Crystallin Research Articles

Characterizing the Role of Palindromic Strand Exchange in Alpha Crystallin Oligomerization

The small heat-shock protein (sHSP) alpha-crystallin is a major component of the eye lens where it performs critical functions in maintaining lens transparency. Alpha-crystallin acts as a molecular chaperone to prevent stress-damaged and aging proteins from forming light-scattering aggregates. Further, a high concentration of alpha-crystallin contributes to a large and uniform refractive index across the lens tissue. Despite being expressed in such high concentrations, alpha-crystallin does not form aggregates itself, a property facilitated by the assembly of alpha-crystallin into polydisperse, dynamic oligomers that frustrate aggregation and crystallization. Structural studies suggest that one mechanism underlying this polydisperity may be the ability of a palindromic sequence centered on the sHSP IXI motif in the alphaB-crystallin isoform c-terminus to bind bidirectionally to other monomers. Strand exchanges formed in this manner would result in varied oligomeric structures while maintaining near identical residue interactions. We are using Förster resonance energy transfer (FRET) to measure the ability of the palindromic sequence to bind in differential orientations in solution and to determine any intrinsic directional bias. These data will help determine whether bidirectional strand exchange is important for αB-crystallin function by assaying how strand binding influences chaperone activity. Our work will build upon the proposed mechanism of alpha-crystallin polydisperity and clarify the importance of bidirectional strand exchange for alpha-crystallin function.

Clarifying Alpha Crystallin Chaperone Function by using an Insulin B-Chain Aggregation Model

The crystallin family of proteins exist in high concentrations in the lens of the eye by are also found throughout the body. In the lens, alpha-crystallin isoforms A and B exist in a ratio of 2 to 3. Their role in the eye is to provide a structural matrix and prevent protein misfolding. The inducible aggregation of the insulin-B chain serves as a model to the protein aggregation that occurs in the human lens over time. This protein aggregation contributes to lens opacity, which is the beginning of cataract formation. In this study, we explore the kinetic and thermodynamics of such system in order to understanding the mechanism of alpha-crystallin chaperone function in insulin aggregation. Insulin aggregation can be measured through light scattering. Our preliminary result suggest a mechanism for aggregation in which a B-chain insulin dimer formation precedes aggregation and that alpha crystallin and insulin monomer form a 2 to 1 complex that inhibits this aggregation. Thermodynamic and kinetic constants will be presented for the purified alpha chrystallins as well as mixtures and bulk protein.

Rewarding Incremental Innovation in Cancer Research

The landmark decision to rejection of patent to anti-cancer drug 'Glivec' by Indian Supreme Court has catalysed range of debates around importance of innovation in research and development. The patent plea by Novartis for beta crystalline for of imanitib (i.e. glivec) was considered as an attempt to extend period of market exclusivity by filing patent for minor modification of molecule for which US patent was already been granted on 1996. Novartis has firmly stated court's ruling as a setback to patent protection for its years of research invested in development of more stable and bioavailable form of imanitib, and offended by this decision they have issued host of statements about not investing in drug discovery RD however after reviewing history of imanitib and its subsequent refinement into glivec, one cannot avoid thinking about it as a typical case of patent evergreening (1-3). The cause of disagreement in this patent war is section 3(d) of Indian Patent Act, which limits scope of incremental innovation by recognizing only those incremental inventive steps, which are associated with significant increase in efficacy of drug; this section was added with an intention to curb evergreening. The importance of controlling patent evergreening cannot be denied, especially for developing and underdeveloped nations, which cannot afford to buy patented drugs at premium price to meet their health care requirements and are heavily dependent on generic version of the drug, as is evident from the impact of availability of low cost generic version of anti-retroviral drugs. At the same time, we cannot deny importance of incremental innovation in making effective and safer drug.

Effects of α-crystallin on bioactivity of iNOS in the activated retinal microglial cells

Objective To investigate effects of α-crystallin on proliferation of lipopolysaccharide (LPS)-activated retinal microglia and bioactivity of iNOS.Methods The retinal microglial cells cultured in vitro were analyzed and their purity was identified by cell immunofluorescence and flow cytometry.After microglia cells being intervened using LPS and α-crystallin at various concentrations,influence of α-crystallin on activity of LPS-activated retinal microglia was detected by MTT method and level of NO was measured by RT-PCR to observe changes of iNOS expression in microglia.Results Purity of primary cultured microglial cells was 94.15％ by GSA-IB4 immunohistochemical identification and 93.34％ by CD11b flow cytometry.α-crystallin of 10-4g/L awakened activity of microglia induced by 10-6g/L LPS (P ＜ 0.01).Expressions of iNOS protein and mRNA showed significant decrease in combined treatment group (P ＜ 0.05).Conclusion In clinical condition,α-crystallin decreases the harm of microglial cells on retinal ganglial cells (RGCs) after optical nerve injury by inhibiting the microglia cells to produce NO and iNOS. Key words: Alpha-crystallins; Microglia; Retina; Cell culture techniques

Differential Expression Patterns of Crystallin Genes during Ocular Development of Olive Flounder (Paralichthys olivaceus).

Olive flounder Paralichthys olivaceus is one of the most widely cultured fish species in Korea. Although olive flounder receive attention from aquaculture and fisheries and extensive research has been conducted eye morphological change in metamorphosis, but little information was known to molecular mechanism and gene expression of eye development- related genes during the early part of eye formation period. For the reason of eyesight is the most important sense in flounder larvae to search prey, the screening and identification of expressed genes in the eye will provide useful insight into the molecular regulation mechanism of eye development in olive flounder. Through the search of an olive flounder DNA database of expressed sequence tags (EST), we found a partial sequence that was similar to crystallin beta A1 and gamma S. Microscopic observation of retinal formation correspond with the time of expression of the crystallin beta A1 and gamma S gene in the developmental stage, these result suggesting that beta A1 and gamma S play a vital role in the remodeling of the retina during eye development. The expression of crystallin beta A1 and gamma S were obviously strong in eye at all tested developing stage, it is also hypothesized that crystallin acts as a molecular chaperone to prevent protein aggregation during maturation and aging in the eye.

IgG and IgM Autoantibody Differences in Discoid and Systemic Lupus Patients

Systemic lupus (SLE) patients with discoid lupus (DLE) were reported to have milder disease. To test this observation, we employed sandwich arrays containing 98 autoantigens to compare autoantibody profiles of SLE subjects without DLE (DLE−SLE+) (N=9), SLE subjects with DLE (DLE+SLE+) (N=10), DLE subjects without SLE (DLE+SLE−) (N=11), and healthy controls (N=11). We validated differentially expressed autoantibodies using immunoassays in DLE−SLE+ (N=18), DLE+SLE+ (N=17), DLE+SLE− (N=23), and healthy subjects (N=22). Arrays showed 15 IgG autoantibodies (ten against nuclear antigens) and four IgM autoantibodies that were differentially expressed (q-value<0.05). DLE−SLE+ subjects had higher IgG autoantibodies against dsDNA, ssDNA, dsRNA, histone H2A and H2B, and SS-A (52 kDa) than all other groups including DLE+SLE+ subjects (p<0.05). Immunoassays measuring anti-dsDNA, -ssDNA, and -SS-A (52 kDa) IgG autoantibodies showed similar trends (p<0.05). Healthy and DLE+SLE−subjects expressed higher IgM autoantibodies against alpha beta crystallin, lipopolysaccharide, heat shock cognate 70, and desmoglein-3 than DLE+SLE+ and DLE−SLE+ subjects. IgG:IgM ratios of autoantibodies against nuclear antigens progressively rose from healthy to DLE−SLE+ subjects. In conclusion, lower IgG autoantibodies against nuclear antigens in DLE+SLE+ versus DLE−SLE+ subjects suggest that DLE indicates lower disease severity. Higher IgM autoantibodies against selected antigens in healthy and DLE+SLE−subjects may be non-pathogenic.

Association of High Myopia with Crystallin Beta A4 (CRYBA4) Gene Polymorphisms in the Linkage-Identified MYP6 Locus

BackgroundMyopia is the most common ocular disorder worldwide and imposes tremendous burden on the society. It is a complex disease. The MYP6 locus at 22 q12 is of particular interest because many studies have detected linkage signals at this interval. The MYP6 locus is likely to contain susceptibility gene(s) for myopia, but none has yet been identified.Methodology/Principal FindingsTwo independent subject groups of southern Chinese in Hong Kong participated in the study an initial study using a discovery sample set of 342 cases and 342 controls, and a follow-up study using a replication sample set of 316 cases and 313 controls. Cases with high myopia were defined by spherical equivalent ≤ -8 dioptres and emmetropic controls by spherical equivalent within ±1.00 dioptre for both eyes. Manual candidate gene selection from the MYP6 locus was supported by objective in silico prioritization. DNA samples of discovery sample set were genotyped for 178 tagging single nucleotide polymorphisms (SNPs) from 26 genes. For replication, 25 SNPs (tagging or located at predicted transcription factor or microRNA binding sites) from 4 genes were subsequently examined using the replication sample set. Fisher P value was calculated for all SNPs and overall association results were summarized by meta-analysis. Based on initial and replication studies, rs2009066 located in the crystallin beta A4 (CRYBA4) gene was identified to be the most significantly associated with high myopia (initial study: P = 0.02; replication study: P = 1.88e-4; meta-analysis: P = 1.54e-5) among all the SNPs tested. The association result survived correction for multiple comparisons. Under the allelic genetic model for the combined sample set, the odds ratio of the minor allele G was 1.41 (95% confidence intervals, 1.21-1.64).Conclusions/SignificanceA novel susceptibility gene (CRYBA4) was discovered for high myopia. Our study also signified the potential importance of appropriate gene prioritization in candidate selection.

Traumatology of the optic nerve and contribution of crystallins to axonal regeneration

Within a few decades, the repair of long neuronal pathways such as spinal cord tracts, the optic nerve or intracerebral tracts has gone from being strongly contested to being recognized as a potential clinical challenge. Cut axonal stumps within the optic nerve were originally thought to retract and become irreversibly necrotic within the injury zone. Optic nerve astrocytes were assumed to form a gliotic scar and remodelling of the extracellular matrix to result in a forbidden environment for re-growth of axons. Retrograde signals to the ganglion cell bodies were considered to prevent anabolism, thus also initiating apoptotic death and gliotic repair within the retina. However, increasing evidence suggests the reversibility of these regressive processes, as shown by the analysis of molecular events at the site of injury and within ganglion cells. We review optic nerve repair from the perspective of the proximal axon stump being a major player in determining the successful formation of a growth cone. The axonal stump and consequently the prospective growth cone, communicates with astrocytes, microglial cells and the extracellular matrix via a panoply of molecular tools. We initially highlight these aspects on the basis of recent data from numerous laboratories. Then, we examine the mechanisms by which an injury-induced growth cone can sense its surroundings within the area distal to the injury. Based on requirements for successful axonal elongation within the optic nerve, we explore the models employed to instigate successful growth cone formation by ganglion cell stimulation and optic nerve remodelling, which in turn accelerate growth. Ultimately, with regard to the proteomics of regenerating retinal tissue, we discuss the discovery of isoforms of crystallins, with crystallin beta-b2 (crybb2) being clearly upregulated in the regenerating retina. Crystallins are produced and used to promote the elongation of growth cones. In vivo and in vitro, crystallins beta and gamma additionally promote the growth of axons by enhancing the production of ciliary neurotrophic factor (CNTF), indicating that they also act on astrocytes to promote axonal regrowth synergistically. These are the first data showing that axonal regeneration is related to crybb2 movement within neurons and to additional stimulation of CNTF. We demonstrate that neuronal crystallins constitute a novel class of neurite-promoting factors that probably operate through an autocrine and paracrine mechanism and that they can be used in neurodegenerative diseases. Thus, the post-injury fate of neurons cannot be seen merely as inevitable but, instead, must be regarded as a challenge to shape conditions for initiating growth cone formation to repair the damaged optic nerve.

The Thermal Structural Transition of Alpha-Crystallin Modulates Subunit Interactions and Increases Protein Solubility

BackgroundAlpha crystallin is an oligomer composed of two types of subunits, alpha-A and alpha-B crystallin, and is the major constituent of human lens. The temperature induced condensation of alpha-crystallin, the main cause for eye lens opacification (cataract), is a two step-process, a nucleation followed by an aggregation phase, and a protective effect towards the aggregation is exhibited over the alpha crystallin phase transition temperature (Tc = 318.16 K).Methods/ResultsTo investigate if a modulation of the subunit interactions over Tc could trigger the protective mechanism towards the aggregation, we followed, by using simultaneously static and dynamic light scattering, the temperature induced condensation of alpha-crystallin. By developing a mathematical model able to uncouple the nucleation and aggregation processes, we find a previously unobserved transition in the nucleation rate constant. Its temperature dependence allows to determine fundamental structural parameters, the chemical potential (Δμ) and the interfacial tension (γ) of the aggregating phase, that characterize subunit interactions.Conclusions/General SignificanceThe decrease of both Δμ and γ at Tc, and a relative increase in solubility, reveal a significative decrease in the strenght of alpha-crystallin subunits interactions, which protects from supramolecolar condensation in hypertermic conditions. On the whole, we suggest a general approach able to understand the structural and kinetic mechanisms involved in aggregation-related diseases and in drugs development and testing.

Cataract-linked γD-crystallin mutants have weak affinity to lens chaperones α-crystallins

To test the hypothesis that α-crystallin chaperone activity plays a central role in maintenance of lens transparency, we investigated its interactions with γ-crystallin mutants that cause congenital cataract in mouse models. Although the two substitutions, I4F and V76D, stabilize a partially unfolded γD-crystallin intermediate, their affinities to α-crystallin are marginal even at relatively high concentrations. Detectable binding required further reduction of γD-crystallin stability which was achieved by combining the two mutations. Our results demonstrate that mutants and possibly age-damaged γ-crystallin can escape quality control by lens chaperones rationalizing the observation that they nucleate protein aggregation and lead to cataract. Structured summary of protein interactionsgammaD Crystallin and alphaB Crystallin bind by molecular sieving (View interaction)gammaD Crystallin and alphaB Crystallin bind by fluorescence technology (View interaction)gammaD Crystallin and alphaA Crystallin bind by fluorescence technology (View interaction)gammaD Crystallin and alphaA Crystallin bind by molecular sieving (View interaction)

Elucidating Molecular Constraints that Effect Alpha Crystallin Oligomerization, Stability, and Chaperone Function

Alpha Crystallin is the major protein component of the human lens and plays an important role in the prevention of cataracts. α-Crystallin (αX) oligomers consist of two isoforms, αX-A and αX-B which share high sequence similarity and define the common α-Crystallin fold found in many small heat shock proteins (sHSPs). αX-A and αX-B are hypothesized to play two important roles within the lens. First, αX-A and αX-B belong to a group of proteins called Crystallins (α, β, and γ) that are very stable proteins that play a role in preserving a uniform density within the lens, which allows it to focus light. The Crystallin proteins’ ability to form diverse and stable oligomers results in a glass-like rather than crystalline organization to the lens protein material, which also aids in the long-term stability of this high-density protein organ. Second, αX-A and αX-B both function as sHSPs that bind to misfolded proteins, preventing formation of large, insoluble protein aggregates (the beginning of cataracts). Our lab is investigating the molecular interactions between αX-A and αX-B that result in its stability, diverse oligomerization, and chaperone function. To this end we are using a model, inducible misfolding protein (insulin B-chain) to study chaperone function by light scatter under various conditions. We are also using random and targeted modification of αX-A and αX-B to simulate long-term protein damage and degradation observed in aged lenses. Focus on the C-terminal strand exchange observed in recent crystal structures and proposed to aid in αX-A and αX-B polydisperse oligomerization is additionally aiding experimental design. We hope to identify specific molecular interactions that result in αX-A and αX-B’s chaperone function, and determine how those interactions relate to stability and self-oligomerization.

Temperature-dependent structural and functional properties of a mutant (F71L) αA-crystallin: Molecular basis for early onset of age-related cataract

Previously we identified a novel mutation (F71L) in the αA-crystallin gene associated with early onset of age-related cataract. However, it is not known how the missense substitution translates into reduced chaperone-like activity (CLA), and how the structural and functional changes lead to early onset of the disease. Herein, we show that under native conditions the F71L-mutant is not significantly different from wild-type with regard to secondary and tertiary structural organization, hydrophobicity and the apparent molecular mass of oligomer but has substantial differences in structural and functional properties following a heat treatment. Wild-type αA-crystallin demonstrated increased CLA, whereas the F71L-mutant substantially lost its CLA upon heat treatment. Further, unlike the wild-type αA-subunit, F71L-subunit did not protect the αB-subunit in hetero-oligomeric complex from heat-induced aggregation. Moreover, hetero-oligomer containing F71L and αB in 3:1 ratio had significantly lower CLA upon thermal treatment compared to its unheated control. These results indicate that α-crystallin complexes containing F71L-αA subunits are less stable and have reduced CLA. Therefore, F71L may lead to earlier onset of cataract due to interaction with several environmental factors (e.g., temperature in this case) along with the aging process. Structured summary of protein interactionsalphaA crystallin and alphaA crystallinbind by molecular sieving (View interaction)alphaA crystallin and alphaB crystallinbind by molecular sieving(View interaction)

Protein post-translational modification and age-related cataract

Cataract is the main cause of vision impairment, and age-related cataract is the most common type of cataract. Protein post-translational modification is the enzymatic processing of a polypeptide chain after translation from messenger RNA and after peptide bond formation has occurred. Deamidation, glycation and oxidation are age-related processes. They can change the structure or solubility of crystallin and result in the opacity of lens while acetylation and methylation may be related to the protection of lens protein. Although much effort has been directed towards slowing progression or preventing the occurrence of cataract, the management of cataract remains surgical. This review deals with the relations between different kinds of protein post-translational modification and age-related cataract as well as some protective methods aiming at the process of post-translational modification.

1H, 13C, and 15N assignments of cataract-related variant gamma-S-G18V crystallin

1H, 13C, and 15N assignments of cataract-related variant gamma-S-G18V crystallin

Implication of MAPK in sodium salicylate-induced heat shock protein 27 expression in human lens eplthelial Cells in vitro

Background Heat shock proteins (HSPs) are highly conserved proteins that are induced in cells when confronted with a wide variety of proteotoxic stresses.HSP27 has a high degree of similarity with α-crystallin protein.The abnormality of HSP27 structure and expression are closely related to the formation of cataracts.Our previous study showed sodium salieylate has the protective effect on H2O2-induced lens damage.Objective This study was to investigate the roles of MAPK signal pathway in sodium salicylate-induced the expression of HSP27 in human lens epithelial cells (LECs) in vitro.Methods Human LECs were incubated in the fresh media containing sodium salicylate at different concentrations (0-55 mmol/L) for different times (1-5 hours) and allowed to be recovered in fresh medium without sodium salicylate for 1-24 hours with or without pretreatment with P38MAPK inhibitor (SB203580), ERK1/2 inhibitor (PD98059) and JNK/SAPK inhibitor ( SP600125). The expressions of P38MAPK, EBK1/2, JNK/SAPK, phosphorylated P38MAPK, phosphorylated ERK1/2, phosphorylated JNK/SAPK and HSP27 were detected by Western blot. HSP27 mRNA was detected by RT-PCR. The expression of HSP27 was also detected by immunohistochemistry. Results There was only weak expression of HSP27 in normal human LECs.After stimulation of 35-55 mmol/L sodium salicylate was removed and human LECs were cultured again for 6 hours,the expression of HSP27 in LECs were significantly increased ( F= 509. 953,P<0. 01). HSP27 was absent expressed in human LECs in 55 mmol/L sodium salicylate stimulation for 1-5 hours groups, but LECs were re-cultured for 3,6 hours after removed the stimulation, the expression of HSP27 was elevated (F = 452. 534, P<0. 01). Activation of P38 M APK occurred after sodium salicylate stimulation 30 minutes and 1 hour ( F = 865.68, P<0. 01). However, ERK 1/2 was expressed after sodium salicylate was eliminated for 1-6 hours ( F = 388.84, P<0. 01). JNK/SAPK was inactived by sodium salicylate. The expression of HSP27 could be down-regulated with the pretreatment of SB203580 and PD98059. Conclusion Sodium salicylatc can induce the expression of HSP27 in human (LECs) . The effects are mediated,at least in part ,through the activation of P38MAPK and ERK1/2 signaling pathway . Key words: Sodium salicylate; Human lens epithelial cells; Mitogen-activated protein kinase; Heat shock proteins

Polymorphisms of the gamma crystallin A and B genes among Indian patients with pediatric cataract

Previous familial studies have reported co-segregation of mutation in gamma crystallin A and B CRYGA and CRYGB genes with childhood cataract. We investigated association of nucleotide variations in these genes in subjects with and without pediatric cataract from India. The study included 195 pediatric subjects including healthy children with no ocular defects and pediatric cataract cases. Subjects were genotyped by PCR-RFLP method for exonic and intronic genetic variations in CRYGA and CRYGB. The association of these polymorphisms with cataract was estimated by two way contingency tables and the risk allele was also analyzed for their functional impact using in silico tools. Results : No significant difference was observed between cases and control subjects for the frequencies of SNPs G198A (Intron A), T196C (Exon 3) of CRYGA and G449T (Exon 2) of CRYGB gene. -47C allele of rs2289917 in CRYGB showed the strongest association with cataract (Odd Ratio-OR=3.34, 95% Confidence Interval-CI 95% =1.82-6.12, P=0.00007). In silico analyses revealed that this polymorphism lies in a phylogenetically conserved region and impacts binding of a transcription factor, viz. progesterone receptor (PR) to CRYGB promoter. rs2289917 risk allele showed a strong association with increased vulnerability for pediatric cataract. The findings suggest that this association may be a secondary phenomenon related to genetic variation playing critical role in lens development during perinatal and/or pediatric growth. Present exploratory study provides a basis for further defining the role of PR as a regulator of CRYG locus in lens formation/transparency.

Tight binding of proteins to membranes from older human cells

The lens is an ideal model system for the study of macromolecular aging and its consequences for cellular function, since there is no turnover of lens fibre cells. To examine biochemical processes that take place in the lens and that may also occur in other long-lived cells, membranes were isolated from defined regions of human lenses that are synthesised at different times during life, and assayed for the presence of tightly bound cytosolic proteins using quantitative iTRAQ proteomics technology. A majority of lens beta crystallins and all gamma crystallins became increasingly membrane bound with age, however, the chaperone proteins alpha A and alpha B crystallin, as well as the thermally-stable protein, βB2 crystallin, did not. Other proteins such as brain-associated signal protein 1 and paralemmin 1 became less tightly bound in the older regions of the lens. It is evident that protein-membrane interactions change significantly with age. Selected proteins that were formerly cytosolic become increasingly tightly bound to cell membranes with age and are not removed even by treatment with 7 M urea. It is likely that such processes reflect polypeptide denaturation over time and the untoward binding of proteins to membranes may alter membrane properties and contribute to impairment of communication between older cells.

Human retinal proteomics study of adults and two-month old infants

Objective To identify proteins that have expressed in human eyes from adults and two-month old infants by proteomics approach, so as to build a two-dimensional gel electrophoresis (two-DE) reference map for human retina. The difference of proteomics between the retinas of adults and two-month old infants are also studied. Methods Human retina tissues were collected from donor eyes (nine adults and two infants). Proteins were separated by two-DE. The gels were analyzed by image software. Protein spots were excised from the gels and detected by matrix assisted laser desorption ionization time off light mass spectrometry (MALDI-TOF-MS). Results A total of 1179 spots and 1295 spots were detected respectively on two-DE gels of Coomassie-stained adults and two-month old infants retina, of which 1039 spots were matched in the position. Five spots up-regulated were successfully identified. Human serum albumin and 4 guanylate kinase 1 (GUK1) were identified in adult retina. β2-tubulin, transaldolasel and alpha A-crystallin were identified in infant retina. Conclusion The two-DE reference map for retina proteomics is successfully established. This study provides an evidence of changes in retinal protein levels between adults and infants and biochemical pathways for future studies of human retina development. Key words: Proteomics; Retina/physiology; Adult; Infant

Molecular cloning of a small heat shock protein (sHSPII) from the cattle tick Rhipicephalus ( Boophilus) annulatus salivary gland

Immunoscreening of a cDNA expression library of the Rhipicephalus ( Boophilus) annulatus tick with purified rabbit anti- R annulatus salivary glands antigens polyclonal antibodies led to the identification of a 661 bp sequence. The sequence includes an open reading frame of 543 bp encoding a protein of 180 amino acids with calculated molecular weight of 20.51 kDa, isoelectric point of 9.071 and with no signal sequence. Comparison of the deduced amino acids with protein data bank showed that the identified polypeptide belongs to the alpha crystallin small heat shock proteins superfamily and shows sequence similarity of 62% and 55% to Ixodes scapularis fed tick salivary gland protein and Ornithodoros parkeri alpha-crystallin protein, respectively. Accordingly, this protein was called Ra-sHSPII. The Ra-sHSPII protein was expressed in E. coli under T7 promotor of the pET-30b vector, purified under denaturation conditions and the immunogenicity and cross-reactivity of the recombinant Ra-sHSPII were evaluated. Direct ELISA showed that the Ra-sHSPII is a strong immunogen. In immunoblotting assay the anti-rRa-sHSPII antisera reacted specifically with purified rRa-sHSPII, with several proteins in R. annulatus whole tick, larval and gut protein extracts in addition to Hyalomma dromedarii and Ornithodoros moubata whole tick protein extracts, as examples of hard and soft tick species, respectively. The rRa-sHSPII protein confers thermal protection to other proteins in vitro as found in other sHSPs. E. coli cell extracts containing the protein were protected from heat-denatured precipitation when heated up to 100 °C, whereas extracts from cells not expressing the protein were heat-sensitive at 60 °C.

Non‐3D domain swapped crystal structure of truncated zebrafish alphaA crystallin

In previous work on truncated alpha crystallins (Laganowsky et al., Protein Sci 2010; 19:1031-1043), we determined crystal structures of the alpha crystallin core, a seven beta-stranded immunoglobulin-like domain, with its conserved C-terminal extension. These extensions swap into neighboring cores forming oligomeric assemblies. The extension is palindromic in sequence, binding in either of two directions. Here, we report the crystal structure of a truncated alphaA crystallin (AAC) from zebrafish (Danio rerio) revealing C-terminal extensions in a non three-dimensional (3D) domain swapped, "closed" state. The extension is quasi-palindromic, bound within its own zebrafish core domain, lying in the opposite direction to that of bovine AAC, which is bound within an adjacent core domain (Laganowsky et al., Protein Sci 2010; 19:1031-1043). Our findings establish that the C-terminal extension of alpha crystallin proteins can be either 3D domain swapped or non-3D domain swapped. This duality provides another molecular mechanism for alpha crystallin proteins to maintain the polydispersity that is crucial for eye lens transparency.