Crystallin Research Articles

Changes in the C-terminal region of alpha-A crystallin during human cataractogenesis

Reverse phase chromatography was used to purify alpha-A crystallin from total protein of human cataractous and normal lenses, followed by tryptic digestion and quantitation of the peptides corresponding to the intact C-terminal region of the protein. Relative to alpha-A crystallin from normal lenses, alpha-A crystallin from cataractous lenses contained decreased amounts of the expected C-terminal tryptic peptides. In an alternative approach, antiserum specific for the C-terminal region of the protein was used to quantitatively probe Western blots of total proteins from cataractous and normal lenses. The results demonstrated a decrease in the amount of this antiserum binding to the C-terminal region of alpha-A crystallin from human cataracts. Together, these studies show that relative to alpha-A crystallin from normal lenses, there is a decrease in the amount of the intact C-terminal region during the process of human cataractogenesis.

Increase in the Intramolecular Disulfide Bonding of Alpha-A Crystallin During Aging of the Human Lens

Aging of the normal human lens is accompanied by oxidation of protein sulfhydryl groups to disulfide groups. Although this has been known for many years, very little is known about the exact amino acid residues involved. In addition, almost nothing is known concerning the temporal sequence of this oxidative process over the lifetime of the individual. To address these two concerns for alpha-A crystallin, the polypeptide was purified from total proteins of the human lens, followed by digestion with lys-C endoprotease. Mass spectral analysis of the resulting fragments demonstrated that the two cysteine residues (cysteine-131 and cysteine-142) are present as a mixture of an intramolecular disulfide bond and free sulfhydryl groups. Reverse phase chromatography was used to resolve and quantitate the relative amounts of the two forms present in alpha-A crystallin from normal lenses of different age. Even in very young lenses (4 months and 5 months of age) there is significant oxidation of the two cysteine residues. However, the oxidative state of these two residues does not significantly change during the next ∼27 years of age, after which there is an increase in the relative amount of intramolecular disulfide bonding. Together, these results have identified and quantitated the relative change in the oxidative state of two specific cysteine residues of alpha-A crystallin in human lenses of different age, and have established that age-dependent oxidation of these two residues occurs primarily during the later part of life.

Kinetic study of racemization of aspartyl residues in model peptides of αA‐crystallin

We have reported that two aspartyl (Asp-151 and Asp-58) residues in alpha A-crystallin in human eye lens were inverted to the D-isomer and isomerized to beta-aspartyl residues with age. We report here the kinetics of the Asp racemization of three model peptides corresponding to fragments of alpha A-crystallin: IQTGLD151ATHAER (T18 peptide), TVLD58SGISEVR (T6 peptide) and HFSPED84LTVK (T10 peptide, as a control). The rate constants of the racemization of Asp residues in these peptides were measured at pH 7.0, at five temperatures: 50, 60, 70, 80 and 90 degrees C. From the Arrhenius equation, we estimated the activation energy (E) of racemization and the time required for the Asp D/L ratio to approximate to 1.0 (D/L ratio of Asp = 0.99) at body temperature. For the peptide T18, E = 21.4 kcal/mol and t = 13.5 yr. For the peptide T6, E = 26.8 kcal/mol and r = 49.5 yr. For the control peptide T10, E = 28.3 kcal/mol and t = 78.1 yr. The racemization rate of Asp in these three peptides is parallel to that of Asp residues in alpha A-crystallin. The racemization rate of Asp in the T18 peptide was very rapid compared to that in the other peptides. This result also reflects the racemization rate in native alpha A-crystallin.

Oxidation of Cysteine Residues from Alpha-A Crystallin during Cataractogenesis of the Human Lens

Although it has been known for many years that opacification of the human lens is accompanied by oxidation of cysteine sulfhydryl groups to half-cystine residues, nothing is known concerning the exact amino acid sequences involved in this oxidative process. Since alpha-A crystallin is one of the major proteins of the lens, and since a decrease in its molecular chaperone activity has been implicated in possible mechanisms of cataract formation, alpha-A crystallin was purified from total lens proteins by reverse phase chromatography, followed by digestion with lys-C endoprotease. Mass spectral analysis of the digest indicated that in normal transparent lenses, cys-131 and cys-142 from alpha-A crystallin are present as a mixture of cysteine sulfhydryl and half-cystine disulfide groups, while identical analysis from cataractous lenses demonstrated undetectable levels of the cysteine sulfhydryl group. Together, these results demonstrate for the first time, the involvement of specific cysteine residues in the oxidative mechanism of human lens opacification.

Differential Phosphorylation of Alpha-A Crystallin in Human Lens of Different Age

Previous studies have demonstrated that a major site of in vivo phosphorylation of alpha-A crystallin from human lens is serine-122. To determine the relative degree of this phosphorylation in alpha-A crystallin from human lenses of different age, alpha-A crystallin was purified from total lens proteins, followed by sequential digestion with lys-C and asp-N endoproteases. Mass spectral analysis of the asp-N peptide fragments that contained serine-122 demonstrated undetectable levels of phosphorylation from infant human lenses (41 days, 2 months and 4 months of age). Identical analysis of alpha-A crystallin from older lenses (12, 15, 40 and 73 years of age) indicated significant phosphorylation of serine-122, demonstrating that phosphorylation of the serine-122 residue of alpha-A crystallin does not occur during the aging process, but is rather a developmentally regulated event in the human lens.

Conformational Properties of Substrate Proteins Bound to a Molecular Chaperone α-Crystallin

alpha-Crystallin, the major protein of the ocular lens, acts as a molecular chaperone by suppressing the nonspecific aggregation of damaged proteins. To investigate the mechanism of the interaction between alpha-crystallin and substrate proteins, we prepared a tryptophan-free mutant of human alpha A-crystallin and assessed the conformation of thermally destabilized proteins captured by this chaperone using fluorescence spectroscopy. The fluorescence emission characteristics of bound substrates (rhodanese and gamma-crystallin) and the results of fluorescence quenching experiments indicate that the proteins captured by alpha-crystallin are characterized by a very low degree of unfolding. In particular, the structure of rhodanese bound to alpha A-crystallin appears to be considerably more native-like compared to that of the enzyme bound to the chaperonin GroEL. We postulate that alpha-crystallin (and likely other small heat shock proteins) recognize preferentially the aggregation-prone conformers that occur very early on the denaturation pathway. With its ability to capture and stabilize these early non-native structures, alpha-crystallin appears to be uniquely well suited to chaperone the transparency properties of the ocular lens.

First Finding of ?-Crystallin Outside the Archosaurian Lineage

First Finding of ?-Crystallin Outside the Archosaurian Lineage

The presence of a glucose-derived Maillard reaction product in the human lens

Pyrraline is an advanced Maillard reaction product formed by the non-enzymatic reaction initiated by glucose with lysine residues on proteins. This reaction involves an intermediate, 3-deoxyglucosone, concentration of which is shown to be elevated in plasma and lenses during diabetes. Bovine lens alpha crystallins incubated with 3-deoxyglucosone showed that pyrraline formation was a major modification and its quantification by two different methods revealed time-dependent accumulation. Pyrraline was quantified in normal, senile cataractous and diabetic lenses. Although a wide variation was observed, the mean value in cataractous lenses (mean ± S.E.: 48.4 ± 12.67 pmol/mg protein) was higher than in age-matched normal lenses (30.9 ± 10.26 pmol). Surprisingly, in diabetic lenses, the mean value was lower than normal lenses (28.4 ± 15.3 pmol). These results suggest that glucose-specific advanced Maillard products occur in the human lens and such modifications may play a role in lens aging and cataract formation.

Phase separation in aqueous solutions of lens gamma-crystallins: special role of gamma s.

We have studied liquid-liquid phase separation in aqueous ternary solutions of calf lens gamma-crystallin proteins. Specifically, we have examined two ternary systems containing gamma s--namely, gamma IVa with gamma s in water and gamma II with gamma s in water. For each system, the phase-separation temperatures (Tph (phi)) alpha as a function of the overall protein volume fraction phi at various fixed compositions alpha (the "cloud-point curves") were measured. For the gamma IVa, gamma s, and water ternary solution, a binodal curve composed of pairs of coexisting points, (phi I, alpha 1) and (phi II, alpha II), at a fixed temperature (20 degrees C) was also determined. We observe that on the cloud-point curve the critical point is at a higher volume fraction than the maximum phase-separation temperature point. We also find that typically the difference in composition between the coexisting phases is at least as significant as the difference in volume fraction. We show that the asymmetric shape of the cloud-point curve is a consequence of this significant composition difference. Our observation that the phase-separation temperature of the mixtures in the high volume fraction region is strongly suppressed suggests that gamma s-crystallin may play an important role in maintaining the transparency of the lens.

Expression of recombinant alpha Ains-crystallin and not alpha A-crystallin inhibits bacterial growth.

alpha A-Crystallin and alpha Ains-crystallin are derived from the alpha A-crystallin gene via alternative splicing. They are identical except for the presence of a polypeptide, 23 amino acids long, encoded by the 'insert' exon. Evolutionary logic would suggest that the insertion of a 23 amino acid peptide in the middle of alpha A-crystallin, a protein evolving more slowly than either histone H1, cytochrome c or hemoglobin, would lead to appreciable structural and functional changes. However, based on physico-chemical studies, it is presently believed that alpha A-crystallin and alpha Ains-crystallin are functionally equivalent and that the presence of the 'insert' peptide in alpha Ains-crystallin is inconsequential. We report here that the independent expression of recombinant alpha Ains-crystallin, and not alpha A-crystallin, inhibits growth of the bacterial host. These observations were confirmed in coexpression experiments, wherein both the proteins were expressed in the same cell. Interestingly, growth inhibition is reversible. Importantly, the data demonstrate that it is catalytic amounts and not the gross accumulation of alpha Ains-crystallin which causes growth inhibition. Given the prior knowledge that alpha A-crystallin and alpha Ains-crystallin differ by a peptide of 23 amino acids, these data suggest that the "insert peptide' in alpha Ains-crystallin imparts properties on this protein that are different from alpha A-crystallin.

α-Crystallin: Molecular Chaperone and Heat Shock Protein

The relationship of alpha-crystallin with the family of small heat shock proteins has led to the discovery that the basic subunit alpha B-crystallin can, like other heat shock proteins, protect cells against heat stress. Here we show that the acidic subunit alpha A-crystallin, which in contrast to alpha B-crystallin is expressed mainly in the eye lens, shares this property. Furthermore we have investigated the in vitro molecular chaperone-like behavior of the natural mutant alpha A ins-crystallin that has a large insert peptide and occurs in rodents. We have found the chaperone-like activity of the mutant to be diminished compared to that of the wild type alpha A-crystallin.

Cataractogenesis in Transgenic Mice Containing the HIV-1 Protease Linked to the Lens αA-Crystallin Promoter

Several lines of transgenic mice were generated with either active or inactive forms of the human immunodeficiency virus type 1 (HIV-1) protease gene under the control of the mouse lens alpha A-crystallin promoter. Mice bearing the inactive protease coding sequence displayed no gross abnormalities in the lens, while mice with the active protease developed time-dependent bilateral cataracts. One line, TG61, developed cataracts in utero while the second line, TG72, developed cataracts postnatally. TG61 mice, homozygous for the transgene, developed severe microphthalmia and were significantly smaller than the control mice at postnatal day 30. two-dimensional-polyacrylamide gel electrophoresis analysis of the protein profiles of TG72 and TG61 lenses revealed extensive modifications in the lens crystallins. Proteolysis in the homozygous TG72 mouse lenses began at postnatal day 20 with the disappearance or partial loss of beta B1-, beta B3-, and beta A3-crystallins and the appearance of crystallin fragments. Protein leakage and the gradual breakdown of cytoskeletal elements also occurred. In contrast, the opacification of the homozygous TG61 lenses appeared to have been influenced by differentiation and developmental processes. It appears that HIV-1 protease expression activates other proteases, and these enzymes, in concert with HIV-1 protease, are responsible for the protein modifications that eventually result in the opacification of the lens.

The Influence of Some Post-Translational Modifications on the Chaperone-Like Activity of α-Crystallin

We investigated the influence of phosphorylation, glycation, carbamylation and oxidative modification on the capacity of alpha-crystallin to protect beta-crystallins against heat denaturation. Simple modification of lysine residues by early glycation or carbamylation had no effect. However, late (cross-linking) glycation products and oxidative modifications decreased the chaperone-like activity of alpha-crystallin. Homopolymers of alpha A-crystallin had a higher protecting capacity compared with those of alpha B-crystallin. The in vivo phosphorylated forms of especially alpha A- but also alpha B-crystallin revealed a somewhat better protecting ability than the respective non-phosphorylated forms.

Phylogenetic relationships of the hoatzin, an enigmatic South American bird.

The hoatzin (Opisthocomus hoazin) lives in the humid lowlands of northern and central South America, often in riparian habitats. It is a slender bird approximately 65 cm in length, brownish with lighter streaks and buffy tips to the long tail feathers. The small head has a ragged, bristly crest of reddish-brown feathers, and the bare skin of the face is bright blue. It resembles a chachalaca (Ortalis, Cracidae) in size and shape, but its plumage and markings are similar to those of the smaller guira cuckoo (Guira guira). The hoatzin (pronounced Watson) has been a taxonomic puzzle since it was described in 1776. It usually has been viewed as related to the gallinaceous birds, but alliances to other groups have been suggested, including the cuckoos. We present DNA sequence evidence from the 12S and 16S rRNA mitochondrial genes, and from the nuclear gene that codes for the eye lens protein, alpha A-crystallin. The results indicate that the hoatzin is most closely related to the typical cuckoos and that the divergence occurred at or near the base of the cuculiform phylogenetic tree.

A reassessment of mammalian ?A-crystallin sequences using DNA sequencing: Implications for anthropoid affinities of tarsier

alpha A-crystallin, a major structural protein in the ocular lenses of all vertebrates, has been a valuable tool for molecular phylogenetic studies. This paper presents the complete sequence for human alpha A-crystallin derived from cDNA and genomic clones. The deduced amino acid sequence differs at two phylogenetically informative positions from that previously inferred from peptide composition. This led us to examine the same region of the alpha A-crystallin gene in 12 other mammalian species using direct sequencing of PCR-amplified genomic DNA. New sequences were added to the database, and corrections were made to all anthropoid sequences, defining clear synapomorphies for anthropoids as a clade distinct from prosimians. Within the anthropoids there are further synapomorphies delineating hominoids, Old World monkeys, and New World monkeys. Significantly, sequence revisions and the addition of new sequence for a prosimian, the sifaka, eliminate the previous support for the proposed anthropoid affinities of the tarsier inferred from alpha A-crystallin protein sequences. In addition, DNA sequences provide greater resolution of certain relationships. For example, although they are identical in protein sequence, comparison of DNA sequences clearly separates mouse and the common tree shrew, grouping the tree shrew closer to prosimians. These results show that adding DNA sequences to the existing alpha A-crystallin database can enhance its value in resolving phylogenetic relationships.

Age-dependent cleavage at the C-terminal region of lens βB2 crystallin

A previous study has demonstrated that in vivo, the peptide bonds at the C-terminal region of the alpha-A crystallins were cleaved in an age-dependent manner, between the two hydroxyl-containing amino acids in the sequence -PS(T)S (Takemoto, 1995). Bovine beta B2 crystallin also contains the sequence-PSS at its C-terminus. To determine if this specific site of cleavage occurred in other lens proteins besides alpha-A crystallin, beta B2 crystallin was prepared from total proteins of young versus adult bovine lens fiber cells. After cleavage by cyanogen bromide, the C-terminal fragments were characterized by mass spectrometry. The results showed that peptide cleavage also occurred between the two hydroxyl-containing amino acids in the sequence -PSS of beta B2 crystallin from older fiber cells, demonstrating that age-dependent cleavage of this peptide bond occurs in multiple proteins of the aging lens.

Reduced levels of γ-crystallin transcripts during embryonic development of murine Cat2 nop mutant lenses

From previous experiments it is known that the murine dominant cataract mutants carrying the gene Cat2 have a decreased content of gamma-crystallin-specific transcripts in the juvenile lens, when the cataract is completely expressed. Moreover, the mutant locus has been mapped recently to chromosome 1, closely linked to the gamma E-crystallin gene (map distance 0.3 +/- 0.3 cM). In the present paper we describe the phenotypic changes and the gamma-crystallin expression in embryonic lenses of the Cat2nop mutants as an example for the Cat2 allelic series. The technique of in situ hybridization was applied using a probe from the murine gamma D-crystallin gene, and, for control, from the murine alpha A-crystallin gene. Simultaneously, a series of lens sections was examined histologically. The presence of gamma-crystallin mRNA was demonstrated from embryonic day 13.5 (E13.5) onward, but in the mutants to a lower extent than in the wild-type lenses. However, the first morphological abnormality in the mutant lenses was observed as swelling of lens fibers at day E15.5. Progressive degeneration of the lens core followed, leading to a cataracta immatura. The reduced level of gamma-crystallin transcripts is the first alteration observable during the embryonic development of the Cat2 mutant lenses: it precedes the morphological changes. This result represents an additional line of argument that the gamma-crystallin genes may be the target of the mutation in the Cat2 mice.

Enthalpy and Entropy of Hydration of Bovine Crystallins

Transparency of the lens of the eye is the result of a short range order in the packing of crystallin molecules within the fiber cells. Short range order depends on crystallin-crystallin as well as water-crystallin interactions. Light scattering measurements can provide information on the hydration of crystallins. Light scattering intensities were obtained as a function of scattering angle, concentration, and temperature on dilute solutions of beta H, beta L, and gamma fractions of bovine lens crystallins. The temperature dependence of the second virial coefficient was negative for the beta crystallin fractions and positive for the gamma fraction as well as that for alpha crystallin (Wang, X., and Bettelheim, F. A. (1989) Proteins Struct. Funct. Genet. 5, 166-169). The partial molar enthalpy values of the solutions were negative for the beta crystallin fractions, indicating a tendency for homo- and heterodimer and -oligomer association. The enthalpy values were positive for the alpha and gamma fractions. The negative values of the enthalpy of solutions differentiate the beta crystallins from the other crystallins. The partial molar entropy values of solutions of beta L and gamma fractions were identical, those of the oligomeric beta H fraction were higher, whereas those of alpha crystallin were a magnitude larger than those of the smaller crystallin molecules.

Substrate Requirements for Transglutaminases: INFLUENCE OF THE AMINO ACID RESIDUE PRECEDING THE AMINE DONOR LYSINE IN A NATIVE PROTEIN

Thirteen recombinant alpha A-crystallin mutants were constructed that differed in the type of amino acid residue directly preceding the sole amine donor lysine for transglutaminases in this protein. The capacity of these mutants to be cross-linked to amine acceptor substrates by tissue transglutaminase and factor XIII was assessed. Two different biotinylated glutamine-containing oligopeptides were used as amine acceptor probes. It appears that the type of residue preceding the amine donor lysine has a considerable influence on the substrate potential of alpha A-crystallin for transglutaminases. This influence shows qualitatively similar trends for tissue transglutaminase and factor XIII and is irrespective of the amine acceptor probe. In general, glycine or aspartic acid before the amine donor lysine has the strongest adverse effects on substrate reactivity, and proline, histidine, and tryptophan are less favorable. Valine, arginine, and phenylalanine, and to a more variable or somewhat lesser extent also serine, alanine, leucine, tyrosine, and asparagine, have an enhancing effect. This pattern of preference is largely in agreement with that observed for the limited number of characterized amine donor lysines in protein substrates for transglutaminases. It can be concluded that tissue transglutaminase and factor XIII have a rather broad yet clearly differentiated tolerance with respect to the residue preceding the amine donor lysine substrate in native proteins.

The mutation Asp69-->Ser affects the chaperone-like activity of alpha A-crystallin.

alpha-Crystallins are members of the family of small heat-shock proteins. The conformation and mode of action of these 'junior chaperones' are unknown. To investigate the structure and chaperone-like activity, four mutants of bovine alpha A-crystallin were generated by site-directed mutagenesis. In comparison with wild-type alpha A-crystallin, the D69S mutant, in which a highly conserved charged residue has been replaced, forms larger multimers and displays a threefold reduced heat-protection capacity. The conformation and thermal stability of this mutant are not noticeably affected. Three other mutations, replacing hydrophobic by uncharged hydrophilic residues, were aimed at disturbing hydrophobic intersubunit interactions. None of these mutations resulted in major structural perturbations and only minor differences in heat-protective capacity were observed. Although it is assumed that small heat-shock proteins interact with denaturing proteins via their hydrophobic surfaces, this study clearly shows that charged residues in alpha-crystallin can also influence the efficiency of substrate binding.