A novel keratocan mutation causing autosomal recessive cornea plana.

PURPOSE: Mutations in keratocan (KERA), a small leucine-rich proteoglycan, have recently been shown to be responsible for cases of autosomal recessive cornea plana (CNA2). A consanguineous pedigree in which cornea plana cosegregated with microphthalmia was investigated by linkage analysis and direct sequencing. METHODS: Linkage was sought to polymorphic microsatellite markers distributed around the CNA2 and microphthalmia loci (arCMIC, adCMIC, NNO1, and CHX10) using PCR and nondenaturing polyacrylamide gel electrophoresis before KERA was directly sequenced for mutations. RESULTS: Positive lod scores were obtained with markers encompassing the CNA2 locus, the maximum two-point lod scores of 2.18 at recombination fraction theta = 0 was obtained with markers D12S95 and D12S327. Mutation screening of KERA revealed a novel single-nucleotide substitution at codon 215, which results in the substitution of lysine for threonine at the start of a highly conserved leucine-rich repeat motif. Structural modeling predicts that the motifs are stacked into an arched beta-sheet array and that the effect of the mutation is to alter the length and position of one of these motifs. CONCLUSIONS: This report describes a novel mutation in KERA that alters a highly conserved motif and is predicted to affect the tertiary structure of the molecule. Normal corneal function is dependent on the regular spacing of collagen fibrils, and the predicted alteration of the tertiary structure of KERA is the probable mechanism of the cornea plana phenotype.

Vision in the ultraviolet.

Sensitivity to ultraviolet light (UV) is achieved by photoreceptors in the eye that contain a class of visual pigments maximally sensitive to light at wavelengths <400 nm. It is widespread in the animal kingdom where it is used for mate choice, communication and foraging for food. UV sensitivity is not, however, a constant feature of the visual system, and in many vertebrate species, the UV-sensitive (UVS) pigment is replaced by a violet-sensitive (VS) pigment with maximal sensitivity between 410 and 435 nm. The role of protonation of the Schiff base-chromophore linkage and the mechanism for tuning of pigments into the UV is discussed in detail. Amino acid sequence analysis of vertebrate VS/UVS pigments indicates that the ancestral pigment was UVS, with loss of UV sensitivity occurring separately in mammals, amphibia and birds, and subsequently regained by a single amino acid substitution in certain bird species. In contrast, no loss of UV sensitivity has occurred in the UVS pigments of insects.

Identification and functional consequences of a new mutation (E155G) in the gene for GCAP1 that causes autosomal dominant cone dystrophy.

Mutations in the gene for guanylate cyclase-activating protein-1 (GCAP1) (GUCA1A) have been associated with autosomal dominant cone dystrophy (COD3). In the present study, a severe disease phenotype in a large white family was initially shown to map to chromosome 6p21.1, the location of GUCA1A. Subsequent single-stranded conformation polymorphism analysis and direct sequencing revealed an A464G transition, causing an E155G substitution within the EF4 domain of GCAP1. Modeling of the protein structure shows that the mutation eliminates a bidentate amino acid side chain essential for Ca2+ binding. This represents the first disease-associated mutation in GCAP1, or any neuron-specific calcium-binding protein within an EF-hand domain, that directly coordinates Ca2+. The functional consequences of this substitution were investigated in an in vitro assay of retinal guanylate cyclase activation. The mutant protein activates the cyclase at low Ca2+ concentrations but fails to inactivate at high Ca2+ concentrations. The overall effect of this would be the constitutive activation of guanylate cyclase in photoreceptors, even at the high Ca2+ concentrations of the dark-adapted state, which may explain the dominant disease phenotype.

Interactions within the coiled-coil domain of RetGC-1 guanylyl cyclase are optimized for regulation rather than for high affinity.

RetGC-1, a member of the membrane guanylyl cyclase family of proteins, is regulated in photoreceptor cells by a Ca(2+)-binding protein known as GCAP-1. Proper regulation of RetGC-1 is essential in photoreceptor cells for normal light adaptation and recovery to the dark state. In this study we show that cGMP synthesis by RetGC-1 requires dimerization, because critical functions in the catalytic site must be provided by each of the two polypeptide chains of the dimer. We also show that an intact alpha-helical coiled-coil structure is required to provide dimerization strength for the catalytic domain of RetGC-1. However, the dimerization strength of this domain must be precisely optimized for proper regulation by GCAP-1. We found that Arg(838) within the dimerization domain establishes the Ca(2+) sensitivity of RetGC-1 by determining the strength of the coiled-coil interaction. Arg(838) substitutions dominantly enhance cGMP synthesis even at the highest Ca(2+) concentrations that occur in normal dark-adapted photoreceptor cells. Molecular dynamics simulations suggest that Arg(838) substitutions disrupt a small network of salt bridges to allow an abnormal extension of coiled-coil structure. Substitutions at Arg(838) were first identified by linkage to the retinal degenerative disease, autosomal dominant cone rod dystrophy (adCORD). Consistent with the characteristics of this disease, the Arg(838)-substituted RetGC-1 mutants exhibit a dominant biochemical phenotype. We propose that accelerated cGMP synthesis in humans with adCORD is the primary cause of cone-rod degeneration.

The destabilization of human GCAP1 by a proline to leucine mutation might cause cone-rod dystrophy.

Guanylate cyclase activating protein-1 (GCAP1) is required for activation of retinal guanylate cyclase-1 (RetGC1), which is essential for recovery of photoreceptor cells to the dark state. In this paper, experimentally derived observations are reported that help in explaining why a proline-->leucine mutation at position 50 of human GCAP1 results in cone-rod dystrophy in a family carrying this mutation. The primary amino acid sequence of wild-type GCAP1 was mutated using site-directed mutagenesis to give a leucine at position 50. In addition, serine replaced a glutamic acid residue at position 6 to promote N-terminal myristoylation, yielding the construct GCAP1 E6S/P50L. The enzyme was over-expressed in Escherichia coli cells, isolated and purified before being used in assays with RetGC1, characterized by circular dichroism (CD) spectroscopy, and investigated for protease resistance and thermal stability. Assays of cyclic guanosine monophosphate (cGMP) synthesis from guanosine triphosphate by RetGC1 in the presence of E6S/P50L showed that E6S/P50L could activate RetGC1 and displayed similar calcium sensitivity to wild-type GCAP1. In addition, E6S/P50L and wild-type GCAP1 possess similar CD spectra. However, there was a marked increase in the susceptibility to protease degradation and also a reduction in the thermal stability of E6S/P50L as observed by both the cGMP assay and CD spectroscopy. It is therefore suggested that although GCAP1 E6S/P50L has a similar activity and calcium dependency profile to the wild-type GCAP1, its lower stability could reduce its cellular concentration, which would in turn alter [Ca2+] and result in death of cells.

Functional characterization of missense mutations at codon 838 in retinal guanylate cyclase correlates with disease severity in patients with autosomal dominant cone-rod dystrophy.

Three different mutations in codon 838 of GUCY2D, the gene for retinal guanylate cyclase 1, have been linked to autosomal dominant cone-rod dystrophy at the CORD6 locus. To examine the relationship between enzyme activity and disease severity, the three disease-causing substitutions (R838C, R838H and R838S) and four artificial mutations (R838A, R838E, R838L and R838K) were generated. Assay of GCAP1-stimulated cyclase activity in vitro shows that, compared with wild-type, R838E, R838L and R838K possess only low activity, whereas R838A, R838C, R838H and R838S have activity equal or superior to wild-type at low Ca(2+) concentrations. These four latter mutants showed a higher apparent affinity for GCAP1 than did wild-type. The Ca(2+) sensitivity of the GCAP1 activation was also altered with marked residual activity at high Ca(2+), the effect increasing: wild-type < R838C < R838H << R838A < R838S. Within the photoreceptor, this would result in a failure to inactivate cyclase activity at high physiological Ca(2+ )concentrations. Amongst the three disease-associated mutations, the effect correlates directly with disease severity. The wild-type and R838H mutant displayed a difference in pH sensitivity, with the mutant showing a higher specific activity with pH > 6.0. Site 838 is in the dimerization domain that forms a coiled-coil in the active protein. A computer-aided structure prediction of this region indicates that R838 in the wild-type breaks the structure at four helical turns, and there is an increasing tendency for the structure to continue for further turns in the order R838C < R838H,S,K << R838E < R838A < R838L.

Spectral tuning of avian violet- and ultraviolet-sensitive visual pigments.

The violet- and ultraviolet-sensitive visual pigments of birds belong to the same class of pigments as the violet-sensitive (so-called blue) pigments of mammals. However, unlike the pigments from mammals and other vertebrate taxa which, depending on species, have lambda(max) values of either around 430 nm or around 370 nm, avian pigments are found with lambda(max) values spread across this range. In this paper, we present the sequences of two pigments isolated from Humbolt penguin and pigeon with intermediate lambda(max) values of 403 and 409 nm, respectively. By comparing the amino acid sequences of these pigments with the true UV pigments of budgerigar and canary and with chicken violet with a lambda(max) value of 420 nm, we have been able to identify five amino acid sites that show a pattern of substitution between species that is consistent with differences in lambda(max). Each of these substitutions has been introduced into budgerigar cDNA and expressed in vitro in COS-7 cells. Only three resulted in spectral shifts in the regenerated pigment; two had relatively small effects and may account for the spectral shifts between penguin, pigeon, and chicken whereas one, the replacement of Ser by Cys at site 90 in the UV pigments, produced a 35 nm shortwave shift that could account for the spectral shift from 403 nm in penguin to around 370 nm in budgerigar and canary.

Visual pigments and oil droplets in the retina of a passerine bird, the canary Serinus canaria: microspectrophotometry and opsin sequences.

The visual receptors of the passeriform bird Serinus canaria, the canary, have been examined microspectrophotometrically and the sequences of the opsins determined. Rods have a maximum absorbance (lambda max) at 506 nm. Four spectral classes of single cone are present: long-wave-sensitive (LWS) containing a photopigment with lambda max at 569 nm, middle-wave-sensitive (MWS) with lambda max at 505 nm, short-wave-sensitive (SWS) with lambda max at 442 nm, and ultraviolet-sensitive (UVS) with lambda max at about 366 nm. Double cones possess the 569-nm pigment in both members. Typical combinations of photopigment and oil droplet occur in most cone classes. An ambiguity exists in the oil droplet of the single LWS cones. In some birds, LWS cones are paired with an R-type droplet, whereas in the majority of canaries the LWS pigment is paired with a droplet similar to the P-type of double cones. Mechanisms of spectral tuning within each opsin class are discussed.

Recombinant expression, purification, and characterization of three isoenzymes of aspartate aminotransferase from Arabidopsis thaliana.

Five different genes encoding isoenzymes of aspartate aminotransferase (AAT) have been identified in the plant Arabidopsis thaliana. cDNA sequences encoding three of these AAT isoenzymes, asp1 (mitochondrial), asp2 (cytosolic), and asp5 (plastid), were manipulated into bacterial expression vectors and the recombinant proteins expressed were purified from liquid culture using conventional methods. Yields of the purified isoenzymes varied from 11.5 mg/g wet wt cells (AAT5) to 0.95 mg/g wet wt cells (AAT2), an improvement of more than 1000-fold over typical yields of native isoenzymes obtained from plant tissues of other species. Analysis of the recombinant proteins on denaturing PAGE gels indicated subunit Mrs of between 44 and 45 K. Kinetic parameters (Km and kcat) obtained for all four substrates (aspartate, alpha-ketoglutarate, glutamate, and oxaloacetate) were consistent with values obtained for native AAT isoenzymes from other plant species. Further characterization of the purified recombinant enzymes alongside native enzymes from A. thaliana leaf tissue on AAT activity gels confirmed the identity of asp1 and asp2 as the mitochondrial and cytosolic AAT genes but indicated that asp5 may encode an amyloplastic rather than the chloroplastic enzyme.

The molecular basis for UV vision in birds: spectral characteristics, cDNA sequence and retinal localization of the UV-sensitive visual pigment of the budgerigar (Melopsittacus undulatus).

Microspectrophotometric (msp) studies have shown that the colour-vision system of many bird species is based on four pigments with absorption peaks in the red, green, blue and UV regions of the spectrum. The existence of a fourth pigment (UV) is the major difference between the trichromacy of humans and the tetrachromacy of such birds, and recent studies have shown that it may play a determining role in such diverse aspects of behaviour as mate selection and detection of food. Avian visual pigments are composed of an opsin protein covalently bound via a Schiff-base linkage to the chromophore 11-cis-retinal. Here we report the cDNA sequence of a UV opsin isolated from an avian species, Melopsittacus undulatus (budgerigar or small parakeet). This sequence has been expressed using the recombinant baculovirus system; the pigment generated from the expressed protein on addition of 11-cis-retinal yielded an absorption spectrum typical of a UV photopigment, with lambdamax 365+/-3 nm. This is the first UV opsin from an avian species to be sequenced and expressed in a heterologous system. In situ hybridization of this sequence to budgerigar retinas selectively labelled a sub-set of UV cones, representing approx. 9% of the total cone population, that are distributed in a semi-regular pattern across the entire retina.

Visual pigments and oil droplets from six classes of photoreceptor in the retinas of birds.

Microspectrophotometric examination of the retinal photoreceptors of the budgerigar (shell parakeet), Melopsittacus undulatus (Psittaciformes) and the zebra finch, Taeniopygia guttata (Passeriformes), demonstrate the presence of four, spectrally distinct classes of single cone that contain visual pigments absorbing maximally at about 565, 507, 430-445 and 360-380 nm. The three longer-wave cone classes contain coloured oil droplets acting as long pass filters with cut-offs at about 570, 500-520 and 445 nm, respectively, whereas the ultraviolet-sensitive cones contain a transparent droplet. The two species possess double cones in which both members contain the long-wave-sensitive visual pigment, but only the principal member contains an oil droplet, with cut-off at about 420 nm. A survey of the cones of the pigeon, Columba livia (Columbiformes), confirms the presence of the three longer-wave classes of single cone, but also reveals the presence of a fourth class containing a visual pigment with maximum absorbance at about 409 nm, combined with a transparent droplet. No evidence was found for a fifth, ultraviolet-sensitive receptor. In the chicken, Gallus gallus (Galliformes), the cone class with a transparent droplet contains "chicken violet" with maximum absorbance at about 418 nm. The rods of all four species contain visual pigments that are spectrally similar, with maximum absorbance between about 506 and 509 nm. Noticeably, in any given species, the maximum absorbance of the rods is spectrally very similar to the maximum absorbance of the middle-wavelength-sensitive cone pigments.

Characterisation of the ultraviolet-sensitive opsin gene in the honey bee, Apis mellifera.

The cDNA sequence of the ultraviolet-sensitive opsin in the honey-bee, Apis mellifera, with associated 5' and 3' untranslated regions, is presented. The analysis of genomic structure reveals seven introns in the coding region of the gene, with six at novel positions for an insect opsin gene. The equivalent site to the counterion in vertebrate opsins is occupied by a Tyr residue. This contrasts with the presence of Phe at this site in the ultraviolet-sensitive opsins of Drosophila sps. A comparison of the amino acid sequence within the seven alpha-helical transmembrane regions of insect ultraviolet/blue-sensitive opsins identifies substitution at five sites that involve either replacement of a polar with a non-polar residue, or a change in charge. Such changes are known to result in spectral shifts in vertebrate pigments. Phylogenetic analysis indicates that the ultraviolet-sensitive pigments represent an ancient class of insect opsins.

The rod and green cone opsins of two avian species, the budgerigar, Melopsittacus undulatus, and the mallard duck, Anas platyrhynchus.

The genes for the rod and rod-like green cone opsins in two avian species, the budgerigar, Melopsittacus undulatus, and the mallard duck, Anas platyrhynchus, are identified on the basis of amino acid identity with the equivalent chicken sequences and their placement into a single phylogenetic clade with the rod and rod-like green cone opsin genes from other vertebrate species. Since the two bird species studied are taxonomically quite distinct, this would indicate that this rod-like green cone opsin gene, although absent in mammals, is common in the Aves. The two avian pigments differ consistently at site 122, consistent with the reported role of this site in determining the rate of metarhodopsin II formation and decay in rod and cone pigments. Candidate sites are identified to compensate for the known spectral effects of substitution at this site.

Chloroplastic aspartate aminotransferase from Arabidopsis thaliana: an examination of the relationship between the structure of the gene and the spatial structure of the protein.

A clone encoding a plastid isoenzyme of aspartate amino-transferase (AAT5) was isolated from an Arabidopsis genomic library and its complete sequence determined. The gene for AAT5 (asp5) contains an open reading frame of 2447 bp comprising 11 exons separated by introns ranging in length from 74 to 207 bp. The upstream regulatory region contains a putative TATA box and multiple copies of two sequence motifs, CTCTT and AAAGAT, previously associated with nodule-specific gene activity in legumes. The deduced primary amino acid sequence of the protein product of asp5 was used to generate a three-dimensional structure of the AAT5 protein by using the computer program Sybyl: Biopolymer Composer and known AAT structures on the protein databases. Both the mature protein and its precursor protein containing a putative N-terminal transit peptide were modelled. The resulting structure of the precursor protein indicated that the transit peptide might also inhibit dimerization of the protein until after its translocation across the chloroplast membrane. The derived structure of the mature protein was then analysed in terms of its component elements of secondary structure, and the positions on the polypeptide back-bone corresponding to intron insertion sites were determined. It is observed that the introns tend to map to regions between structural subdomains of the protein and also map to sites on the surface of the molecule. The asp5 gene in Arabidopsis is thus consistent with Gilbert's exon-shuffling theory of gene evolution [Gilbert (1985) Science 228, 823-824]. A high degree of conservation of intron insertion sites between AAT genes from different plants and animals is observed, particularly within the part of the gene encoding a large beta-sheet structure that forms the structural and functional core of the protein. This beta-sheet structure is thus believed to compromise an ancient and very highly conserved moiety of the molecule.

Isolation, characterisation and expression of a cDNA clone encoding plastid aspartate aminotransferase from Arabidopsis thaliana.

A clone encoding aspartate aminotransferase (AAT, EC 2.6.1.1) was isolated from an Arabidopsis thaliana leaf cDNA library. This clone contains a 1365 bp open reading frame encoding a polypeptide of 49.8 kDa, designated Ataat1. The clone was shown to contain a chloroplastic isoenzyme as an in organellar protein import assay demonstrated that a radiolabelled transcription/translation product of 49.8 kDa was imported into viable pea chloroplasts and was subsequently processed to yield a mature protein of 45 kDa. The open reading frame corresponding to the predicted mature AAT was manipulated into an expression construct (pEC14). Transformed Escherichia coli cells containing pEC14 expressed up to 16 times more AAT activity than vector only controls, thus demonstrating conclusively that the clone encoded AAT.

Random amplified polymorphic DNA (RAPD) markers for genetic analysis inAllium.

RAPD analysis was applied to onion (Allium cepa) and otherAllium species in order to assess the degree of polymorphism within the genus and to investigate if this approach was suitable for genetic studies of onion. Seven cultivars ofA. cepa, including shallot, and single cultivars of Japanese bunching onion (A. fistulosum), chive (A. schoenoprasum), leek (A. ampeloprasum), and a wild relative of onion (A. roylei), were evaluated for variability using a set of 20 random 10-mer primers. Seven out of the twenty primers revealed scorable polymorphisms between cultivars ofA. cepa and these will be further evaluated for use in genetic mapping. Wide variations in banding profiles between species were observed with nearly every primer tested. These were assessed for use in systematic studies within the genus. Ninety-one band positions were scored (+/-) for all the cultivars studied. Genetic distances between each of the cultivars were calculated and cluster analysis was used to generate a dendrogram showing phylogenetic relationships between them. The resulting analysis was in broad agreement with previous classifications of the species studied, confirming the validity of the method. However, amongst the species studied, it placedA. roylei as the closest relative ofA. cepa, questioning the current classification of the former species in the section Rhizideum.