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     

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     

A natively unfolded βγ-crystallin domain from Hahella chejuensis

To date, very few βγ-crystallins have been identified and structurally characterized. Several of them have been shown to bind Ca(2+) and thereby enhance their stability without any significant change in structure. Although Ca(2+)-induced conformational changes have been reported in two putative βγ-crystallins from Caulobacter crescentus and Yersinia pestis, they are shown to be partially unstructured, and whether they acquire a βγ-crystallin fold is not known. We describe here a βγ-crystallin domain, hahellin, its Ca(2+) binding properties and NMR structure. Unlike any other βγ-crystallin, hahellin is characterized as a pre-molten globule (PMG) type of natively unfolded protein domain. It undergoes drastic conformational change and acquires a typical βγ-crystallin fold upon Ca(2+) binding and hence acts as a Ca(2+)-regulated conformational switch. However, it does not bind Mg(2+). The intrinsically disordered Ca(2+)-free state and the close structural similarity of Ca(2+)-bound hahellin to a microbial βγ-crystallin homologue, Protein S, which shows Ca(2+)-dependent stress response, make it a potential candidate for the cellular functions. This study indicates the presence of a new class of natively unfolded βγ-crystallins and therefore the commencement of the possible functional roles of such proteins in this superfamily.     

Complete backbone assignment of a Ca2+-binding protein of the βγ-crystallin superfamily from Methanosarcina acetivorans, at two denaturant concentrations

We report here almost complete backbone assignment of a Ca²⁺-binding protein of the βγ-crystallin superfamily from Methanosarcina acetivorans, at two denaturant (GdmCl) concentrations, using double and triple resonance experiments. These NMR assignments will be useful to understand the unfolding path of this protein. Ravi P. Barnwal and Geetika Agarwal have contributed equally.     

Sequence specific 1H, 13C, and 15N resonance assignments of Hahellin from Hahella chejuensis, a putative member of the βγ-crystallin superfamily

The sequence specific ¹H, ¹³C, and ¹⁵N resonance assignments of Hahellin, a putative member of βγ-crystallin family, from Hahella Chejuensis, have been accomplished by NMR spectroscopy. The resonance assignments reveal that the protein adopts predominantly a β-sheet conformation as in the case of βγ-crystallin folds.     

Iterative cloning, overexpression, purification and isotopic labeling of an engineered dimer of a Ca(2+)-binding protein of the βγ-crystallin superfamily from Methanosarcina acetivorans

βγ-Crystallins are a large superfamily of proteins found in vertebrate eye lens. They are hetero-dimers (linked in tandem by a specific peptide) and are shown to bind calcium. The monomers possess two β-strand rich greek-key motifs. Recently, a structurally closest member to the family of lens βγ-crystallins has been described, for the first time, from the archaea Methanosarcina acetivorans, which is named as M-crystallin. Unlike lens βγ-crystallins, M-crystallin exits as a monomer. Here, we synthesized a dimeric gene of M-crystallin in which two monomers are linked by a 10-amino acid residue coding sequence. The linker sequence in the target protein is long and flexible enough to reduce the proximity between the individual crystallins in the dimer. This methodology would be highly beneficial in designing polyproteins (two or more proteins linked in tandem to aid mechanical stretching studies) that are regularly used in single-molecule force spectroscopy. The dimer of M-crystallin was overexpressed in Escherichia coli BLR(DE3) strain. The overexpressed protein containing an N-terminal hexa-histidine tag was purified using nickel affinity chromatography and then by size-exclusion chromatography. Further, a method to purify isotopically ((15)N) labeled protein with high yield for NMR studies is reported. The uniformly (15)N-labeled M-crystallin dimer thus produced has been characterized by recording sensitivity enhanced 2D [(15)N-(1)H] HSQC and other optical spectroscopy techniques. Observation of only one set of peaks in the HSQC, along with the structural characterization using optical spectroscopy, suggests that the domains in the dimer possess similar structure as that of the monomer.     

Decoding the molecular design principles underlying Ca(2+) binding to βγ-crystallin motifs

Numerous proteins belonging to the recently expanded βγ-crystallin superfamily bind Ca(2+) at the double-clamp N/D-N/D-X(1)-X(2)-S/T-S motif. However, there have been no attempts to understand the intricacies involving Ca(2+) binding, such as the determinants of Ca(2+)-binding affinity and their contributions to gain in stability. This work is an in-depth analysis of understanding the modes and determinants of Ca(2+) binding to βγ-crystallin motifs. We have performed extensive naturally occurring substitutions from related proteins on the βγ-crystallin domains of flavollin, a low-affinity Ca(2+)-binding protein, and clostrillin, a moderate-affinity protein. We monitored the consequences of these modifications on Ca(2)(+) binding by isothermal titration calorimetry, thermal stability and conformational and crystal structure analyses. We demonstrate that Ca(2)(+) binding to the two sites of a βγ-domain is interdependent and that the presence of Arg at the fifth position disables a site. A change from Thr to Ser, or vice versa, influences Ca(2+)-binding affinity, highlighting the basis of diversity found in these domains. A subtle change in the first site has a greater influence on Ca(2)(+) binding than a similar alteration in the second site. Thus, the second site is more variable in nature. Replacing an acidic or hydrophobic residue in a binding site alters the Ca(2+)-binding properties drastically. While it appears from their binding site sequence that these domains have evolved randomly, our examination illustrates the subtlety in the design of these modules. Decoding such design schemes would aid in our understanding of the functional themes underlying differential Ca(2)(+) binding and in predicting these in emerging sequence information.     

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.     

Backbone 1H, 13C and 15N resonance assignments of an intrinsically unstructured βγ-crystallin from Hahella chejuensis

The sequence specific backbone ¹H, ¹³C and ¹⁵N resonance assignments of an intrinsically unstructured βγ-crystallin from Hahella chejuensis are reported. The secondary structure chracterization of the unstructured protein reveals that large fraction of residues exhibits β-strand propensity, as in the case of the Ca²⁺-bound structured protein.     

Preliminary X-ray crystallographic study of the turkey lens protein, δ-crystallin

The structural protein, δ-crystallin, has been purified and crystallized from adult turkey lens. The crystals are orthorhombic and normally belong to space group P2₁2₁2 with unit cell dimensions $\text{a = 99.9(2)}\text{A}\text{?}\text{, b = 133.4(3)}\text{A}\text{?}\text{and}\text{c = 69.1(2)}\text{A}\text{?}$. This corresponds to two molecules of molecular weight approximately 200,000 per unit cell. A second crystal form has also been found in which b and c increase to 135.4(3) Å and 140.0(3) Å. respectively, indicating four molecules per unit cell.     

Binding of γ-crystallin substrate prevents the binding of copper and zinc ions to the molecular chaperone α-crystallin

α-Crystallin is a small heat shock protein and molecular chaperone. Binding of Cu2+ and Zn2+ ions to α-crystallin leads to enhanced chaperone function. Sequestration of Cu2+ by α-crystallin prevents metal-ion mediated oxidation. Here we show that binding of human γD-crystallin (HGD, a natural substrate) to human αA-crystallin (HAA) is inversely related to the binding of Cu2+/Zn2+ ions: The higher the amount of bound HGD, the lower the amount of bound metal ions. Thus, in the aging lens, depletion of free HAA will not only lower chaperone capacity but also lower Cu2+ sequestration, thereby promoting oxidation and cataract.     

How α-crystallin prevents the aggregation of insulin

Using steady-state, polarized, and phase-modulation fluorometry, the dithiothreitol-induced denaturation of insulin and formation of its complex with alpha-crystallin in solution were studied. Prevention of the aggregation of insulin by alpha-crystallin is due to formation of chaperone complexes, i.e. interaction of chains of the denatured insulin with alpha-crystallin. The conformational changes in alpha-crystallin that occur during complex formation are rather small. It is unlikely that N-termini are directly involved in the complex formation. The 8-anilino-1-naphthalenesulfonate (ANS) is not sensitive to the complex formation. ANS emits mainly from alpha-crystallin monomers, dimers, and tetramers, but not from oligomers or aggregates. The possibility of highly sensitive detection of aggregates by light scattering using a spectrofluorometer with crossed monochromators is demonstrated.     

The βγ-crystallin domain of Lysinibacillus sphaericus phosphatidylinositol phospholipase C plays a central role in protein stability

Applied Microbiology and Biotechnology - βγ-crystallin has emerged as a superfamily of structurally homologous proteins with representatives across all domains of life. A major portion of...     

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.     

Further studies on the sub-units of α-crystallin

1. A new procedure is described for the purification of alpha-crystallin, including: preparative zone electrophoresis, density-gradient centrifugation and gel filtration. The total amino acid composition of highly purified samples prepared according to this procedure has been determined. 2. Evidence is presented for the presence of intermediates in the urea-induced splitting of alpha-crystallin into sub-units. A possible mechanism for this splitting is proposed. 3. The recombination of sub-units has been studied by polyacrylamide-gel electrophoresis and ultracentrifugal analysis. As judged from these criteria, only a partial recovery of starting material was obtained. 4. The origin of the minor bands in the electrophoretic pattern of alpha-crystallin on 7m-urea-polyacrylamide gel has been investigated. No evidence was found that their presence is due to carbamoylation or sulphide-disulphide interchange. They probably arise from isomerization. 5. The mean molecular weight of the sub-units was calculated to be 24000 (Archibald's method). Determination of the sedimentation-diffusion equilibrium revealed a value of 21000 at the meniscus. Assuming that all sub-units contain one cysteine residue/molecule, 23000 can be derived for the mean molecular weight.     

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.     

Degradation of δ-crystallin mRNA in the lens fiber cells of the chicken

δ-Crystallin is the principal protein synthesized in the embryonic chicken lens. After hatching δ-crystallin synthesis decreases and eventually ceases. We have determined when the δ-crystallin messenger RNA (mRNA) disappears from the lens fiber cells during the first year of age by cell-free translation of lens RNA in a reticulocyte lysate, RNA blot (Northern) hybridization, and in situ hybridization. The hybridization was performed with a nick-translated, cloned δ-crystallin cDNA (pδCr2). δ-Crystallin mRNA was present in the lens until 3 months of age and disappeared between the third and fifth month after hatching. The in situ hybridization experiments indicated that the δ-crystallin mRNA was present throughout the lens fiber mass until 1 month after hatching and was greatly reduced in the cortical fiber cells thereafter. In contrast to earlier stages, then, the cortical fiber cells differentiating at the lens equator after about 1 month of age do not accumulate δ-crystallin mRNA. The data also indicate that the maximal half-life of functional δ-crystallin mRNA in the posthatched chicken lens is about 2 months.     

Evolutionary changes ofα-crystallin and the phylogeny of mammalian orders

Summary The sequences of the A chains of the eye lens protein-crystallin from seventeen mammalian species were compared. They showed a generally slow rate of evolution, but with marked variations in different lineages. Most substitutions have occurred in the C-terminal part of the chain, which probably forms part of the surface of the-crystallin aggregate. The ancestral sequence method of Dayhoff revealed interesting indications about the phylogenetic relationships between the eleven mammalian orders that were represented by the investigated species. Most evident was the divergence of marsupial and placental orders. A notable resemblance between the hyrax and elephant sequences was observed, setting them apart from the ungulates, including whale. Primates, rodents, lagomorphs, insectivores and tupaiids seem to derive from a common stem group. These phylogenetic inferences are discussed in relation to current palaeontological and taxonomical opinions, and compared to evidence from other protein sequence data.     

Calcium-induced decrease of the thermal stability and chaperone activity of α-crystallin

α-Crystallin, one of the major proteins in the vertebrate eye lens, acts as a molecular chaperone, like the small heat-shock proteins, by protecting other proteins from denaturing under stress or high temperature conditions. α-Crystallin aggregation is involved in lens opacification, and high [Ca²⁺] has been associated with cataract formation, suggesting a role for this cation in the pathological process. We have investigated the effect of Ca²⁺ on the thermal stability of α-crystallin by UV and Fourier-transform infrared (FTIR) spectroscopies. In both cases, a Ca²⁺-induced decrease in the midpoint of the thermal transition is detected. The presence of high [Ca²⁺] results also in a marked decrease of its chaperone activity in an insulin-aggregation assay. Furthermore, high Ca²⁺ concentration decreases Cys reactivity towards a sulfhydryl reagent. The results obtained from the spectroscopic analysis, and confirmed by circular dichroism (CD) measurements, indicate that Ca²⁺ decreases both secondary and tertiary–quaternary structure stability of α-crystallin. This process is accompanied by partial unfolding of the protein and a clear decrease in its chaperone activity. It is concluded that Ca²⁺ alters the structural stability of α-crystallin, resulting in impaired chaperone function and a lower protective ability towards other lens proteins. Thus, α-crystallin aggregation facilitated by Ca²⁺ would play a role in the progressive loss of transparency of the eye lens in the cataractogenic process.