Shyh-Horng Chiou

Phylogenetic comparison of lens crystallins from the vertebrate and invertebrate ‐ convergent or divergent evolution?

A systematic biochemical comparison has been made of the crystallins isolated from the lenses of five different species belonging to the five major classes of vertebrates. Gel‐permeation chromatography of the lens homogenates on Fractogel TSK HW‐55(S) revealed well‐defined elution patterns with a characteristic distribution of different classes of crystallins from each species. SDS gel electrophoresis and statistical comparison of the amino acid contents indicated that all crystallin groups from different classes share some common subunits and similarity in their amino acid compositions. The results coupled with the relatedness shown in the amino acid compositions of fish γ‐crystallin with those of mammalian γ‐crystallin and the squid crystallin from the invertebrate pointed to the possibility of the existence of a common ancestral protein for all crystallins. This is in favor of the divergent rather than convergent evolution of lens crystallins as commonly assumed in the literature.

Characterization of lens cyrstallins 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 Ω‐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 Ω‐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.

Biochemical comparison of γ‐crystallins from duck and frog eye lenses

A biochemical comparison has been made on the crystallins isolated from duck and frog lenses. Gel‐permeation chromatography of lens homogenates from both classes on Fractogel TSK HW‐55(S) revealed a homogeneous trimeric protein of 120 kDa in the duck lenses and a monomeric protein of 39 kDa in the frog lenses. Both crystallin fractions consist only of an approx. 38‐kDa polypeptide in their subunit structures as determined by SDS gel electrophoresis. These two crystalline were compared with respect to their native molecular masses, subunit structures, peptide mapping and amino acid compositions in order to establish the identity of each crystallin. We have found differences in the protein structures of these two crystallins despite some degree of similarity in their amino acid compositions.

The amino-terminal sequences of four major carp γ-crystallin polypeptides and their homology with frog and calf γ-crystallins

Four major γ‐crystallin subfractions have been isolated from the carp (Cyprinus carpio) and their N‐terminal sequences determined by Edman protein sequencing. Extensive homologies indicative of close relatedness in their primary structure were found in these four γ‐crystallin polypeptides. Comparison of the carp N‐terminal sequences with those of mammalian and amphibian γ‐crystallins also showed a high degree of homology present in their N‐terminal segments despite the dissimilarity of amino acid compositions of fish γ‐crystallins to those of higher classes of vertebrates. The distinct yet closely‐related partial sequences of carp γ‐crystallins could account for the profound microheterogeneity detected in the characterization of carp crystallins, suggesting the presence of a multigene family for γ‐crystallin in the lowest class of vertebrates, i.e. the fish.

Sequence comparison of γ-crystallins from the reptilian and other vertebrate species

Lens crystalline were isolated from homogenates of reptilian eye lenses (Caiman crocodylus apaporiensis) by gel‐permeation chromatography and characterized by gel electrophoresis, and amino acid and N‐terminal sequence analyses. Four fractions corresponding to α‐, δ/ε/β/‐, β‐ and γ‐crystallins were identified on the basis of their electrophoretic patterns as revealed by SDS gel electrophoresis. Comparison of the amino acid contents of reptilian crystallins with those of mammals suggests that each orthologous class of crystallins from the evolutionarily distant species still exhibits similarity in their amino acid compositions and probably sequence homology as well. All fractions except that of γ‐crystallin were found to be N‐terminally blocked. N‐terminal sequence analysis of the purified γ‐crystallin subfractions showed extensive homology between the reptilian γ‐crystallin polypeptides themselves and also those from other vertebrate species, suggesting the existence of a multigene family and their close relatedness to γ‐crystallins of other vertebrates.

Biochemical comparison of lens crystallins from three reptilian species

Lens crystallins were isolated from the homogenates of reptilian eye lenses derived from three different species by gel-permeation chromatography and characterized by gel electrophoresis, amino-acid analysis, N-terminal sequence analysis and circular dichroism. Four fractions corresponding to alpha-, delta/epsilon/beta-, beta- and gamma-crystallins were obtained for the crystallins from caiman lenses, whereas delta- and gamma-crystallin fraction were present in lesser amounts or missing in the turtle and snake lenses, respectively. The native molecular masses for these purified fractions and their polypeptide compositions were determined by gel filtration and SDS-gel electrophoresis, respectively, revealing the typical subunit compositions for each classified crystallin. The spectra of circular dichroism indicate a predominant beta-sheet structure in alpha-, beta- and gamma-crystallins, and a major contribution of alpha-helical structure in delta/epsilon-crystallin fraction, which bears a resemblance to the secondary structure of delta-crystallin from the chicken lenses. Comparison of the amino-acid contents of each orthologous class of reptilian crystallins with those of evolutionary distant species still exhibited similarity in their amino-acid compositions. N-terminal sequence analysis of the crystallin fractions revealed that all fractions except that of gamma-crystallin are N-terminally blocked. Extensive sequence similarity between the reptilian gamma-crystallin polypeptides and those from other vertebrate species were found, which establish the close relatedness of gamma-crystallins amongst the major classes of vertebrates.

Biochemical characterization of crystallins from pigeon lenses: structural and sequence analysis of pigeon δ-crystallin☆

Crystallins from pigeon eye lenses were isolated and purified by gel-permeation chromatography and characterized by gel electrophoresis, amino-acid composition and sequence analysis. Alpha- and beta-crystallins could be obtained in relatively pure forms by single-step size-exclusion chromatography whereas an extra step of ion-exchange chromatography was needed for the separation of delta-crystallin from the beta-crystallin fraction. In contrast to most characterized vertebrate species, a large amount of glycogen is eluted as a high molecular form in the first peak of the gel filtration column. Pigeon delta-crystallin, similar to duck and reptilian delta-crystallins, exists as a tetrameric structure of about 200 kDa in the native form and is composed of one major subunit of 50 kDa with heterogeneous isoelectric points spreading in a range of 4.7 to 6.8. In contrast to those obtained from duck, goose and caiman, delta-crystallin isolated from the pigeon lens possessed very little argininosuccinate lyase activity. However, pigeon delta-crystallin can still cross-react with the antibody against enzymically active duck delta-crystallin as revealed by the sensitive immunoblotting technique. It was also shown that the delta-crystallin content of the total pigeon soluble proteins decreased with the age of the animal. Structural analysis of purified delta-crystallin fraction was made with respect to its amino-acid composition and protein primary sequence. N-terminal sequence analysis indicated the presence of blocked amino-termini in all crystallin fractions of pigeon lenses. Therefore, a sequence analysis of PCR (polymerase chain reaction) amplified delta-crystallin cDNA was employed to deduce the protein sequence of this crystallin. Structural comparison of delta-crystallin sequences from pigeon, chicken and duck lenses casts some doubts on the recent claim that His-89-->Gln mutation in the chicken delta-crystallin may account for the loss of argininosuccinate lyase activity in this avian species, as compared to high enzymic activity in the duck crystallin (Barbosa et al. (1991) J. Biol. Chem. 266, 5286-5290).

A re-evaluation of the molecular size of duck ε-crystallin and its comparison with avian lactate dehydrogenases

Abstract A biochemical comparison of e-crystallin isolated from the duck lens and lactate dehydrogenase of chicken heart has been made in order to establish the structural and functional identities of these two proteins. The native molecular weight of e-crystallin was re-examined by combining sedimentation and gel-filtration data. It was found that e-crystallin is 150 kDa in contrast to the 120 kDa reported previously for this crystallin. Subunit cross-linking experiments corroborated that lactate dehydrogenase and e-crystallin both exist as tetramers of four identical subunits in their native quaternary structures. Amino acid compositions plus N-terminal analyses revealed no differences between the two proteins. Duck e-crystallin exhibited high enzymatic activity of lactate dehydrogenases even after a long period of storage, and showed characteristic thermostability at 50°C for several hours. Comparison of the enzyme activity of duck lens homogenate with those of heart, liver and muscle tissues revealed that duck lens is a much richer source than other tissues for the isolation and characterization of this important enzyme which appears also as a structural protein in the lens.

Sequence analysis of pigeon δ-crystallin gene and its deduced primary structure Comparison of avian δ-crystallins with and without endogenous argininosuccinate lyase activity

δ‐Crystallin is a major lens protein present in the avian and reptilian lenses. To facilitate the cloning of the δ‐crystallin gene, cDNA was constructed from the poly(A)+ RNA of pigeon lenses, amplified by the polymerase chain reaction (PCR). The PCR product was then subcloned into pUC19 vector and transformed into E. coli strain JM109. Plasmids purified from the positive clones were prepared for nucleotide sequencing by the dideoxynucleotide chain‐termination method. Sequencing two clones, containing 1.4 kb DNA inserts coding for δ‐crystallin allowed the construction of a complete, full‐length reading frame of 1,417 bp covering a deduced protein sequence of 466 amino acids, including the universal translation‐initiating methionine. The pigeon δ‐crystallin shows 88, 83 and 69% sequence identity to duck δ2, chicken δ1 crystallins and human argininosuccinate lyase respectively. It is also shown that, in contrast to duck δ2 crystallin which has a high argininosuccinate lyase activity, pigeon δ‐crystallin appears to contain very low activity of this enzyme, despite the fact that they share a highly homologous structure. A structural comparison of δ‐crystallins with or without enzymatic activity suggested several amino acid replacements which may account for the loss of argininosuccinate lyase activity in the lenses of certain avian species.