Vertebrate-like βγ-crystallins in the ocular lenses of a copepod

The diverse crystallins are water-soluble proteins that are responsible for the optical properties of cellular lenses of animal eyes. While all vertebrate lenses contain physiological stress-related - and -crystallins, some also contain taxon-specific, often enzyme-related crystallins. To date, the - and -crystallins have been found only in vertebrate lenses. Here we report lenses from an invertebrate, the pontellid copepod Anomalocera ornata, accumulate -crystallin family members as judged by immunocytochemistry, western immunoblotting and microsequencing. Our data provide the first example of -crystallin members in an invertebrate lens, establishing that the use of this protein family as lens crystallins is not confined to vertebrates.     

The βγ-crystallins: Native state stability and pathways to aggregation

The βγ-crystallins are among the most stable and long-lived proteins in the human body. With increasing age, however, they transform to high molecular weight light-scattering aggregates, resulting in cataracts. This occurs despite the presence in the lens of high concentrations of the a-crystallin chaperones. Aggregation of crystallins can be induced in vitro by a variety of stresses, including acidic pH, ultraviolet light, oxidative damage, heating or freezing, and specific amino acid substitutions. Accumulating evidence points to the existence of specific biochemical pathways of protein: protein interaction and polymerization. We review the methods used for studying crystallin stability and aggregation and discuss the sometimes counterintuitive relationships between factors that favor native state stability and those that favor non-native aggregation. We discuss the behavior of βγ-crystallins in mixtures and their chaperone ability; the consequences of missense mutations and covalent damage to the side-chains; and the evolutionary strategies that have shaped these proteins. Efforts are ongoing to reveal the nature of cataractous crystallin aggregates and understand the mechanisms of aggregation in the context of key models of protein polymerization: amyloid, native-state, and domain-swapped. Such mechanistic understanding is likely to be of value for the development of therapeutic interventions and draw attention to unanswered questions about the relationship between a protein’s native state stability and its transformation to an aggregated state.     

Physicochemical characterization of α-crystallins from bovine lenses: Hydrodynamic and conformational properties

A detailed investigation of hydrodynamic and conformational behavior has been made of the HMα-crystallin and α-crystallins of bovine lens. Results from this study indicated that HMα (high-molecular-weight α-crystallin) and α (low-molecular-weight α-crystallin) possess considerable size and charge heterogeneities in their native structures and subunit polypeptides, respectively. Sedimentation velocity showed a heterogeneous polydisperse system of HMα with an average sedimentation coefficient of about 50 S and a more homogeneous system of α-crystallin of 20 S. Viscosity and circular dichroism studies pointed to a compact and globular shape of dominant β-sheet conformation for α-crystallin, yet a highly asymmetrical and aggregated form for HMα. The conformational stability of α-crystallin was investigated in the presence of various denaturants. The evidence presented shows that hydrogen bonding is the main force in maintaining the quaternary structure of compact native α-crystallin. Conformational flexibility of α-crystallin demonstrated in the equilibrium unfolding study indicated a multistep transition that made the extraction of thermodynamic data from the heat denaturation study difficult. Temperature perturbation on α-crystallin suggested the possible involvement of hydrophobic interaction in the aggregation process, leading to the formation of HMα from α-crystallin. The comparison of conformational properties between HMα and α-crystallin strongly indicated that HMα is a denatured form of α-crystallin.     

Physicochemical characterization of β-crystallins from bovine lenses: Hydrodynamic and aggregation properties

A detailed investigation of the hydrodynamic and aggregation behaviors has been made on the -crystallins of bovine lens. Results from this study indicated that H (high-molecular-weight -crystallin) and L (low-molecular-weight -crystallin) exhibited considerable heterogeneity in their native structures and subunit polypeptides. Low-speed sedimentation equilibrium showed a heterogeneous paucidisperse system in each -crystallin fraction. Viscosity and circular dichroism studies pointed to a compact and globular shape and the presence of -sheet and -turns in these crystallins. Dissociation of H by urea and guanidinium HCl followed by reassociation during gel-filtration chromatography produced an elution pattern with two fractions corresponding to L crystallin and high-molecular-weight aggregates without the formation of native H. By contrast, under similar treatment, about 60% L reassociated into the correct native structure and the rest into high-molecular-weight fractions. Amino acid analyses of H and L and their corresponding subunit polypeptides demonstrated the close similarity of these crystallins. Trace element analyses indicated that both Ca and Mg are present in H and L crystallins and may be involved in maintaining the native quarternary structures of these proteins.     

Characterization of the βγ-crystallin domains of βγ-CAT, a non-lens βγ-crystallin and trefoil factor complex, from the skin of the toad Bombina maxima

βγ-CAT is a naturally existing 72-kDa complex of a non-lens βγ-crystallin (α-subunit, CAT-α) and a trefoil factor (β-subunit, CAT-β) that contains a non-covalently linked form of αβ₂ and was isolated from the skin secretions of the toad Bombina maxima. The N-terminal region of CAT-α (CAT-αN, residues 1–170) contains two βγ-crystallin domains while the C-terminal region (CAT-αC) has sequence homology to the membrane insertion domain of the Clostridium perfringens epsilon toxin. To examine the biochemical characteristics of the βγ-crystallin domains of βγ-CAT, CAT-αN, CAT-αC and CAT-β were expressed in Escherichia coli. Co-immunoprecipitation of the naturally assembled βγ-CAT confirmed that the CAT-α and CAT-β complex always exists. Furthermore, recombinant CAT-β bound recombinant CAT-αN. Ca²⁺-binding motifs were identified in CAT-αN, and recombinant CAT-αN was able to bind the calcium probe terbium. However, the conformation of CAT-αN was not significantly altered upon Ca²⁺ binding. βγ-CAT possesses strong hemolytic activity toward human erythrocytes, and treatment of erythrocytes with βγ-CAT resulted in a rapid Ca²⁺ influx, eventually leading to hemolysis. However, in the absence of extracellular Ca²⁺, no significant hemolysis was detected, even though the binding and oligomerization of βγ-CAT in the erythrocyte membrane was observed. Our data demonstrate the binding of CAT-β (a trefoil factor) to CAT-αN (βγ-crystallin domains) and provide a basis for the formation of a βγ-crystallin and trefoil factor complex in vivo. Furthermore, the βγ-crystallin domains of βγ-CAT are able to bind Ca²⁺, and βγ-CAT-induced hemolysis is Ca²⁺ dependent.     

Evolutionary remodeling of βγ-crystallins for domain stability at cost of Ca2+ binding

The topologically similar βγ-crystallins that are prevalent in all kingdoms of life have evolved for high innate domain stability to perform their specialized functions. The evolution of stability and its control in βγ-crystallins that possess either a canonical (mostly from microorganisms) or degenerate (principally found in vertebrate homologues) Ca2+-binding motif is not known. Using equilibrium unfolding of βγ-crystallin domains (26 wild-type domains and their mutants) in apo- and holo-forms, we demonstrate the presence of a stability gradient across these members, which is attained by the choice of residues in the (N/D)(N/D)XX(S/T)S Ca2+-binding motif. The occurrence of a polar, hydrophobic, or Ser residue at the 1st, 3rd, or 5th position of the motif is likely linked to a higher domain stability. Partial conversion of a microbe-type domain (with a canonical Ca2+-binding motif) to a vertebrate-type domain (with a degenerate Ca2+-binding motif) by mutating serine to arginine/lysine disables the Ca2+-binding but significantly augments its stability. Conversely, stability is compromised when arginine (in a vertebrate-type disabled domain) is replaced by serine (as a microbe type). Our results suggest that such conversions were acquired as a strategy for desired stability in vertebrate members at the cost of Ca2+-binding. In a physiological context, we demonstrate that a mutation such as an arginine to serine (R77S) mutation in this motif of γ-crystallin (partial conversion to microbe-type), implicated in cataracts, decreases the domain stability. Thus, this motif acts as a "central tuning knob" for innate as well as Ca2+-induced gain in stability, incorporating a stability gradient across βγ-crystallin members critical for their specialized functions.     

Explosive Expansion of βγ-Crystallin Genes in the Ancestral Vertebrate

In jawed vertebrates, βγ-crystallins are restricted to the eye lens and thus excellent markers of lens evolution. These βγ-crystallins are four Greek key motifs/two domain proteins, whereas the urochordate βγ-crystallin has a single domain. To trace the origin of the vertebrate βγ-crystallin genes, we searched for homologues in the genomes of a jawless vertebrate (lamprey) and of a cephalochordate (lancelet). The lamprey genome contains orthologs of the gnathostome βB1-, βA2- and γN-crystallin genes and a single domain γN-crystallin-like gene. It contains at least two γ-crystallin genes, but lacks the gnathostome γS-crystallin gene. The genome also encodes a non-lenticular protein containing βγ-crystallin motifs, AIM1, also found in gnathostomes but not detectable in the uro- or cephalochordate genome. The four cephalochordate βγ-crystallin genes found encode two-domain proteins. Unlike the vertebrate βγ-crystallins but like the urochordate βγ-crystallin, three of the predicted proteins contain calcium-binding sites. In the cephalochordate βγ-crystallin genes, the introns are located within motif-encoding region, while in the urochordate and in the vertebrate βγ-crystallin genes the introns are between motif- and/or domain encoding regions. Coincident with the evolution of the vertebrate lens an ancestral urochordate type βγ-crystallin gene rapidly expanded and diverged in the ancestral vertebrate before the cyclostomes/gnathostomes split. The β- and γN-crystallin genes were maintained in subsequent evolution, and, given the selection pressure imposed by accurate vision, must be essential for lens function. The γ-crystallin genes show lineage specific expansion and contraction, presumably in adaptation to the demands on vision resulting from (changes in) lifestyle.     

Metamorphic change in EP37 expression: members of the βγ-crystallin superfamily in newt

 EP37 is an epidermis-specific protein found in the developing embryo of the Japanese newt, Cynops pyrrhogaster. Our previous study predicted the presence of genes homologous to EP37, which show temporary shared expression at the turn of metamorphosis. In this study, we isolated and characterized three cDNAs encoding novel EP37 homologues; two from the skin of an adult newt and the other from swimming larva. Conceptual translation of the open reading frames of these cDNAs predicted proteins carrying βγ-crystallin motifs and putative calcium-binding sites, both of which are features shared by the originally identified EP37 (EP37L1), as well as a spore coat protein of Myxococcus xanthus, protein S. Immunoblot analyses and immunohistochemical studies indicated that two of the EP37 proteins, EP37L1 and EP37L2, are exclusively expressed in the epidermis (skein cells) including the figures of Eberth at premetamorphic stages. During and after metamorphosis, the expression of EP37 proteins was mainly observed in cutaneous glands, and a molecular transition to the adult types of EP37, EP37A1 and EP37A2, occurred. These observations suggest that EP37 proteins play an important role in construction of integumental tissues and adaptation to the aquatic or amphibious environment. Received: 6 September 1996 / Accepted: 30 October 1996     

Protein interactions in the calf eye lens: interactions between β-crystallins are repulsive whereas in γ-crystallins they are attractive

Non-specific interactions in beta- and gamma-crystallins have been studied by solution X-ray scattering and osmotic pressure experiments. Measurements were carried out as a function of protein concentration at two ionic strengths. The effect of temperature was tested between 7 degrees C and 31 degrees C. Two types of interactions were observed. With beta-crystallin solutions, a repulsive coulombic interaction could be inferred from the decrease of the normalized X-ray scattering intensity near the origin with increasing protein concentration and from the fact that the osmotic pressure increases much more rapidly than in the ideal case. As was previously observed with alpha-crystallins, such behaviour is dependent upon ionic strength but is hardly affected by temperature. In contrast, with gamma-crystallin solutions, the normalized X-ray scattering intensity near the origin increases with increasing protein concentration and the osmotic pressure increases less rapidly than in the ideal case. Such behaviour indicates that attractive forces are predominant, although we do not yet know their molecular origin. Under our experimental conditions, the effect of temperature was striking whereas no obvious contribution of the ionic strength could be seen, perhaps owing to masking by the large temperature effect. The relevance of the different types of non-specific interactions for lens function is discussed.     

Parachordeumium amphiurae: a cuckoo copepod?

The impact of the endoparasitic copepod Parachordeumium amphiurae (=Amphiurophilus amphiurae of Goudey-Perrière, 1979) on a host population of the brooding, hermaphroditic ophiuroid Amphipholis squamata in intertidal rock pools in South Devon, UK was assessed. In August 1986 the parasite prevalence was 20.6% and the mean population intensity of infection 0.286 parasites per brittlestar. Female parasites inhabited the genital bursae of hosts and most were found in hosts that were potentially reproductively active. In such hosts brooding capacity was reduced by copepod parasitism. Infected brittlestars showed a reduction in numbers of brooded developmental stages when compared with uninfected hosts of similar size (but the mean size of brooded development stages was not affected). For the population sampled, the overall reduction in brooding capacity was about 44%. The association between Amphipholis and Parachordeumium was compared with normal brood parasitism.     

Reconstruction of the shape and optics of the lenses in the abathochroal‐eyed trilobite Neocobboldia chinlinica

Until now, the structure and optics of the calcite lenses in abathochroal trilobite eyes have not been investigated. So, the relationship of the abathochroal eye to other types of trilobite eyes has remained unclear. We have reconstructed the exact shape and optics of the lenses in the eodiscid trilobite Neocobboldia chinlinica to determine the mechanism of its abathochroal eye. The distal lens surface has a convex profile, while on the proximal lens surface there is a small central bulge, resulting in an undulating profile. Due to this bulge, the curvature and refractive power of the central region of the lens are greater than those of the peripheral zone. Consequently, the lens is bifocal. However, Neocobboldia could not take advantage of this bifocal property of its tiny lenses because of the diffraction of light and the infinite depth of field in object space. For the same reason, it is also sure that the undulating lower surface of the abathochroal lens did not evolve as a Huygensian profile, correcting for spherical aberration, as suggested earlier. This undulation is a result of the presence of the central bulge, the evolutionary significance of which remains enigmatic. On the basis of our results, we have outlined an evolutionary scenario for development of the optics of the lenses in trilobite eyes.     

The effects of short-term pH decrease on the reproductive output of the copepod Acartia bifilosa – a laboratory study

This laboratory study reports some reproductive responses of the copepod Acartia bifilosa to rapid variations in pH. The imposed changes mimic those that copepods could experience due to coastal upwelling, changed mixing conditions or vertical migration. We measured effects of low pH on egg production, hatching and early nauplii development (H₀: no effects on response variables between low and ambient pH). On treatment with low pH, we found positive effects on egg production rate and nauplii development time. The positive response to low pH could be an initial stress response or show that A. bifilosa is tolerant to the experimental pH values. The result suggests that A. bifilosa is adapted to pH changes as it performs daily migrations between the depths with differing pH. It could also be advantageous for population development if eggs hatch at high speed and so reduce the possibility that they will sink into anoxic and low pH waters.     

Cloning and tissue expression of the mouse ortholog of AIM1, a βγ-crystallin superfamily member

We report the isolation of the murine ortholog of AIM1, a human gene whose expression is associated with the reversal of tumorigenicity in an experimental model of melanoma. Mouse and human AIM1 are more than 90% identical in amino acid sequence in the βγ-crystallin repeats and the C-terminal domain, and more than 75% identical in the extended N-terminal domain. Consistent with the isolated cDNA representing the authentic AIM1 ortholog, linkage analysis localized mouse Aim1 to proximal mouse Chromosome (Chr) 10 in a conserved linkage group with genes localized to human Chr band 6q21. Searches of EST databases identified a second AIM1-like gene in both mouse and human, suggesting the existence of a gene family. Northern analysis demonstrates Aim1 is expressed most abundantly in adult skin, lung, heart, liver, and kidney and is temporally regulated during embryogenesis. Aim1 is expressed highly in the shaft region of the hair follicles and the presumptive ectoderm, but not at detectable levels in melanocytes or melanocyte precursor cells. Received: 18 February 1998 / Accepted: 8 May 1998     

Cholesterol-derived bile acids enhance the chaperone activity of α-crystallins

Human lens membranes contain the highest cholesterol concentration of any known biological membranes, but it significantly decreases with age. Oxygenation of cholesterol generates numerous forms of oxysterols (bile acids). We previously showed that two forms of the bile acid components—ursodeoxycholic acid (UDCA) and tauroursodeoxycholic acid (TUDCA)—suppressed lens epithelial cell death and alleviated cataract formation in galactosemic rat lenses. We investigated whether these compounds also suppress the thermal aggregation of human lens crystallins. Total water-soluble (WS) proteins were prepared from human lenses, and recombinant human crystallins (αA-, αB-, βB2-, and γC-crystallin) were generated by a prokaryotic expression system and purified by liquid chromatography. The light scattering of proteins in the presence or absence of UDCA or TUDCA was measured using a spectrofluorometer set at Ex/Em = 400/400 nm. Protein blot analysis was conducted for detection of α-crystallins in the human lens WS proteins. High concentrations of UDCA and TUDCA significantly suppressed thermal aggregation of total lens WS proteins, which contained a low level of αA-/αB-crystallin. Spectroscopic analysis with each recombinant human lens crystallin indicated that the bile acids did not suppress the thermal aggregation of γC-, βB2-, αA-, or αB-crystallin. Combination of α-crystallin and bile acid (either UDCA or TUDCA) suppressed thermal aggregation of each individual crystallin as well as a non-crystallin protein, insulin. These results suggest that UDCA or TUDCA protects the chaperone activity of α-crystallin. It is believed that these two naturally occurring intermediate waste products in the lens enhance the chaperone activity of α-crystallin. This finding may lead to the development of UDCA and TUDCA as anticataract agents.     

Conformational heterogeneity and dynamics in a βγ-crystallin from Hahella chejuensis

Most of the βγ-crystallins are structural proteins with high intrinsic stability, which gets enhanced by Ca(2+)-binding in microbial members. Functions of most of these proteins are yet to be known. However, a few of them were reported to be involved in Ca(2+)-dependent and stress-related functions. Hahellin, a microbial homolog, is a natively unfolded protein that acquires a well-folded structure upon Ca(2+) binding. Although the structure of βγ-crystallin domains is well understood, the dynamical features are yet to be explored. We have investigated for the first time the equilibrium dynamics, conformational heterogeneity and associated low-lying free-energy states of hahellin in its Ca(2+)-bound form using NMR spectroscopy to understand the dynamics of a βγ-crystallin domain. Hahellin shows large conformational heterogeneity with nearly 40% of the residues, some of which are part of Ca(2+)-binding loops, accessing alternative states. Further, out of the two Greek key motifs, which together constitute the βγ-crystallin domain, the second Greek key motif is floppy as compared to its relatively rigid counterpart. Taken together, we believe that these characteristics might be of importance to understand the stability and functions of βγ-crystallin domains.     

Role of βγ-Dimers of GTP-Binding Proteins in Processes of Hormonal Signal Transduction

This review summarizes and analyzes data on structural and functional peculiarities of - and -subunits of heterotrimeric G proteins and their participation in -dimer in processes of hormonal signal transduction to the cell. The molecular mechanisms are discussed, which are responsible for formation of the -dimer complex and its interaction with the wide spectrum of signal and structural proteins, such as G protein -subunit, serpentine type receptors, kinases of G protein-coupled receptors, adenylyl cyclase, phospholipase C2, phosphatidyl-inositol-3-kinase, Raf-kinase, potassium and calcium channels, microtubular and RGS-proteins. The characteristics of - and -subunits are given for living organisms of different phylogenetic levels (lower eukaryotes, fungi, plants, vertebrates, and invertebrates). It is concluded that the -dimer is the most important regulatory link in various signal systems to carry out switch from one signal cascade to another and thereby to determine the final cellular response to the signal impulse.     

Succession of pelagic copepod species in coastal waters off northern Chile: the influence of the 1997–98 El Niño

A 2-year time series (17-d interval) of zooplankton, sampled from June 1996 through January 1998, along with oceanographic data, allowed the study of species abundances and diversity during non-El Niño and El Niño conditions at Mejillones (23° S), northern Chile. A total of 28 species was identified, of which the most abundant were the calanoids Paracalanus parvus, Calanus chilensis, Acartia tonsa, and Centropages brachiatus, the cyclopoid Oithona similis, and the poecilostomatoids Oncaea conifera and Corycaeus typicus. El Niño Southern Oscillation (El Niño) was first detected in the area in May 1997 and persisted until March 1998. We divided the time series into two parts: non-El Niño and El Niño conditions. The number of species significantly increased during El Niño conditions, but total abundance of copepods was greater during non-El Niño conditions. Although some expatriate species appeared during El Niño, such as the harpacticoid Microsetella norvegica and Eucalanus subtenuis, the diversity index of Hulburt was not significantly different between the two periods. Despite the dramatic alteration of oceanographic conditions during the El Niño period, we concluded that the community of pelagic copepods was not subject to drastic changes either in total abundance or in structure.