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.

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.

Enzyme activity of macrophage migration inhibitory factor toward oxidized catecholamines.

Macrophage migration inhibitory factor (MIF) is a relatively small, 12.5-kDa protein that is structurally related to some isomerases and for which multiple immune and catalytic roles have been proposed. MIF is widely expressed in tissues with particularly high levels in neural tissues. Here we show that MIF is able to catalyze the conversion of 3,4-dihydroxyphenylaminechrome and norepinephrinechrome, toxic quinone products of the neurotransmitter catecholamines 3,4-dihydroxyphenylamine and norepinephrine, to indoledihydroxy derivatives that may serve as precursors to neuromelanin. This raises the possibility that MIF participates in a detoxification pathway for catecholamine products and could therefore have a protective role in neural tissues, which as in Parkinson's disease, may be subject to catecholamine-related cell death.

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.

Cutting edge: role of macrophage migration inhibitory factor in inhibiting NK cell activity and preserving immune privilege.

The absence of MHC class I Ags on the corneal endothelium, which lines the anterior chamber of the eye, makes this cell layer potentially vulnerable to lysis by NK cells. However, aqueous humor (AH), which bathes the corneal endothelium, contains a 12-kDa protein which inhibits the NK-mediated lysis of corneal endothelial cells. An amino acid sequence analysis of AH revealed that this factor shared >90% homology with macrophage migration inhibitory factor (MIF). The NK inhibitory effect of AH was neutralized with anti-human MIF Ab. Moreover, mouse rMIF produced a similar inhibition of NK cell activity. However, neither rMIF nor AH inhibited the CTL-mediated Lysis of allogeneic cells. rMIF prevented the release of perforin granules by NK cells but not CTLs. Although MIF displays proinflammatory properties, these results indicate that it can also inhibit at least one immune effector element, NK cells, and thereby contribute to immune privilege in the eye.

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.

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.

Expression of the duck alpha-enolase/tau-crystallin gene in transgenic mice.

In many vertebrates, metabolic enzymes have been directly recruited to an additional structural role as crystallins in the eye lens. In some species the glycolytic enzyme alpha-enolase (alpha ENO) attains high concentrations in the lens, as tau-crystallin (tau CRY). A line of transgenic mice was constructed containing the entire duck alpha ENO/tau CRY gene with 5'- and 3'-flanking regions and all introns. Full-sized duck alpha ENO mRNA was expressed in the transgenic mice with the same pattern as the endogenous mouse alpha ENO isozyme. Although there was no evidence for tissue preference, the concentration of enolase increased markedly in transgenic lens as well as in other tissues. In spite of this, transgenic lenses were transparent and the animals were normal in appearance. The increase in enolase levels in the transgenic lens mimics the stepped increase that might occur in the early stages of enzyme crystallin recruitment. These results demonstrate that lens transparency is sufficiently robust to be refractory to some increase in metabolic enzyme concentration without the need for compensatory adaptation.

A macrophage migration inhibitory factor is expressed in the differentiating cells of the eye lens.

A discrete 10-kDa polypeptide (10K) is expressed from early stages in the embryonic chicken lens. Since this has potential as a marker for lens cell development, chicken 10K and its homologues from mouse and human lenses were identified by protein sequencing and cloning. Surprisingly, lens 10K proteins appear to be identical to a lymphokine, macrophage migration inhibitory factor (MIF), originally identified in activated human T cells. Using microdissection and PCR techniques, we find that expression of 10K/MIF is strongly correlated with cell differentiation in the developing chicken lens. Northern blot analysis shows that 10K/MIF is widely expressed in mouse tissues. These results suggest that proteins with MIF activity may have roles beyond the immune system, perhaps as intercellular messengers or part of the machinery of differentiation itself. Indeed, partial sequence of other small lens proteins identifies another MIF-related protein (MRP8) in calf lens. The relatively abundant expression of MIF in lens may have clinical significance, with the possibility of involvement in ocular inflammations that may follow damage to the lens.

Carbonyl-metabolizing enzymes and their relatives recruited as structural proteins in the eye lens.

The refractive properties of the eye lens are determined by abundant soluble structural proteins known as crystallins. While some crystallins are common to most vertebrates, others are abundant only in groups of related species. These taxon-specific crystallins all turn out to be enzymes, apparently recruited by modification of gene expression without prior gene duplication. They include eta-crystallin, accounting for up to 25% of protein in elephant shrew lenses and apparently identical to cytoplasmic aldehyde dehydrogenase; rho-crystallin from frog lenses, a member of the same superfamily as aldose and aldehyde reductases; and zeta-crystallin, found in guinea pig and camel lenses, which is structurally related to alcohol dehydrogenase (ADH). Unlike ADH, zeta-crystallin requires NADPH rather than NAD+/NADH as cofactor. Molecular modelling of zeta-crystallin shows that amino-acid changes around the co-factor binding site are responsible for this change in affinity. Purified guinea pig lens zeta-crystallin has a substrate preference for orthoquinones which are reduced by a single electron transfer mechanism. cDNA sequencing of zeta-crystallin suggests that the expression in lens as a crystallin depends on a different gene promoter from that used predominantly in liver. The putative guinea pig zeta-crystallin lens promoter has now been assayed for function in transfection studies. Elements with positive and negative effects on transcription, at least one of which has tissue preferred function, have been defined. When introduced into transgenic mice this promoter exhibits tissue-specific expression in the lens. This is the first identification of a lens-specific, alternative promoter in an enzyme crystallin gene.

The cDNA RPE1 and monoclonal antibody HMB-50 define gene products preferentially expressed in retinal pigment epithelium.

As a first step towards identifying genes expressed specifically in the retinal pigment epithelium (RPE), we generated a bovine RPE cDNA library. In preliminary characterization of this library, we isolated a clone, RPE1, that was identified as the bovine homologue of a human cDNA thought to encode the HMB-50 melanoma antigen. Sequence analysis suggests RPE1 encodes a membrane-anchored glycoprotein. In Northern blot analysis of RNA from bovine tissues, the 2.5 kb RPE1 transcript was detected only in the RPE. Southern blot analysis suggested the presence of a single bovine rpe1 gene. In normal human eyes, HMB-50 immunoreactivity was also restricted to the RPE; no immunoreactivity was detected in other intraocular pigmented cells. Thus, RPE1 and HMB-50 define gene products preferentially expressed in the RPE at the RNA and protein levels, respectively.

mu-crystallin is a mammalian homologue of Agrobacterium ornithine cyclodeaminase and is expressed in human retina.

mu-Crystallin is the major component of the eye lens in several Australian marsupials. The complete sequence of kangaroo mu-crystallin has now been obtained by cDNA cloning. The predicted amino acid sequence shows similarity with ornithine cyclodeaminases encoded by the tumor-inducing (Ti) plasmids of Agrobacterium tumefaciens. Until now, neither ornithine cyclodeaminase nor any structurally related enzymes have been observed in eukaryotes. RNA analysis of kangaroo tissues shows that mu-crystallin is expressed at high abundance in lens, but outside the lens mu-crystallin is preferentially expressed in neural tissues, retina, and brain. An almost full-length cDNA for mu-crystallin was cloned from human retina. In human tissues, mu-crystallin mRNA is present in neural tissue, muscle, and kidney. This pattern of expression and relationship to an enzyme involved in unusual amino acid metabolism suggests the interesting possibility that mammalian mu-crystallins could be enzymes participating in processes such as osmoregulation or the metabolism of excitatory amino acids.

Vertebrate lens alpha-crystallins are modified by O-linked N-acetylglucosamine.

Crystallins are structural proteins responsible for establishing the remarkable optical properties of the lens. Yet many of these highly conserved proteins are also expressed in nonocular tissues, where they have alternative functions apparently unrelated to their structural role in the lens. Here we report that lens alpha-crystallins, some of which function as heat-shock proteins in other tissues, are modified with O-linked N-acetylglucosamine (O-GlcNAc). An in vitro enzymatic assay that transfers [3H]Gal to terminal GlcNAc moieties labels alpha A and alpha B crystallins in lens homogenates from man, rhesus monkey, rat, cow, and rhea (an ostrich-like bird). O-Linkage of the saccharide is demonstrated by sensitivity to base-catalyzed beta-elimination and resistance to peptide:N-glycosidase F treatment. Chromatographic analyses of the beta-elimination products and fast atom bombardment-mass spectrometry of [3H]Gal-labeled tryptic peptides confirm the saccharide structure. Isoelectric focusing of [3H]Gal-labeled bovine lens proteins reveals the presence of O-GlcNAc on all four alpha-crystallin subunits, A1, A2, B1, and B2. Electrospray mass spectrometry of bovine alpha-crystallin demonstrates the presence of a single O-GlcNAc substitution on alpha A2. Gas-phase protein sequencing and fast atom bombardment-mass spectrometry of the major radiolabeled tryptic peptide from bovine alpha-crystallin reveal that GlcNAc is attached to the alpha A subunits at serine 162. This post-translational modification may play an important role in the molecular organization of lens alpha-crystallin.

Expression of duck lens delta-crystallin cDNAs in yeast and bacterial hosts. Delta 2-crystallin is an active argininosuccinate lyase.

The major soluble protein in the lenses of most birds and reptiles is delta-crystallin. In chickens and ducks the delta-crystallin gene has duplicated, and in the duck both genes contribute to the protein in the lens, while in the chicken lens there is a great preponderance of the delta 1 gene product. Purified delta-crystallin has previously been shown to possess the enzymatic activity of argininosuccinate lyase. In order to determine the enzymatic properties of the two duck delta-crystallins their corresponding cDNA molecules were placed in yeast and bacterial expression plasmids. In Saccharomyces cerevisiae, the activity of each crystallin was assessed by transformation of the expression plasmids into a strain deficient for argininosuccinate lyase activity. The ability of the resulting yeast to grow on arginine deficient medium was used as a measure of enzymatic activity. Yeast expressing the duck delta 2-crystallin protein grew rapidly, while those expressing delta 1-crystallin failed to grow. Enzyme activity measurements confirmed the presence of activity in the delta 2-crystallin-expressing yeast, and no detectable activity could be demonstrated in the delta 1-crystallin-expressing yeast. Northern blotting of RNA from the transformed yeast revealed equal levels of mRNA species from the two constructs. For further analysis, the delta 2-crystallin cDNA was placed in the bacterial expression plasmid, pET-3d. The delta 2-crystallin protein produced in Escherichia coli was purified to homogeneity and analyzed to determine the kinetic properties. A Km of 0.35 mM was determined for argininosuccinate and a Vm of 3.5 mumols/min/mg was determined. These data demonstrate that, following duplication of the primordial argininosuccinate lyase gene, one of the genes maintained its role as an enzyme (delta 2-crystallin) while also serving as a crystallin and the other has evolved to specialize as a structural protein in the lens (delta 1-crystallin), presumably losing most or all of its catalytic capacity.

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.

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.