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.     

N-terminal sequences of gamma-crystallins from the amphibian lens and their homology with gamma-crystallins of other major classes of vertebrates

gamma-Crystallins were isolated from the homogenate of frog eye lenses (Rana catesbeiana) by exclusion gel chromatography and further purified by cation-exchange chromatography. They were the only group of crystallins possessing free amino groups amenable to sequence analysis by Edman degradation. Comparison of the amino acid contents of the purified subfractions of gamma-crystallins indicated their close relatedness in amino acid compositions and probably sequence homology as well. The amino-terminal sequence analysis of the purified gamma-crystallin subfractions showed extensive homology between these amphibian gamma-crystallin polypeptides themselves and also those from other vertebrate species, suggesting the existence of a multigene family and their close relatedness to gamma-crystallins of other vertebrates. The sequence comparison of the gamma-crystallin polypeptides from all major classes of vertebrates has provided strong support for the divergent evolution of gamma-crystallin family.     

Biochemical characterization of crystallins from frog lenses

Lens crystallins were isolated from the homogenate of frog (Rana catesbeiana) eye lenses by gel permeation chromatography and characterized by gel electrophoresis, amino acid analysis and circular dichroism. Four well-defined fractions corresponding to alpha/beta-, beta-, frog 39.5 kDa and gamma-crystallins comprising the relative weight percentages in the total soluble cytoplasmic proteins of 18%, 15%, 14% and 48% respectively were obtained. The native molecular masses for each purified fraction were determined to be 432, 207, 40 and 23 kDa, respectively. The polypeptide compositions as determined by SDS-gel electrophoresis revealed the typical subunit structures of mammalian crystallins with the exception of 39.5 kDa monomeric crystallin, which has not been shown in other classes of vertebrate lenses. The spectra of circular dichroism indicate a predominant beta-sheet structure in all four fractions, which also bears a resemblance to the secondary structure of mammalian crystallins. Comparison of the amino acid compositions of frog crystallins with those of mammalian and fish crystallins suggests that gamma-crystallin from the frog is more closely related to that of porcine than fish crystallins, and the frog 39.5 kDa, frog beta- and lamprey 48 kDa crystallins are probably mutually interrelated.     

Comparison of the gamma-crystallins isolated from eye lenses of shark and carp. Unique secondary and tertiary structure of shark gamma-crystallin

gamma-Crystallin isolated from the shark of cartilaginous fishes was compared with the cognate gamma-crystallin from the carp of bony fishes. Distinct differences in amino acid compositions, primary, secondary and tertiary structures were found. The most salient features of shark gamma-crystallin lie in the fact that this crystallin possessed a significant alpha-helical structure in the peptide backbone as revealed by circular dichroism study, in contrast to those orthologous gamma-crystallins from other vertebrate species including bony fishes which all show a predominant beta-sheet secondary structure. The tertiary structure as reflected in the intrinsic microenvironments of various aromatic amino acids in the native crystallins also shows unambiguous differences between these two classes of gamma-crystallins. N-Terminal sequence analysis corroborates the structural differences between shark and carp gamma-crystallins. gamma-Crystallin from the more primitive shark seems to be more in line with the main evolutionary phylogeny leading to the modern mammalian gamma-crystallin.     

Homology models of human gamma-crystallins: structural study of the extensive charge network in gamma-crystallins

The lens is composed of highly stable and long-lived proteins, the crystallins which are divided into alpha-, beta-, and gamma-crystallins. Human gamma-crystallins belong to the betagamma superfamily. A large number of gamma-crystallins have been sequenced and have been found to share remarkable sequence homology with each other. Some of the gamma-crystallins from various sources have also been elucidated structurally by X-ray crystallographic or NMR spectroscopic experiments. Their three-dimensional structures are also similar having consisted of two domains each possessing two Greek key motifs. In this study we have constructed the comparative or homology models of the four major human gamma-crystallins, gammaA-,gammaB-, gammaC-, and gammaD-crystallins and studied the charge network in these crystallins. Despite an overall structural similarity between these crystallins, differences in the ion pair formation do exist which is partly due to the differences in their primary sequence and partly due to the structural orientation of the neighboring amino acids. In this study, we present an elaborate analysis of these charged interactions and their formation or loss with respect to the structural changes.     

Rat lens gamma-crystallins. Characterization of the six gene products and their spatial and temporal distribution resulting from differential synthesis

We have isolated, purified and characterized six individual gamma-crystallin polypeptides present in the rat lens. Comparison of their amino acid compositions with the known structure of the six gamma-crystallin genes permits a one-to-one correspondence to be made between each protein synthesized and the encoding gene. This demonstrates that each of the six genes is actually expressed in vivo. Two classes of three gamma-crystallins each, which we have designated classes gamma ABC and gamma DEF, are known to exist, on the basis of internal sequence homology. We have measured the temperature-dependent phase-separation characteristics of solutions of the six purified gamma-crystallins, and find that the three members of the gamma DEF class (gamma 2-2, gamma 3-1 and gamma 4-1) are all cryo-proteins with relatively high phase-separation temperatures, whereas the three gamma ABC crystallins (gamma 1-1, gamma 1-2 and gamma 2-1) do not show phase separation above -7 degrees C. We have measured the spatial distribution in rat lens of each of the alpha-, beta- and gamma-crystallins as a function of age from 1 to 420 days, using size-exclusion and ion-exchange high-pressure liquid chromatography (HPLC). Our findings in the cortical layer permit us to establish the differential synthesis of each of the crystallins during lens development. Particular attention has been devoted to the spatial and temporal distribution of the six individual gamma-crystallins. Up to birth, synthesis of the three components of the gamma DEF class predominates, and in particular that of gamma 2-2. In subsequent development the three components of the gamma ABC class assume a greater proportion of monomeric crystallins synthesized, while beta s-crystallin synthesis predominates in late development. Our analysis of different layers within single lenses provides novel information on spatial gradients of the water-soluble and water-insoluble protein fractions as a function of age. We consider the consequences of these findings for lens transparency and opacity in both rat and mouse lens. We show that the high concentrations of gamma DEF-crystallins appear to be responsible for the opacity known to occur in young rat lenses. We conclude from these observations that close control of the differential synthesis of gamma-crystallins plays an important role in maintaining lens transparency during development.     

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.     

A novel crystallin from octopus lens

Lens crystallins were isolated from cephalopods, octopus and squid. Two protein fractions were obtained from the octopus in contrast to only one crystallin from the squid. The native molecular mass for these purified fractions and their polypeptide compositions were determined by gel filtration, sedimentation analysis, and SDS-gel electrophoresis. Octopod and decapod lenses share one common major squid-type crystallin of 29 kDa, with one additional novel crystallin present only in the octopus lens. This newly-characterized crystallin (termed omega-crystallin) exists as a tetrameric protein of 230 kDa, consisting of 4 identical subunits of approx. 59 kDa. It is distinct from the previously known crystallins both in amino acid composition and subunit structure. N-terminal sequence analysis indicated that the omega-crystallin is N-terminally blocked, whereas the major octopus crystallin is identical to the reported squid crystallin with regard to the first 25 residues of protein sequence. Sequence similarity between this major cephalopod crystallin and glutathione S-transferase were found, which suggested some enzymatic role of crystallins inside the cephalopod lens.     

Purification and characterization of an extracellular beta-1,4-mannanase from a marine bacterium, Vibrio sp. strain MA-138.

A beta-mannanase (EC 3.2.1.78) from Vibrio sp. strain MA-138 was purified by ammonium sulfate precipitation and several chromatographic procedures including gel filtration, adsorption, and ion-exchange chromatographies. The final ion-exchange chromatography Mono Q yielded one major active fraction and three minor active fractions. The major active fraction was purified to homogeneity on the basis of native polyacrylamide gel electrophoresis (PAGE). This purified enzyme was identified as a glycoprotein by periodic acid-Schiff staining and a monomeric protein with a molecular mass of 49 kDa by sodium dodecyl sulfate-PAGE. The pI of the enzyme was 3.8. The purified enzyme exhibited maximal activity at pH 6.5 and 40 degrees C and hydrolyzed at random the internal beta-1,4-mannosidic linkages in beta-mannan to give various sizes of oligosaccharides. The first 20 N-terminal amino acid sequence of the purified enzyme showed high homology with the N-terminal region of beta-mannanase from Streptomyces lividans 66.     

The protein sequence homology of γ-crystallins among major vertebrate classes and their DNA sequence homology to heat-shock protein genes

A systematic characterization of lens crystallins from five major classes of vertebrates was carried out by exclusion gel filtration, cation-exchange chromatography and N-terminal sequence determination. All crystallin fractions except that of γ-crystallin were found to be N-terminally blocked. γ-Crystallin is present in major classes of vertebrates except the bird, showing none, or decreased amounts, of this protein in chicken and duck lenses, respectively. N-Terminal sequence analysis of the purified γ-crystallin polypeptides showed extensive homology between different classes of vertebrates, supporting the close relatedness of this family of crystallin even from the evolutionarily distant species. Comparison of nucleotide sequences and their predicted amino acid sequences between γ-crystallins of carp and rat lenses and heat-shock proteins demonstrated partial sequence homology of the encoded polypeptides and striking homology at the gene level. The unexpected strong homology of complementary DNA (cDNA) lies in the regions coding for 40 N-terminal residues of carp γ-II, rat γ2-1, and the middle segments of 23,000- and 70,000-M _r heat-shock proteins. The optimal alignment of DNA sequences along these two segments shows about 50% homology. The percentage of protein sequence identity for the corresponding aligned segments is only 20%. The weak sequence homology at the protein level is also found between the invertebrate squid crystallin and rat γ-crystallin polypeptides. These results pointed to the possibility of unifying three major classes of vertebrate crystallins into one α/β/γ superfamily and corroborated the previous supposition that the existing crystallins in the animal kingdom are probably mutually interrelated, sharing a common ancestry.     

Structural homology of lens crystallins. A method to detect protein structural homology from primary sequences

The X-ray crystallographic structure of bovine gamma-crystallin shows four similar folding motifs each composed of about 42 residues arranged as four topologically sequential, anti-parallel beta-strands. Since the beta and gamma-crystallin sequences show good homology, proposals for a four-motif beta-crystallin model have been made. The other bovine eye-lens protein species, alpha-crystallins, are not homologous to beta or gamma-crystallin in primary structure. In the present work, smoothed plots of amino acid sequence number versus a residue characteristic (e.g. hydrophobicity) were calculated for the various crystallins. Cross-correlation coefficients were then determined between pairs of crystallin plots for various registers of the curves. The correlation plots were then combined for several characteristics and for pairwise comparisons between beta or gamma-crystallin and the alpha-crystallins. The resulting plots showed four peaks separated by about 42 residues for the alpha-crystallins, suggesting that they also possess a four-motif beta-barrel structure. The physical parameter comparison technique appears generally applicable in suggesting a structural and functional relationship amongst proteins that show no primary sequence homology.     

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.     

beta s-Crystallin: structure and evolution of a distinct member of the beta gamma-superfamily

The nucleotide sequence of the cDNA of bovine lens beta s-crystallin has been determined, and the derived amino acid sequence has been confirmed by amino acid compositions and partial sequences of the tryptic peptides of this monomeric protein. beta s-Crystallin has a length of 177 residues, corresponding to a mol. wt. of 20 773, and a blocked N-terminal serine. Comparison of beta s with the known sequences of other beta- and gamma-crystallins, and computer construction of a phylogenetic tree of these sequences, shows beta s to be more closely related to the monomeric gamma-crystallins than to the oligomeric beta-crystallins. Also the tertiary structure of beta s modelled by interactive computer graphics on the coordinates of gamma II-crystallin, revealed similarities with the gamma-crystallins which might explain its monomeric behavior: the presence of a very short N-terminal 'arm' as compared with the beta-crystallins; a distribution of charged residues on the surface as in the gamma-crystallins; and finally the nature of certain residues of its inter-domain contacts. beta s-Crystallin seems to be an old and isolated offshoot of the gamma-family, and, considering its ancient origin, might well be present in other, non-mammalian, vertebrate classes.     

Glycation by ascorbic acid oxidation products leads to the aggregation of lens proteins

Previous studies from this laboratory have shown that there are striking similarities between the yellow chromophores, fluorophores and modified amino acids released by proteolytic digestion from calf lens proteins ascorbylated in vitro and their counterparts isolated from aged and cataractous lens proteins. The studies reported in this communication were conducted to further investigate whether ascorbic acid-mediated modification of lens proteins could lead to the formation of lens protein aggregates capable of scattering visible light, similar to the high molecular aggregates found in aged human lenses. Ascorbic acid, but not glucose, fructose, ribose or erythrulose, caused the aggregation of calf lens proteins to proteins ranging from 2.2 x 10(6) up to 3.0 x 10(8 )Da. This compared to proteins ranging from 1.8 x 10(6) up to 3.6 x 10(8 )Da for the water-soluble (WS) proteins isolated from aged human lenses. This aggregation was likely due to the glycation of lens crystallins because [U-(14)C] ascorbate was incorporated into the aggregate fraction and because NaCNBH(3), which reduces the initial Schiff base, prevented any protein aggregation. Reactions of ascorbate with purified crystallin fractions showed little or no aggregation of alpha-crystallin, significant aggregation of beta(H)-crystallin, but rapid precipitation of purified beta(L)- and gamma-crystallin. The aggregation of lens proteins can be prevented by the binding of damaged crystallins to alpha-crystallin due to its chaperone activity. Depending upon the ratios between the components of the incubation mixtures, alpha-crystallin prevented the precipitation of the purified beta(L)- and gamma-crystallin fractions during ascorbylation. The addition of at least 20% of alpha-crystallin by weight into glycation mixtures with beta(L)-, or gamma-crystallins completely inhibited protein precipitation, and increased the amount of the high molecular weight aggregates in solution. Static and dynamic light scattering measurements of the supernatants from the ascorbic acid-modified mixtures of alpha- and beta(L)-, or gamma-crystallins showed similar molar masses (up to 10(8 )Da) and hydrodynamic diameter (up to 80( )nm). These data support the hypothesis, that if the lens reducing environment is compromised, the ascorbylation of lens crystallins can significantly change the short range interactions between different classes of crystallins leading to protein aggregation, light scattering and eventually to senile cataract formation.     

Evolutionary conservatism of the molecular structure of gamma-crystallins of vertebrates

Homology of 18 amino acid sequences of lens gamma-crystallins of several vertebrates: frog, mouse, rat, calf and human being--has been considered. Pair sequence homology varies in the range from 57 to 100%, the mean value is equal to 74%. The spatial structures have been determined only for two calf gamma-crystallins. The protein molecule consists of four-fold repeated "motifs" (patterns) which are joint in two domains. After comparison of 18 gamma-crystallin sequences it was found that "motifs" domains and whole protein molecules have about 10, 30 and 58% conservative residues, respectively, that seem to be related to the evolution of these structural units. Structure analysis shows that almost all the conservative residues have an important structural meaning and play a basic role in the domain and molecular structure organization. This result allows us to make a conclusion about the homology of spatial structures of all considered gamma-crystallins of vertebrates.     

Thermodynamics of thermal and athermal denaturation of gamma-crystallins: changes in conformational stability upon glutathione reaction

The conformational stabilities of bovine lens gamma-crystallin fractions II, IIIA, IIIB, and IVA and those modified with glutathione were compared by studying the thermal and guanidine hydrochloride (Gdn-HCl) denaturation behavior. The conformational state was monitored by both far-UV CD and fluorescence measurements. All the gamma-crystallins studied showed a sigmoidal order-disorder transition with varied melting temperatures. The thermal denaturation of these proteins is reversible up to a temperature 3 or 4 degrees C above T 1/2; above this temperature, irreversible aggregation occurs. The validity of a two-state approximation of both thermal and Gdn-HCl denaturation was tested for all four crystallins, and the presence of one or more intermediates was evident in the unfolding of IVA. delta GDH2O values of these crystallins range from 4 to 9 kcal/mol. Upon glutathione treatment IVA showed the maximum decrease in T 1/2 by approximately 9 degrees C and in delta GDH2O value by 29%; the smallest decrease in T 1/2 was for IIIA by 2 degrees C and in delta GDH2O by 15%. We have demonstrated that the glutathione reaction can dramatically reduce the conformational stability of gamma-crystallins and, thus, that the thermodynamic quantities of the unreacted crystallins can be used to evaluate the stability of these proteins when modified during cataract formation.     

Structural characterization of antigens encoded by rabbitRLA-11 histocompatibility genes

Products of rabbitRLA-11 histocompatibility genes were isolated from rabbit lymphoid tumor cells (RL-5) and characterized. The tumor cells were grown in culture with radioactive amino acids and were lysed by treatment with the detergent NP-40. Glycoprotein molecules were isolated by affinity chromatography using immobilized lentil lectin. Chromatography on purified sheep anti-rabbit beta-2-microglobulin yielded a 43,000 dalton glycoprotein fraction, designated RLA-11gp, which was noncovalently associated with beta-2-microglobulin. N-terminal amino acid sequence analysis using radiochemical methods allowed assignment of 28 of the N-terminal 35 residues. The data revealed 89% homology of the RLA-11gp N-terminus with that of the human HLA-B7 and 82% with the mouse H-2K^b histocompatibility antigens. Comparison of the RLA-11 gp N-terminal sequence data obtained in this work to sequence data reported for major histocompatibility complex antigens of other species revealed no amino acid substitutions unique to the rabbit antigens.     

Immunochemical study of the water-soluble lens proteins in the embryo of Xenopus laevis with the mutation of periodic albinism]

The crystallins of ap mutants of Xenopus laevis have been studied in comparison with those of normal embryos and adults using the complex of immunochemical methods (immunoelectrophoresis, immunodiffusion, immunoadsorption, immunofluorescence, isoelectrofocusing with immunoidentification). The analysis was carried out with antisera to electrophoretic fractions of the mutant lens. 11 organ-specific antigens were found in the lens of both the normal and mutant animals. These proteins are heterogenous by electrophoretic mobility, isoelectrical point, antigenic and species specificity. Each class of crystallins contains antigens which are specific: a) for amphibians only, b) for lower vertebrates, c) for vertebrates in general. No qualitative differences were found between crystallins of the normal and mutant animals. Immunofluorescence analysis has shown that crystalins appear in the normal and mutant embryos practically at the same time. No significant differences in the appearance of specific immunofluorescence between the normal and mutant embryos were found (with various antisera). gamma-crystallins and, perhaps, a part of the primary lens fibers. Alpha-crystallins appear later. gamma-crystallins are first identified the synthesis of which manifests itself at the advanced developmental stages. The quantitative predominance of some beta--gamma-crystallins in the mutant lens detected by us (electrophoresis, isoelectrofocusing) is not related to their earlier synthesis in the embryogenesis.     

Gamma-crystallin genes in carp: cloning and characterization.

The carp gamma-crystallin gene family was found to be composed of at least three members: gamma m1, gamma m2 and gamma m3. The encoded products are very similar to other known gamma-crystallins but with their own peculiarities: (1) they all have a high methionine content: 12.4%, 14% and 8.4% in gamma m1, gamma m2 and gamma m3, respectively; and (2) the amino acid sequences are aberrant in the region before connecting peptides and its corresponding region in motif 4. Their protein structures might remain the same as those of other gamma-crystallins since they retain all the conserved amino acid residues essential for maintaining the loops in the protein structures.