Expression analysis of human pterygium shows a predominance of conjunctival and limbal markers and genes associated with cell migration.

PURPOSE: Pterygium is a vision-impairing fibrovascular lesion that grows across the corneal surface and is associated with sunlight exposure. To increase our understanding of the cells types involved in pterygium, we have used expressed sequence tag analysis to examine the transcriptional repertoire of isolated pterygium and to identify marker genes for tissue origin and cell migration. METHODS: An unnormalized unamplified cDNA library was prepared from 15 pooled specimens of surgically removed pterygia as part of the NEIBank project. Gene expression patterns were compared with existing data for human cornea, limbus, and conjunctiva, and expression of selected genes was verified by immunofluorescence localization in normal eye ocular surface and in pterygium. RESULTS: Sequence analysis of 2,976 randomly selected clones produced over 1,800 unique clusters, potentially representing single genes. The most abundant complementary DNAs from pterygium include clusterin, keratins 13 (Krt13) and 4 (Krt4), S100A9/calgranulin B, and spermidine/spermine N1-acetyltransferase (SAT1). Markers for both conjunctiva (such as keratin 13/4 and AQP3) and corneal epithelium (such as keratin 12/3 and AQP5) were present. Immunofluorescence of Krt12 and 13 in the normal ocular surface showed specificity of Krt12 in cornea and Krt13 in conjunctival and limbal epithelia, with a fairly sharp boundary at the limbal-corneal border. In the pterygium there was a patchy distribution of both Krt12 and 13 up to a normal corneal epithelial region specific for Krt12. Immunoglobulins were also among the prominently expressed transcripts. Several of the genes expressed most abundantly in excised pterygium, particularly S100A9 and SAT1, have roles in cell migration. SAT1 exerts its effects through control of polyamine levels. IPENSpm, a polyamine analogue, showed a significant ability to reduce migration in primary cultures of pterygium. A number of genes highly expressed in cornea were not found in pterygium (several small leucine-rich proteoglycan family members) or were expressed at considerably lower levels (ALDH3A1 and decorin). CONCLUSIONS: The expression pattern of keratins and other markers in pterygium most closely resemble those of conjunctival and limbal cells; some corneal markers are present, notably Krt12, but at lower levels than equivalent conjunctival markers. Our data are consistent with the model of pterygium developing from the migration of conjunctival- and limbal-like cells into corneal epithelium. Identification of genes with roles in cell migration suggests potential therapeutic targets. In particular, the ability of polyamine analogues to reduce migration in primary cultures of pterygium presents a possible approach to slowing pterygium growth.

Tau-crystallin/alpha-enolase: one gene encodes both an enzyme and a lens structural protein.

tau-Crystallin has been a major component of the cellular lenses of species throughout vertebrate evolution, from lamprey to birds. Immunofluorescence analysis of the embryonic turtle lens, using antiserum to lamprey tau-crystallin showed that the protein is expressed throughout embryogenesis and is present at high concentrations in all parts of the lens. Partial peptide sequence for the isolated turtle protein and deduced sequences for several lamprey peptides all revealed a close similarity to the glycolytic enzyme enolase (E.C. 4.2.1.11). A full-sized cDNA for putative duck tau-crystallin was obtained and sequenced, confirming the close relationship with alpha-enolase. Southern blot analysis showed that the duck genome contains a single alpha-enolase gene, while Northern blot analysis showed that the message for tau-crystallin/alpha-enolase is present in embryonic duck lens at 25 times the abundance found in liver. tau-Crystallin possesses enolase activity, but the activity is greatly reduced, probably because of age-related posttranslational modification. It thus appears that a highly conserved, important glycolytic enzyme has been used as a structural component of lens since the start of vertebrate evolution. Apparently the enzyme has not been recruited for its catalytic activity but for some distinct structural property. tau-Crystallin/alpha-enolase is an example of a multifunctional protein playing two very different roles in evolution but encoded by a single gene.

Recruitment of enzymes as lens structural proteins.

Crystallins, the principal components of the lens, have been regarded simply as soluble, structural proteins. It now appears that the major taxon-specific crystallins of vertebrates and invertebrates are either enzymes or closely related to enzymes. In terms of sequence similarity, size, and other physical characteristics delta-crystallin is closely related to argininosuccinate lyase, tau-crystallin to enolase, and SIII-crystallin to glutathione S-transferase; moreover, it has recently been demonstrated that epsilon-crystallin is an active lactate dehydrogenase. Enzymes may have been recruited several times as lens proteins, perhaps because of the developmental history of the tissue or simply because of evolutionary pragmatism (the selection of existing stable structures for a new structural role).

Lens crystallins: gene recruitment and evolutionary dynamism.

In a novel evolutionary process, enzymes and stress proteins have undergone direct gene recruitment as eye lens crystallins in a number of independent events. This may have allowed a dynamic response to changing visual environment during evolution. In spite of their diversity, many crystallins may share an origin in essential developmental processes such as cell elongation.

Tandem sequence repeats in transmembrane channel proteins.

The flow of ions and small molecules out of and between cells is mediated by various classes of transmembrane proteins. One group of putative channel proteins, including the abundant lens protein MIP, is widely distributed from prokaryotes to vertebrates. This article suggests that these proteins contain a structural twofold repeat and may have arisen by gene duplication. Such a model has implications for the tertiary structures of these important proteins.

Lens-specific gene recruitment of zeta-crystallin through Pax6, Nrl-Maf, and brain suppressor sites.

Zeta-Crystallin is a taxon-specific crystallin, an enzyme which has undergone direct gene recruitment as a structural component of the guinea pig lens through a Pax6-dependent mechanism. Tissue specificity arises through a combination of effects involving three sites in the lens promoter. The Pax6 site (ZPE) itself shows specificity for an isoform of Pax6 preferentially expressed in lens cells. High-level expression of the promoter requires a second site, identical to an alphaCE2 site or half Maf response element (MARE), adjacent to the Pax6 site. A promoter fragment containing Pax6 and MARE sites gives lens-preferred induction of a heterologous promoter. Complexes binding the MARE in lens nuclear extracts are antigenically related to Nrl, and cotransfection with Nrl elevates zeta-crystallin promoter activity in lens cells. A truncated zeta promoter containing Nrl-MARE and Pax6 sites has a high level of expression in lens cells in transgenic mice but is also active in the brain. Suppression of the promoter in the brain requires sequences between -498 and -385, and a site in this region forms specific complexes in brain extract. A three-level model for lens-specific Pax6-dependent expression and gene recruitment is suggested: (i) binding of a specific isoform of Pax6; (ii) augmentation of expression through binding of Nrl or a related factor; and (iii) suppression of promoter activity in the central nervous system by an upstream negative element in the brain but not in the lens.

The duck gene for alpha B-crystallin shows evolutionary conservation of discrete promoter elements but lacks heat and osmotic shock response.

The gene for alpha Beta-crystallin from a bird (the domestic duck, Anas platyrhynchos) has been cloned and sequenced to allow comparison with its mammalian homologues. The duck gene has the same general structure as those of humans and rodents although, unlike those of mammals, the duck gene has two polyadenylation signals at the 3' end. The most interesting comparisons are in the 5'flanking promoter regions. In contrast to the broad conservation of promoter sequence among mammals, only two significant blocks and a few smaller elements have been conserved during evolution in the more distantly related avian gene. Block 1 (-350/-308) corresponds to alpha BE-2, a functional element defined in the mouse gene. Further downstream, block 2 (-98/-65) shows 27/33 identity among all three species but does not correspond to any previously defined element. Other regions are less well-conserved. In particular, putative heat-shock response elements of the mammalian alpha B-crystallin genes are absent from the duck gene. In contrast to the heat and osmotic stress-inducibility of mouse alpha B-crystallin in NIH 3T3 cells, duck alpha B-crystallin showed no inducibility in duck cells in culture. Thus, although high expression in lens is common to alpha B-crystallin genes in birds and mammals, other modes of expression appear to be taxon-specific.

Linkage and expression of the argininosuccinate lyase/delta-crystallin genes of the duck: insertion of a CR1 element in the intergenic spacer.

delta-Crystallin is the major component of the lenses of most birds and reptiles. In the chicken there are two closely linked, tandemly oriented genes. Almost all of the delta-crystallin of the embryonic chicken lens is produced by the 5' delta 1 gene. This high lens activity has been attributed to an enhancer in intron 3. The 3' delta 2 gene encodes the enzyme argininosuccinate lyase (ASL) which is expressed at a low level in the chicken lens. Both chicken delta-crystallin genes are also expressed slightly in heart and brain, with ASL/delta 2 predominating over delta 1. In the duck (Anas platyrhynchos), ASL/delta 2-crystallin serves as both enzyme and crystallin, resulting in very high levels of ASL activity in the lens. Here we show by genomic cloning that the ASL/delta- crystallin locus is highly conserved between duck and chicken, with the two duck delta-crystallin genes closely linked in tandem. The 4.6 kbp intergenic spacer in the duck locus is 79% identical to the 4 kbp chicken spacer, except for the existence of a 615 bp CR1 element, highly reiterated in the duck genome, 1.8 kbp upstream of the duck ASL/delta 2 gene. The CR1 sequence is a truncated LINE element containing the 3' half of an open reading frame for a retroviral pol-like reverse transcriptase. Sequence analysis revealed (i) that intron 3 of the duck ASL/delta 2 gene is very similar (80%) to intron 3 of the chicken delta 1 and ASL/delta 2 genes, especially in the region of the chicken delta 1 enhancer core (93% identical) and (ii) that the 3' boundary of exon 2 of the duck ASL/delta 2 gene has undergone a recent splice-site slippage event, resulting in a two amino acid insertion in the encoded polypeptide. Finally, reverse transcription/polymerase chain reaction experiments established that both delta-crystallin genes are equally expressed to a high level in the embryonic duck lens; by contrast, both delta-crystallin genes produce a low amount of mRNA in the heart and brain of the embryonic duck, with the enzymatically active ASL/delta 2 being preferentially expressed.

Zeta-crystallin: a lens-specific promoter and the gene recruitment of an enzyme as a crystallin.

A novel enzyme with quinone oxidoreductase activity has undergone gene recruitment in certain mammals, acquiring a second function as the lens structural protein zeta-crystallin. Here we show that recruitment of this enzyme crystallin can be explained by the lens specificity of an alternative promoter which does not require host-specific factors. The strong lens preference of this promoter is apparent in both cultured cell transfections and in transgenic mice. While proximal regions of the promoter have some activity in the brain of transgenic mice this is abolished by the addition of more distal regions. The minimal active promoter is differentially footprinted by extracts from lens and non-lens cells. Deletion within the major region footprinted in lens, ZPE (zeta protected element), abolishes promoter function. This is the first example of a lens-specific promoter in an enzyme crystallin gene and the first demonstration of gene recruitment by this mechanism.

An avian alpha B-crystallin. Non-lens expression and sequence similarities with both small (HSP27) and large (HSP70) heat shock proteins.

alpha B-crystallin is multifunctional, serving as both a major structural protein in the lens and a small heat-shock protein (shsp) in other tissues in mammals. Cloning and Northern analysis show similarly that alpha B-crystallin mRNA is present in all mature tissues examined in a bird (Anas platyrhynchos), although there are some differences in the pattern of transcripts seen. Interestingly, sequence analysis not only shows that duck alpha B-crystallin is a member of the shsp family, as expected, but that this family shares more distant similarity with another heat shock protein family, the highly conserved HSP70s of both eukaryotes and prokaryotes. This raises the possibility that large and small hsps may share structural and perhaps functional features.

The P23T cataract mutation causes loss of solubility of folded gammaD-crystallin.

Mutations in the human gammaD-crystallin gene have been linked to several types of congenital cataracts. In particular, the Pro23 to Thr (P23T) mutation of human gammaD crystallin has been linked to cerulean, lamellar, coralliform, and fasciculiform congenital cataracts. We have expressed and purified wild-type human gammaD, P23T, and the Pro23 to Ser23 (P23S) mutant. Our measurements show that P23T is significantly less soluble than wild-type human gammaD, with P23S having an intermediate solubility. Using synchrotron radiation circular dichroism spectroscopy, we have determined that the P23T mutant has a slightly increased content of beta-sheet, which may be attributed to the extension of an edge beta-strand due to the substitution of Pro23 with a residue able to form hydrogen bonds. Neither of the point mutations appears to have reduced the thermal stability of the protein significantly, nor its resistance to guanidine hydrochloride-induced unfolding. These results suggest that insolubility, rather than loss of stability, is the primary basis for P23T congenital cataracts.

X-ray analysis of the eye lens protein gamma-II crystallin at 1.9 A resolution.

We report the X-ray structure analysis and refinement at 1.9 A resolution of calf gamma-II crystallin, a lens-specific protein. The sequence of Croft (1972) has been modified to give a polypeptide chain of 174 residues (cf. 165). The protein has a symmetrical, hierarchical structure of two globular domains each comprising two similar "Greek key" motifs, consecutive along the polypeptide chain, and related by a pseudo 2-fold axis. The two domains pack together with a single connection and are related by a further pseudo 2-fold axis which bisects the angle between the intra-domain dyads. Forty-two pairs of C alpha positions for the two most similar motifs have root-mean-square separation at best fit of 0.69 A. The N and C-terminal domains gave root-mean-square separation of 0.89 A for 82 pairs of C alpha atoms at best fit. In each domain the two Greek key motifs form a pair of four-stranded antiparallel beta-pleated sheets, each sheet composed of three stands from one motif and one from the other. The sheets pack together in a wedge shape, closed at the top by the loops connecting the third and fourth strands of each motif. The first two strands of each motif form an extended beta-hairpin which is folded on to the beta-sheet. The packing of each motif into the globular domains involves a staggered bilayer of side-chains between each pair of beta-sheets which does not preserve the pseudo 2-fold axes observed in the C alpha position topology. In the core of each domain there are interactions between polarizable aromatic groups and sulphur-containing residues which may contribute to stability and may also serve to protect aromatic side-chains from ultraviolet light damage in the lens. At the surface of the molecule over half the ionic side-chains are closely paired, which probably stabilizes the tertiary fold and may reduce the water bound. Crystal lattice interactions are described which may be similar to those occurring in vivo in the lens between crystallins. Seven cysteine residues have been identified in the structure and these may have a role in the thermodynamic stability of the molecule, its intermolecular interactions under the normal reducing conditions of the lens, and also in the aggregation and cross-linking which occur in some forms of cataract. Three of these residues, Cys18, Cys23 and Cys74, form a cluster in the N-terminal domain.(ABSTRACT TRUNCATED AT 400 WORDS)

Expression pattern of macrophage migration inhibitory factor during embryogenesis.

Although macrophage migration inhibitory factor (MIF) was originally identified as a lymphokine that inhibits the migration of macrophages, its ubiquitous expression suggests it may have a role beyond the immune system. Here we report a detailed characterization of MIF expression during mouse embryogenesis. The MIF expression pattern was found to parallel tissues specification and organogenesis.

Domain exchange experiments in duck delta-crystallins: functional and evolutionary implications.

Delta-crystallin, the major soluble protein component of the avian and reptilian eye lens, is homologous to the urea cycle enzyme argininosuccinate lyase (ASL). In duck lenses there are two delta crystallins, denoted delta1 and delta2. Duck delta2 is both a major structural protein of the lens and also the duck orthologue of ASL, an example of gene recruitment. Although 94% identical to delta2/ASL in the amino acid sequence, delta1 is enzymatically inactive. A series of hybrid proteins have been constructed to assess the role of each structural domain in the enzymatic mechanism. Five chimeras--221, 122, 121, 211, and 112, where the three numbers correspond to the three structural domains and the value of 1 or 2 represents the protein of origin, delta1 or delta2, respectively--were constructed and thermodynamically and kinetically analyzed. The kinetic analysis indicates that only domain 1 is crucial for restoring ASL activity to delta1 crystallin, and that amino acid substitutions in domain 2 may play a role in substrate binding. These results confirm the hypothesis that only one domain, domain 1, is responsible for the loss of catalytic activity in delta1. The thermodynamic characterization of human ASL (hASL) and duck delta1 and delta2 indicate that delta crystallins are slightly less stable than hASL, with the delta1 being the least stable. The deltaGs of unfolding are 57.25, 63.13, and 70.71 kcal mol(-1) for delta1, delta2, and hASL, respectively. This result was unexpected, and we speculate that delta crystallins have adapted to their structural role by adopting a slightly less stable conformation that might allow for enhanced protein-protein and protein-solvent interactions.

Gene conversion and splice-site slippage in the argininosuccinate lyases/delta-crystallins of the duck lens: members of an enzyme superfamily.

Argininosuccinate lyase(ASL)/delta-crystallin is a prominent example of an enzyme-crystallin with roles as both a catalyst and a major structural component of the eye lens in birds and reptiles. In chicken it appears that gene duplication and separation of function may have occurred with one gene product acting primarily as a crystallin and one primarily as an enzyme. However, two delta-crystallin-encoding genes are abundantly expressed in the lens of the embryonic duck (Anas platyrhynchos) which has extremely high ASL activity. Here the isolation and sequence analysis of full length cDNA clones for both duck delta-crystallins are described. The two delta-crystallins are highly similar (94% identical in predicted aa sequence), probably as a result of gene conversion. However, the cDNA for duck delta 2-crystallin contains an in-frame insertion of two codons, probably the result of a recent intron boundary slippage. ASL/delta-crystallin belongs to a superfamily of lyases, including fumarases, aspartases and adenylosuccinate lyase which possess some highly conserved blocks of aa sequence. There may be some clues to the tertiary structures of these conserved motifs in otherwise unrelated proteins for which three-dimensional structures are known.

Structure and expression of the duck alpha-enolase/tau-crystallin-encoding gene.

In the duck, the glycolytic enzyme, alpha-enolase (alpha ENO) and the lens structural protein, tau-crystallin (tau CRY), are products of the same gene, an example of protein multi-functionality. We report that duck alpha ENO/tau CRY mRNA levels are developmentally regulated: alpha ENO/tau CRY mRNA levels in the lens increase over those in the liver by embryonic day 14 and, within the lens, are higher in the lens epithelium than in fiber cells. We determined the structure of the duck alpha ENO/tau CRY-encoding gene (alpha ENO/tau CRY), sequenced 1 kb of 5'-flanking region, and demonstrated that this region contains a functional promoter. The gene is 13 kb in size and is composed of twelve exons; the exon organization is identical to that of mammalian enolase-encoding genes. A fragment of 5'-flanking region (-803/+3) containing three CCAAT boxes and a TATA box was able to activate transcription of a heterologous reporter gene when transfected into cultured lens cells. However, in spite of greater quantities of alpha ENO/tau CRY mRNA and protein in the lens, the promoter was equally active in primary cultures of embryonic lens, liver and fibroblast cells. Since the cultured cells unexpectedly lost the restricted pattern of alpha ENO/tau CRY mRNA levels observed in vivo, evaluation of the promoter's tissue specificity was precluded.

DNA sequence motifs are associated with aberrant homologous recombination in the mouse macrophage migration inhibitory factor (Mif) locus.

Homologous recombination is a precise genetic event that can introduce specific alteration in the genome. A planned targeted disruption by homologous recombination of the macrophage migration inhibitory factor (Mif) locus in mouse embryonic stem (ES) cells yielded the targeted clones, some of which had genomic rearrangements inconsistent with the expected homologous recombination event. A detailed characterization of the recombination breakpoints in two of these clones revealed several sequence motifs with possible roles in recombination. These motifs included short regions of sequence identity that may promote DNA alignment, multiple 5'-AAGG/TTCC-3' tetrameres, topoisomerase I consensus sites, and AT-rich sequences that can promote DNA cleavage and recombination. A retrovirus-like intracisternal-A particle (IAP) family sequence was also identified upstream of the Mif gene, and the LTR of this IAP was involved in one of the recombinations. Identification and characterization of such sequence motifs will be valuable for the gene targeting experiments.

Schiff's base formation in the lens protein gamma-crystallin.

Uniquely among the soluble lens-specific proteins, gamma-crystallin is capable of binding the strongly chromophoric aldehyde retinal. A role for gamma-crystallin in protecting lens components from toxic aldehydes resulting from membrane oxidation is proposed and a molecular model of the probable interaction site is presented. The sequence of a tetrapeptide at this site is identical to that of the retinal binding site of bacteriorhodopsin.

Sequence analysis of bovine retinal S-antigen. Relationships with alpha-transducin and G-proteins.

S-Antigen is a major soluble protein of the retina and pineal. It is capable of inducing experimental autoimmune uveitis (EAU) in laboratory animals and also seems to play an important role in the visual cycle. The results of partial cDNA sequence analysis reveal interesting homologies with alpha-transducin, a GTP-binding protein of retina and other purine nucleotide-binding proteins. In particular S-antigen shows over 50% identity to the proposed pertussis toxin ADP-ribosylation site of alpha-transducin. It also contains the Gly-X-X-X-X-Gly-Lys pattern common to phosphoryl binding sites. A possible relationship between S-antigen and purine nucleotide-binding proteins is discussed. There is also evidence for a repetitious beta-structure in the C-terminal half of S-antigen, with a monoclonal antibody epitope in a helical region at the C-terminus.

Aldose reductase and p-crystallin belong to the same protein superfamily as aldehyde reductase.

Aldose reductase (EC 1.1.1.21) has been implicated in a variety of diabetic complications. Here we present the first primary sequence data for the rat lens enzyme, obtained by amino acid and cDNA analysis. We have found structural similarities with another NADPH-dependent oxidoreductase: human liver aldehyde reductase (EC 1.1.1.2). The identity between these two enzymes is 50%. Both enzymes share approx. 40-50% homology with p-crystallin, a major lens protein present only in the frog, Rana pipiens. We propose that aldose reductase, aldehyde reductase and p-crystallin are members of a superfamily of related proteins.