Genetic markers for identification of Patagonian toothfish and Antarctic toothfish

Fillet samples of the toothfish Dissostichus eleginoides and D. mawsoni can be distinguished readily by muscle proteins revealed by isoelectric focusing and mitochondrial DNA markers. The proteins also distinguish toothfish from other species marketed under similar trade names.     

Interaction of inflammation mediators (myeloperoxidase and defensin) with mixed monolayers from lens lipids and crystallins]

Accumulation of inflammation mediators (myeloperoxidase and defensin) on lens membranes was studied in a model system. For this purpose ability of these proteins to interact with mixed monolayers from the lens lipids and lens proteins (crystallins) was appreciated.     

Physicochemical characterization of lens crystallins from the carp and biochemical comparison with other vertebrate and invertebrate crystallins

Lens crystallins were isolated from the homogenate of carp (Cyprinus carpio) eye lenses by gel permeation chromatography and characterized by gel electrophoresis, immunodiffusion, amino acid analysis, circular dichroism, and protein sequence analysis. Three well-defined fractions corresponding to alpha/beta-, beta-, and gamma-crystallins were obtained in relative weight percentages of 26, 22, and 52%. The native molecular masses of the purified fractions were determined to be 410, 60, and 20 kDa, respectively. The polypeptide compositions as determined by SDS gel electrophoresis revealed the substantial presence of beta-crystallin polypeptides in the alpha-crystallin fraction; this is also evident in the fractionation of amphibian crystallins but is not common in the case of higher classes of vertebrates. The circular dichroism spectra indicate a predominant beta-sheet structure in all three fractions, albeit with some contribution of alpha-helical structure in the gamma-crystallin, the amino acid composition of which bears a resemblance to that of squid crystallin. Sequence comparison of carp gamma-crystallin with frog and calf gamma-crystallins indicates a high degree of homology in their N-terminal segments despite the dissimilarity of amino acid compositions and weak immunological cross-reactivity.     

Characterization of lens crystallins and their mRNA from the carp lenses

Crystallins from carp eye lenses have been isolated and characterized by gel permeation chromatography, SDS-gel electrophoresis, immunodiffusion and amino acid analysis. gamma-Crystallin is the most abundant class of crystallins and constitutes over 55% of the total lens cytoplasmic proteins. It is immunologically distinct from the alpha- and beta-crystallins isolated from the same lens and its antiserum shows a very weak cross-reaction to total pig lens antigens. Comparison of the amino acid compositions of carp gamma-crystallin with those of bovine gamma-II, haddock gamma- and squid crystallins indicates that gamma-crystallin from the carp is very closely related to that of the haddock, and probably also related to the invertebrate squid crystallin. In vitro translation of total mRNAs isolated from carp lenses confirms the predominant existence of gamma-crystallin. The genomic characterization of carp crystallin genes should provide some insight into the mechanism of crystallin evolution in general.     

Physicochemical characterization of a crystallin from the squid lens and its comparison with vertebrate lens crystallins

A crystallin was isolated from the homogenate of the Squid (Loligo pealii) lens by gel filtration on a Sepharose CL-6B (2.5 X 170 cm) column. Biochemical characterization showed it is a dimeric protein with a molecular weight of (5.1 +/- 0.4) X 10(4) and a Stokes' radius of 26A. Electrophoresis on a cellulose acetate membrane indicated it is a basic protein with an isoelectric point higher than 8.6. High resolution two-dimensional gel in 8 M urea/2% NP-40 resolved this crystallin into 6 charge isomers, each with a major subunit of molecular weight 29,000 daltons and a minor subunit of 27,000 daltons in a molar ratio of 3:1. The extreme susceptibility of the protein to denaturation and precipitation even at low temperature hampered further characterization of this crystallin under nondenaturing conditions. Amino acid analysis indicated it contains an unusually high content of methionine (12.8 mol%) which may have some bearing on the instability of this crystallin in vitro. Biochemical comparison of the squid crystallin with mammalian lens crystallins shows that it is a crystallin distinguishable from all reported vertebrate lens crystallins. A detailed study of this protein may shed light on the evolution of lens crystallins in general.     

Photoreactions of human lens monomeric crystallins

Human lens beta s- and gamma A-crystallins exhibit very similar tryptophan fluorescence emission maxima (329 nm). gamma A isolated from infant human lenses is photo-oxidized by 300 nm irradiation and forms water-insoluble aggregates; beta s or gamma A from young human lenses form a small amount of water-soluble crosslinked species. At least part of the mechanism of photodamage by 300 nm irradiation is photogeneration of the oxidant H2O2 via the generation of O2- radical, this reaction occurs via photosensitization by the tryptophan photo-oxidation product N-formylkynurenine (N-FK) or related species. These results indicate that even though the tryptophan residues of beta s- and gamma A-crystallins are in hydrophobic (buried) microenvironments as compared to those of the alpha- and beta-crystallins, the photogeneration of N-FK is sufficient to produce O2- and H2O2.     

Comparative study of crystallins from the nucleus and cortex of the bovine ocular lens by the gel filtration and x-ray diffraction methods

Water--soluble proteins (alpha-, beta H-, beta L- and gamma-crystallins) from the bovine lens nucleus and cortex were fractionated and compared by gel filtration on Sephadex G-200. X-ray diffraction patterns from concentrated gels of these proteins were obtained. It allowed to compare qualitatively the structures of different crystallins and also to identify the maxima on X-ray diffraction patterns of the lens intact tissue.     

Protein carboxyl methyltransferase from cow eye lens

Protein carboxyl methyltransferase activity (S-adenosyl-L-methionine: protein carboxyl-0-methyltransferase; E.C. 2.1.1.24) has been detected in crude soluble extracts of cow eye lens. The activity incorporates methyl groups from S-adenosyl-L-methionine into endogenous lens proteins in vitro, and several of these species co-migrate electrophoretically with lens crystallins. A 2600-fold purification of the enzyme free of endogenous substrates was achieved by gel filtration and affinity chromatography. The lens methyltransferase has a native molecular weight of approximately 27,000, and catalyzes the substoichiometric incorporation of highly alkali-labile methyl ester groups into a broad range of protein substrates. The lens enzyme appears to be similar to that found in human erythrocytes, which specifically recognizes and modifies D-aspartic acid residues in aged proteins in a postulated degradative or racemization-repair pathway (McFadden, P.N., and Clarke, S. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 2460-2464).     

Evolution of eye lens crystallins: the stress connection

Crystallins, the structural proteins of the eye lens, ensure the transparency and integrity of the lens throughout life. Recent sequence comparisons have shown that evolution has recruited crystallins among already existing heat-shock proteins and stress-inducible enzymes.     

The cellular eye lens and crystallins of cubomedusan jellyfish

Summary The ultrastructure and major soluble proteins of the transparent eye lens of two cubomedusan jellyfish,Tripedalia cystophora andCarybdea marsupialis, have been examined. Each species has two complex eyes (one large and one small) on four sensory structures called rhopalia. The lenses consist of closely spaced cells with few organelles. The lens is situated next to the retina, with only an acellular layer separating it from the photoreceptors. SDS-PAGE showed that the large lens ofC. marsupialis has only two crystallin polypeptide bands (with molecular masses of approximately 20000 and 35000 daltons), while that ofT. cystophora has three bands (two with a molecular mass near 20000 daltons and one with a molecular mass near 35000 daltons). Interestingly, the small lens ofT. cystophora appears to be markedly deficient in or lack the lower molecular weight proteins. The crystallins behaved as monomeric proteins by FPLC and showed no immunological reaction with antisera of the major squid crystallin, chicken-crystallin or mouse-crystallin in western immunoblots. Very weak reactions were found with antimouse- and-crystallin sera. The 35000 dalton crystallin ofT. cystophora was purified and called J1-crystallin. It contained relatively high leucine (13%) and tyrosine (9%) and low methionine (2%). Several tryptic peptides were sequenced. Weak sequence similarities were found with- and-crystallins, which may account for some of the apparent weak immunological crossreactivity with these vertebrate crystallins. A polyclonal antiserum made in rabbits from a synthetic peptide of J1-crystallin reacted strongly with J1-crystallin ofT. cystophora andC. marsupialis in immunoblots; by contrast, no reaction was obtained with the lower molecular weight crystallins from these jellyfish, with the squid crystallin, or with any crystallins from the frog or human lens. Thus, despite the structural similarities between the cubomedusan, squid and vertebrate lenses, their crystallins appear very different.Abbreviations SDS-PAGE sodium dodecylsulfate-polyacrylamide gel electrophoresis - bp base pairs - PTC phenylisothiocyanate - FPLC fast phase liquid chromatography - NBRF National Biomedical Research Foundation A portion of this work was presented by Joram Piatigorsky at the First Hans Bloemendal Lecture in June 1988 in Nijmegen, The Netherlands     

Isolation and physical characterization of bovine lens crystallins.

The soluble proteins from bovine lens homogenate were separated on Sepharose CL-6B (2 X 200 cm) in 0.05 M tris-NaHSO3 pH 8.2 buffer containing 20 mM EDTA. Five sharp and defined fractions (HM alpha, alpha, beta H, beta L, gamma) were obtained. Each crystallin fraction was further purified by rechromatography on the same column. Each protein fraction was pure as judged by ultracentrifugation and SDS-gel electrophoresis. The molecular weights of the five fractions were 3.04 x 10(6), 5.83 x 10(5), 1.58 x 10(5) , 4.59 x 10(4), 2.14 x 10(4) as determined from sedimentation coefficient and intrinsic viscosity data by Scheraga-Mandelkern equation, which was in close agreement with that obtained by gel filtration. The polypeptide composition of crystallins as determined by SDS-gel electrophoresis revealed one band for high molecular weight alpha (HM alpha) and alpha, three for beta H, two for beta L and one for gamma. The gross CD patterns of crystallins were about the same in the peptide region (200 nm similar to or approximately 250 nm) with a minimum centered at about 217 nm, indicative of a beta-sheet structure in all crystallins. The [theta] values at 217 nm ranged from --1700 to --3700 degrees cm2 per decimole. The CD spectra of these crystallins in the aromatic region (250 nm similar to or approximately 300 nm) were different, reflecting the different contributions of aromatic amino acids to the tertiary structure of crystallins.     

Aspects of body size and gonadal histology in the Antarctic toothfish, Dissostichus mawsoni, from McMurdo Sound, Antarctica

Twenty-eight specimens of the large notothenioid Dissostichus mawsoni were dissected after capture on a set line near the southern limit of its range in McMurdo Sound, Antarctica. Total length (L _T) averaged 127.3 cm (range 90–162 cm) and weight (W ) was 26.7 kg (range 10.4–60.3 kg). The length-weight relationship was W=3.44×10⁻⁵ L _T ^2.85 (n=28, r ²=0.96). Subcutaneous lipid thickness averaged 2.6 mm and showed no difference due to sex, but a significant weak relationship to W and L _T. Hepatosomatic index (I _H) was 1.6% for females and 1.7% for males; gonadosomatic index (I _G) was 0.9% for females and 0.2% for males. Although specimens were large and sexually mature, the histology indicated that the gonads of this November sample were in a resting stage. Testes lacked spermatids and spermatozoa. Oocytes were in the previtellogenic stage (91.2%) or in the first stage of vitellogenesis (8.8%). A few atretic oocytes and empty follicles indicated that some females in this sample had spawned previously. A summary of the life-cycle is also presented. Accepted: 27 September 1999     

A differential scanning calorimetric study of the bovine lens crystallins

Differential scanning calorimetry was performed on the five major lens crystallin fractions [HM-alpha, alpha, beta H, beta L, and (beta s + gamma)] of the bovine lens as well as on more purified forms of alpha- and gamma-crystallins. All were found to be relatively thermally stable although the alpha-crystallin were found to at least partially unfold at an approximately 10 degrees C lower temperature than the beta and gamma fractions. Increasing protein concentration had little effect on gamma-crystallin thermograms but had marked effects on those of the alpha- and beta-crystallins. Increases in the thermal stability with increasing protein concentration for the beta-crystallins can be explained most simply by the known beta L/beta H equilibrium, but, in the case of the alpha-crystallins, excluded volume effects may be an important factor. In both cases, the increased stability at high concentrations could be of physiological relevance. As well as the expected endothermic unfolding transitions, all of the lens crystallins revealed exothermic peaks that correlate with protein precipitation. Interestingly, this phenomenon occurs only after extensive structural alteration in the case of the alpha-crystallins but is present very early in the initial stages of structural perturbation of the beta- and gamma-crystallins.     

Electrophoretic variation in low molecular weight lens crystallins from inbred strains of rats

Analysis of rat lens soluble proteins by analytical isoelectric focusing detected two inherited electrophoretic differences in low molecular weight (LM) crystallins from inbred strains of rats (Rattus norvegicus). The polymorphic lens crystallins were shown to be similar to a genetically variant LM crystallin, LEN-1, previously described in mice (Mus musculus) and encoded on chromosome 1, at a locus linked to Pep-3 (dipeptidase). Linkage analysis demonstrated that the rat crystallin locus was loosely linked to Pep-3 at a recombination distance of 38 +/- 4.5 U. These data suggest the conservation of a large chromosomal region during the evolution of Rodentia and support the hypothesis that the gamma-crystallins are evolving more rapidly than alpha- or beta-crystallins.     

Protein kinase catalytic subunit (PKAcat) from bovine lens: purification, characterization and phosphorylation of lens crystallins

The purification and functional characterization of protein kinase A catalytic subunit (PKAcat) from bovine lens cytosol has been described. Purification to homogeneity has been achieved by using 100 kDa cut-off membrane filtration followed by Sephacryl S-300 chromatography and finally fractionating on High Q anion exchange column. The purified protein migrates as a single band of molecular mass ∼41 kDa on 12.5% SDS-PAGE. Proteomic data from ion trap LC-MS when analyzed through NCBI blast program reveals significant homology (52%) with bovine zeta-crystallin and also some homology with pig casein kinase I alpha chain (38%) and SLA-DR1 beta 1 domain (38%). The search does not indicate homology with any known catalytic subunit of PKA. Inspite of the significant homology with the zeta-crystallin, our protein is different from it in terms of molecular mass. pI value of the kinase (5.3) obtained from 2D analysis is also different from zeta-crystallin (8.5). The protein is found to contain 17% α-helix, 26.5% β-sheet, 21.4% turn and 34.7% random coil. The active catalytic subunit of the bovine lens cAMP-dependent kinase belongs to Type I Cα subtype. The enzyme shows maximum activity at 30 min incubation in presence of 5 mM MgCl₂ and 50 μM ATP. The kinase shows broad substrate specificity. It prefers Ser over Thr as phosphorylating residue. Phosphorylation of crystallin proteins, major protein fraction of bovine lens and phosphorylation of chaperone protein α crystallin by the kinase suggests that the kinase plays some crucial role in regulation of chaperone function within lens.     

Genetic variation among populations of the Antarctic toothfish: evolutionary insights and implications for conservation

Commercial fishing is having an increasingly negative impact on marine biodiversity, with over 70% of the world's fish stocks being fully exploited and, in many cases, overexploited. On top of this, the Commission for the Conservation of Antarctic Marine Living Resources (CCAMLR) has granted commercial fishing permits in the most remote marine environment on earth, the high-latitude Southern Ocean. The primary target of these new commercial fishing ventures is the large pelagic piscivorous predator, the Antarctic toothfish (Dissostichus mawsoni). Unfortunately, little information is available on the demography, genetics, or life history of this large fish. Without such information we have little idea as to the effects of commercial fishing on the population structure and survival of this species. In this study, we focus on patterns of genetic diversity within and between geographically disparate populations of the Antarctic toothfish, using randomly amplified polymorphic DNA markers. Results of our study showed high levels of genetic similarity within and between populations. Despite high levels of genetic similarity, genetic analyses detected significant population structure, including fixed differences among populations, a significant fixation index (Fst) and between-population differentiation via a Mantel test. From a conservation perspective, low levels of genetic diversity may be indicative of relatively small populations that would not be able to withstand heavy commercial fishing pressures. Given that there is evidence for significant genetic structure, it will be important to manage these fisheries in a manner that will help prevent the loss of unique genetic variation from regional overfishing.     

Evolution of crystallins for a role in the vertebrate eye lens

The camera eye lens of vertebrates is a classic example of the re‐engineering of existing protein components to fashion a new device. The bulk of the lens is formed from proteins belonging to two superfamilies, the α ‐crystallins and the β γ ‐crystallins. Tracing their ancestry may throw light on the origin of the optics of the lens. The α ‐crystallins belong to the ubiquitous small heat shock proteins family that plays a protective role in cellular homeostasis. They form enormous polydisperse oligomers that challenge modern biophysical methods to uncover the molecular basis of their assembly structure and chaperone‐like protein binding function. It is argued that a molecular phenotype of a dynamic assembly suits a chaperone function as well as a structural role in the eye lens where the constraint of preventing protein condensation is paramount. The main cellular partners of α ‐crystallins, the β ‐ and γ ‐crystallins, have largely been lost from the animal kingdom but the superfamily is hugely expanded in the vertebrate eye lens. Their structures show how a simple Greek key motif can evolve rapidly to form a complex array of monomers and oligomers. Apart from remaining transparent, a major role of the partnership of α ‐crystallins with β ‐ and γ ‐crystallins in the lens is to form a refractive index gradient. Here, we show some of the structural and genetic features of these two protein superfamilies that enable the rapid creation of different assembly states, to match the rapidly changing optical needs among the various vertebrates.     

Site-specific glycation of lens crystallins by ascorbic acid.

The oxidation of ascorbic acid leads to the formation of several compounds which are capable of reacting with protein amino groups via a Maillard reaction. Radioactivity from [1-14C]ascorbic acid was linearly incorporated into lens crystallins over a 10 day period in the presence of NaCNBH3. This rate of incorporation was 6-7-fold more rapid than that obtained with [14C]glucose under the same conditions. SDS-PAGE showed a linear incorporation into all the crystallin subunits. [1-14C]Ascorbic acid-label led alpha-crystallin was separated into its component A and B subunits, and each was digested with chymotrypsin. HPLC peptide analysis showed a differential labelling of the various lysine residues. Analysis of the peptides by mass spectrometry allowed the identification of the sites and the extent of modification. These values ranged from 6% for Lys-78 to 36% for Lys-11 in the A subunit and from 5% for Lys-82 to an average of 38% for the peptide containing Lys-166, Lys-174 and Lys-175 in the B subunit. Amino acid analysis demonstrated a single modification reaction producing N epsilon-(carboxymethyl)lysine. This agreed with the mass increase of 58 observed for each modified peptide.