protein were studied using affinity chromatography with Pax-6 as ligand, glutathione-S-transferase (GST) pull-down assays, and immunoprecipitations.The authors have shown that Pax-6 is a sequence-specific activator of many crystallin genes, all containing a TATA box, in the lens. Others have shown that lens fiber cell differentiation, characterized by temporally and spatially regulated crystallin gene expression, depends on retinoblastoma protein. In the present study it was shown that Pax-6 interacted with the TBP, the DNA-binding subunit of general transcription complex TFIID. GST pull-down assays indicated that this interaction was mediated by the Pax-6 HD, with a substantial role for its N-terminal arm and first two alpha-helices. The experiments also indicated a binding role for the C-terminal-activation domain of the protein. In addition, the present study showed that the HD of Pax-6 interacted with retinoblastoma protein. Immunoprecipitation experiments confirmed retinoblastoma protein/Pax-6 complexes in lens nuclear extracts.Blending the present results with those in the literature suggests that Pax-6 and retinoblastoma protein participate in overlapping regulatory pathways controlling epithelial cell division, fiber cell elongation, and crystallin gene expression during lens development.
In their report “Ciliary photoreceptors with a vertebrate-type opsin in an invertebrate brain” (29 Oct. 2004, p. 869), D. Arendt et al. offer an interesting perspective on the ancestry of vertebrate and invertebrate opsins and their associated receptors. Here, we offer an even broader
