Production of Recombinant β-Hexosaminidase A, a Potential Enzyme for Replacement Therapy for Tay-Sachs and Sandhoff Diseases, in the Methylotrophic Yeast Ogataea minuta

Human β-hexosaminidase A (HexA) is a heterodimeric glycoprotein composed of α- and β-subunits that degrades GM2 gangliosides in lysosomes. GM2 gangliosidosis is a lysosomal storage disease in which an inherited deficiency of HexA causes the accumulation of GM2 gangliosides. In order to prepare a large amount of HexA for a treatment based on enzyme replacement therapy (ERT), recombinant HexA was produced in the methylotrophic yeast Ogataea minuta instead of in mammalian cells, which are commonly used to produce recombinant enzymes for ERT. The problem of antigenicity due to differences in N-glycan structures between mammalian and yeast glycoproteins was potentially resolved by using α-1,6-mannosyltransferase-deficient (och1Δ) yeast as the host. Genes encoding the α- and β-subunits of HexA were integrated into the yeast cell, and the heterodimer was expressed together with its isozymes HexS (αα) and HexB (ββ). A total of 57 mg of β-hexosaminidase isozymes, of which 13 mg was HexA (αβ), was produced per liter of medium. HexA was purified with immobilized metal affinity column for the His tag attached to the β-subunit. The purified HexA was treated with α-mannosidase to expose mannose-6-phosphate (M6P) residues on the N-glycans. The specific activities of HexA and M6P-exposed HexA (M6PHexA) for the artificial substrate 4MU-GlcNAc were 1.2 ± 0.1 and 1.7 ± 0.3 mmol/h/mg, respectively. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis pattern suggested a C-terminal truncation in the β-subunit of the recombinant protein. M6PHexA was incorporated dose dependently into GM2 gangliosidosis patient-derived fibroblasts via M6P receptors on the cell surface, and degradation of accumulated GM2 ganglioside was observed.     

Secretion of Recombinant Proteins via the Chaperone/Usher Pathway in Escherichia coli

F1 antigen (Caf1) of Yersinia pestis is assembled via the Caf1M chaperone/Caf1A usher pathway. We investigated the ability of this assembly system to facilitate secretion of full-length heterologous proteins fused to the Caf1 subunit in Escherichia coli. Despite correct processing of a chimeric protein composed of a modified Caf1 signal peptide, mature human interleukin-1β (hIL-1β), and mature Caf1, the processed product (hIL-1β:Caf1) remained insoluble. Coexpression of this chimera with a functional Caf1M chaperone led to the accumulation of soluble hIL-1β:Caf1 in the periplasm. Soluble hIL-1β:Caf1 reacted with monoclonal antibodies directed against structural epitopes of hIL-1β. The results indicate that Caf1M-induced release of hIL-1β:Caf1 from the inner membrane promotes folding of the hIL-1β domain. Similar results were obtained with the fusion of Caf1 to hIL-1β receptor antagonist or to human granulocyte-macrophage colony-stimulating factor. Following coexpression of the hIL-1β:Caf1 precursor with both the Caf1M chaperone and Caf1A outer membrane protein, hIL-1β:Caf1 could be detected on the cell surface of E. coli. These results demonstrate for the first time the potential application of the chaperone/usher secretion pathway in the transport of subunits with large heterogeneous N-terminal fusions. This represents a novel means for the delivery of correctly folded heterologous proteins to the periplasm and cell surface as either polymers or cleavable monomeric domains.     

Mildly acidic pH activates the extracellular molecular chaperone clusterin

Many features of the chaperone action of clusterin are similar to those of the intracellular small heat shock proteins (sHSPs) that, like clusterin, exist in solution as heterogeneous aggregates. Increased temperature induces dissociation of some sHSP aggregates and an enhanced chaperone action, suggesting that a dissociated form is the active chaperone species. We recently reported that clusterin aggregates dissociate at mildly acidic pH. To further explore the similarities between clusterin and the sHSPs, we tested the effects of temperature and pH on the structure of clusterin and its chaperone action. Our results demonstrate that increased temperature does not induce dissociation of clusterin aggregates, or other major structural changes, and has little effect on its chaperone action. However, we show that the chaperone action of clusterin is enhanced at mildly acidic pH. Clusterin is the first chaperone shown to be activated by reduced pH. This unique mode of activation appears to result from an increase in regions of solvent-exposed hydrophobicity, which is independent of any major changes in secondary or tertiary structure. We propose a model in which low pH-induced dissociation of clusterin aggregates increases the abundance of the heterodimeric chaperone-active species, which has greater hydrophobicity exposed to solution.     

Evidence that clusterin has discrete chaperone and ligand binding sites.

Clusterin is the first identified extracellular mammalian chaperone and binds to a wide variety of partly unfolded, stressed proteins.Clusterin also binds to many different unstressed ligands including the cell surface receptor low-density lipoprotein receptor-related protein-2 (LRP-2). It is unknown whether clusterin binds to all of these many ligands via one or more binding sites. Furthermore, the region(s) of clusterin involved in these many binding interactions remain(s) to be identified. As part of an investigation of these issues, we expressed recombinant human clusterin in the yeast Pichia pastoris. The resultant protein had variable proteolytic truncations of the C-terminal region of the alpha-chain and the N-terminal region of the beta-chain. We compared the chaperone and ligand binding activities of this recombinant product with those of clusterin purified from human serum. We also tested whether the binding of clusterin to ligands could be inhibited by competitive binding with other clusterin ligands or by anti-clusterin monoclonal antibodies. Collectively, our results indicate that (i) clusterin has three independent classes of binding sites for LRP-2, stressed proteins, and unstressed ligands, respectively, and (ii) the binding sites for LRP-2 and stressed proteins are likely to be in parts of the molecule other than the C-terminal region of the alpha-chain or the N-terminal region of the beta-chain. It has been suggested that, in vivo, clusterin binds to toxic molecules in the extracellular environment and carries these to cells expressing LRP-2 for uptake and degradation. This hypothesis is supported by our demonstration that clusterin has discrete binding sites for LRP-2 and other (potentially toxic) molecules.     

The Heat Shock Response Is Modulated by and Interferes with Toxic Effects of Scrapie Prion Protein and Amyloid β

The heat shock response (HSR) is an evolutionarily conserved pathway designed to maintain proteostasis and to ameliorate toxic effects of aberrant protein folding. We have studied the modulation of the HSR by the scrapie prion protein (PrP^Sc) and amyloid β peptide (Aβ) and investigated whether an activated HSR or the ectopic expression of individual chaperones can interfere with PrP^Sc- or Aβ-induced toxicity. First, we observed different effects on the HSR under acute or chronic exposure of cells to PrP^Sc or Aβ. In chronically exposed cells the threshold to mount a stress response was significantly increased, evidenced by a decreased expression of Hsp72 after stress, whereas an acute exposure lowered the threshold for stress-induced expression of Hsp72. Next, we employed models of PrP^Sc- and Aβ-induced toxicity to demonstrate that the induction of the HSR ameliorates the toxic effects of both PrP^Sc and Aβ. Similarly, the ectopic expression of cytosolic Hsp72 or the extracellular chaperone clusterin protected against PrP^Sc- or Aβ-induced toxicity. However, toxic signaling induced by a pathogenic PrP mutant located at the plasma membrane was prevented by an activated HSR or Hsp72 but not by clusterin, indicating a distinct mode of action of this extracellular chaperone. Our study supports the notion that different pathological protein conformers mediate toxic effects via similar cellular pathways and emphasizes the possibility to exploit the heat shock response therapeutically.     

The herpesvirus accessory protein γ134.5 facilitates viral replication by disabling mitochondrial translocation of RIG-I

RIG-I and MDA5 are cytoplasmic RNA sensors that mediate cell intrinsic immunity against viral pathogens. While it has been well-established that RIG-I and MDA5 recognize RNA viruses, their interactive network with DNA viruses, including herpes simplex virus 1 (HSV-1), remains less clear. Using a combination of RNA-deep sequencing and genetic studies, we show that the γ₁34.5 gene product, a virus-encoded virulence factor, enables HSV growth by neutralization of RIG-I dependent restriction. When expressed in mammalian cells, HSV-1 γ₁34.5 targets RIG-I, which cripples cytosolic RNA sensing and subsequently suppresses antiviral gene expression. Rather than inhibition of RIG-I K63-linked ubiquitination, the γ₁34.5 protein precludes the assembly of RIG-I and cellular chaperone 14-3-3ε into an active complex for mitochondrial translocation. The γ₁34.5-mediated inhibition of RIG-I-14-3-3ε binding abrogates the access of RIG-I to mitochondrial antiviral-signaling protein (MAVS) and activation of interferon regulatory factor 3. As such, unlike wild type virus HSV-1, a recombinant HSV-1 in which γ₁34.5 is deleted elicits efficient cytokine induction and replicates poorly, while genetic ablation of RIG-I expression, but not of MDA5 expression, rescues viral growth. Collectively, these findings suggest that viral suppression of cytosolic RNA sensing is a key determinant in the evolutionary arms race of a large DNA virus and its host.     

Thermo and pH stable ATP-independent chaperone activity of heat-inducible Hsp70 from Pennisetum glaucum

Heat shock proteins (Hsps) are a class of molecular chaperones that play an essential role in preserving cellular functions under stressful conditions. The over production of recombinant proteins often causes cellular stress that results in aggregation/misfolding of proteins, which sometimes leads to the formation of inclusion bodies. Here we report the cloning and characterization of heat-inducible PgHsp70 from Pennisetum glaucum, a heat and drought tolerant plant that showed stability and chaperone activity at elevated temperatures. The predicted amino acid sequence of PgHsp70 revealed a high homology with Hsp70 from other plants, and the overall 3D structure homology modeling is similar to that of the constitutively expressed bovine cytosolic Heat Shock Cognate (HSC)-70. The purified recombinant protein had an apparent molecular mass of 70 kDa and displayed optimal chaperone activity at 50°C, and pH 8.0. Under these conditions, the T_1/2 of PgHsp70 increased from 10 to 15 h in the presence of glycerol. The PgHsp70 exhibited a higher chaperone activity towards glutamate dehydrogenase than alcohol dehydrogenase. The expression of recombinant carbonic anhydrase (CA ) in E. coli in a catalytically active soluble form rather than in inclusion bodies was made feasible by co-expression of PgHsp70. Circular dichroism (CD) studies of the recombinant PgHsp70 did not reveal any discernible changes in the α-helix content, with increase in temperature from 35 to 85°C, thus suggesting a critical role of α-helix content in maintaining the chaperone activity.Key words: abiotic stress, carbonic anhydrase, chaperone, circular dichroism, heat shock proteins (Hsp70), Pennisetum glaucum, pH stable     

Low reduction potential of Ero1alpha regulatory disulphides ensures tight control of substrate oxidation

Formation of disulphide bonds within the mammalian endoplasmic reticulum (ER) requires the combined activities of Ero1α and protein disulphide isomerase (PDI). As Ero1α produces hydrogen peroxide during oxidation, regulation of its activity is critical in preventing ER-generated oxidative stress. Here, we have expressed and purified recombinant human Ero1α and shown that it has activity towards thioredoxin and PDI. The activity towards PDI required the inclusion of glutathione to ensure sustained oxidation. By carrying out site-directed mutagenesis of cysteine residues, we show that Ero1α is regulated by non-catalytic disulphides. The midpoint reduction potential (E°′) of the regulatory disulphides was calculated to be approximately −275 mV making them stable in the redox conditions prevalent in the ER. The stable regulatory disulphides were only partially reduced by PDI (E°′~−180 mV), suggesting either that this is a mechanism for preventing excessive Ero1α activity and oxidation of PDI or that additional factors are required for Ero1α activation within the mammalian ER.     

NADP+ Binding to the Regulatory Subunit of Methionine Adenosyltransferase II Increases Intersubunit Binding Affinity in the Hetero-Trimer

Mammalian methionine adenosyltransferase II (MAT II) is the only hetero-oligomer in this family of enzymes that synthesize S-adenosylmethionine using methionine and ATP as substrates. Binding of regulatory β subunits and catalytic α2 dimers is known to increase the affinity for methionine, although scarce additional information about this interaction is available. This work reports the use of recombinant α2 and β subunits to produce oligomers showing kinetic parameters comparable to MAT II purified from several tissues. According to isothermal titration calorimetry data and densitometric scanning of the stained hetero-oligomer bands on denatured gels, the composition of these oligomers is that of a hetero-trimer with α2 dimers associated to single β subunits. Additionally, the regulatory subunit is able to bind NADP⁺ with a 1∶1 stoichiometry, the cofactor enhancing β to α2-dimer binding affinity. Mutants lacking residues involved in NADP⁺ binding and N-terminal truncations of the β subunit were able to oligomerize with α2-dimers, although the kinetic properties appeared altered. These data together suggest a role for both parts of the sequence in the regulatory role exerted by the β subunit on catalysis. Moreover, preparation of a structural model for the hetero-oligomer, using the available crystal data, allowed prediction of the regions involved in β to α2-dimer interaction. Finally, the implications that the presence of different N-terminals in the β subunit could have on MAT II behavior are discussed in light of the recent identification of several splicing forms of this subunit in hepatoma cells.     

α-Crystallin-Type Heat Shock Proteins: Socializing Minichaperones in the Context of a Multichaperone Network

α-Crystallins were originally recognized as proteins contributing to the transparency of the mammalian eye lens. Subsequently, they have been found in many, but not all, members of the Archaea, Bacteria, and Eucarya. Most members of the diverse α-crystallin family have four common structural and functional features: (i) a small monomeric molecular mass between 12 and 43 kDa; (ii) the formation of large oligomeric complexes; (iii) the presence of a moderately conserved central region, the so-called α-crystallin domain; and (iv) molecular chaperone activity. Since α-crystallins are induced by a temperature upshift in many organisms, they are often referred to as small heat shock proteins (sHsps) or, more accurately, α-Hsps. α-Crystallins are integrated into a highly flexible and synergistic multichaperone network evolved to secure protein quality control in the cell. Their chaperone activity is limited to the binding of unfolding intermediates in order to protect them from irreversible aggregation. Productive release and refolding of captured proteins into the native state requires close cooperation with other cellular chaperones. In addition, α-Hsps seem to play an important role in membrane stabilization. The review compiles information on the abundance, sequence conservation, regulation, structure, and function of α-Hsps with an emphasis on the microbial members of this chaperone family.     

Reconstitution of the recombinant human γ-tubulin ring complex

Cryo-electron microscopy recently resolved the structure of the vertebrate γ-tubulin ring complex (γ-TuRC) purified from Xenopus laevis egg extract and human cells to near-atomic resolution. These studies clarified the arrangement and stoichiometry of γ-TuRC components and revealed that one molecule of actin and the small protein MZT1 are embedded into the complex. Based on this structural census of γ-TuRC core components, we developed a recombinant expression system for the reconstitution and purification of human γ-TuRC from insect cells. The recombinant γ-TuRC recapitulates the structure of purified native γ-TuRC and has similar functional properties in terms of microtubule nucleation and minus end capping. This recombinant system is a central step towards deciphering the activation mechanisms of the γ-TuRC and the function of individual γ-TuRC core components.     

Small heat-shock proteins and clusterin: intra- and extracellular molecular chaperones with a common mechanism of action and function?

Small heat-shock proteins (sHsps) and clusterin are molecular chaperones that share many functional similarities despite their lack of significant sequence similarity. These functional similarities, and some differences, are discussed. sHsps are ubiquitous intracellular proteins whereas clusterin is generally found extracellularly. Both chaperones potently prevent the amorphous aggregation and precipitation of target proteins under stress conditions such as elevated temperature, reduction and oxidation. In doing so, they act on the slow, off-folding protein pathway. The conformational dynamism and aggregated state of both proteins may be crucial for their chaperone function. Subunit exchange is likely to be important in regulating chaperone action; the dissociated form of the protein is probably the chaperone-active species rather than the aggregated state. They both exert their chaperone action without the need for hydrolysis of ATP and have little ability to refold target proteins. Increased expression of sHsps and clusterin accompanies a range of diseases that arise from protein misfolding and deposition of highly structured protein aggregates known as amyloid fibrils, e.g., Alzheimer's, Creutzfeldt-Jakob and Parkinson's diseases. The interaction of sHsps and clusterin with fibril-forming species is discussed along with their ability to prevent fibril formation.     

Effects of glycosylation on the structure and function of the extracellular chaperone clusterin

Clusterin is the first well characterized, constitutively secreted extracellular chaperone that binds to exposed regions of hydrophobicity on non-native proteins. It may help control the folding state of extracellular proteins by targeting them for receptor-mediated endocytosis and intracellular lysosomal degradation. A notable feature of secreted clusterin is its heavy glycosylation. Although carbohydrate comprises approximately 20-25% of the total mass of the mature molecule, its function is unknown. Results from the current study demonstrate that deglycosylation of human serum clusterin had little effect on its overall secondary structure content but produced a small increase in solvent-exposed hydrophobicity and enhanced the propensity of the molecule to aggregate in solution. These changes were associated with increased binding to a variety of ligands but did not substantially impact the ability of clusterin to inhibit heat-induced precipitation of citrate synthase. Evidence suggesting that the normally conjugated sugars are important in the interaction of secreted clusterin with a lectin-type receptor on liver cells is also presented. Bulk expression of fully processed, glycosylated clusterin in mammalian cells is difficult, often producing inappropriately disulfide-bonded high molecular weight aggregates; this has hampered previous studies aimed at identifying those regions of the molecule important in its chaperone action. The current results suggest that it may be possible in the future to study the structure and chaperone function of clusterin using recombinant protein (lacking sugars) conveniently bulk-expressed in bacteria.     

Structural Basis of Pharmacological Chaperoning for Human β-Galactosidase

G_M1 gangliosidosis and Morquio B disease are autosomal recessive diseases caused by the defect in the lysosomal β-galactosidase (β-Gal), frequently related to misfolding and subsequent endoplasmic reticulum-associated degradation. Pharmacological chaperone (PC) therapy is a newly developed molecular therapeutic approach by using small molecule ligands of the mutant enzyme that are able to promote the correct folding and prevent endoplasmic reticulum-associated degradation and promote trafficking to the lysosome. In this report, we describe the enzymological properties of purified recombinant human β-Gal^WT and two representative mutations in G_M1 gangliosidosis Japanese patients, β-Gal^R201C and β-Gal^I51T. We have also evaluated the PC effect of two competitive inhibitors of β-Gal. Moreover, we provide a detailed atomic view of the recognition mechanism of these compounds in comparison with two structurally related analogues. All compounds bind to the active site of β-Gal with the sugar-mimicking moiety making hydrogen bonds to active site residues. Moreover, the binding affinity, the enzyme selectivity, and the PC potential are strongly affected by the mono- or bicyclic structure of the core as well as the orientation, nature, and length of the exocyclic substituent. These results provide understanding on the mechanism of action of β-Gal selective chaperoning by newly developed PC compounds.     

Unmodified Cre recombinase crosses the membrane

Site-specific recombination in genetically modified cells can be achieved by the activity of Cre recombinase from bacteriophage P1. Commonly an expression vector encoding Cre is introduced into cells; however, this can lead to undesired side-effects. Therefore, we tested whether cell-permeable Cre fusion proteins can be directly used for lox-specific recombination in a cell line tailored to shift from red to green fluorescence after loxP-specific recombination. Comparison of purified recombinant Cre proteins with and without a heterologous ‘protein transduction domain’ surprisingly showed that the unmodified Cre recombinase already possesses an intrinsic ability to cross the membrane border. Addition of purified recombinant Cre enyzme to primary bone marrow cells isolated from transgenic C/EBPα^fl/fl mice also led to excision of the ‘floxed’ C/EBPα gene, thus demonstrating its potential for in vivo applications. We conclude that Cre enyzme itself or its intrinsic membrane-permeating moiety are attractive tools for direct manipulation of mammalian cells.     

α-Hemoglobin Stabilizing Protein (AHSP), a Kinetic Scheme of the Action of a Human Mutant,AHSPV56G

A kinetic analysis has been made of the interaction of α-Hb chains with a mutant α-hemoglobin stabilizing protein, AHSP^V56G, which is the first case of an AHSP mutation associated with clinical symptoms of mild thalassemia syndrome. The chaperone AHSP is thought to protect nascent α chains until final binding to the partner β-Hb. Rather than protecting α chains, the mutant chaperone is partially unfolded but recovers its secondary structure via interaction with α-Hb. For both AHSP^WT and AHSP^V56G, the binding to α-Hb is quite rapid relative to the α-β reaction, as expected because the chaperone binding must be quite competitive to complete the α chain folding process before α-Hb binds irreversibly to β-Hb. The main kinetic difference is a dissociation rate of AHSP^V56G·α-Hb some four times faster relative to AHSP·α-Hb. Considering a role of protein folding, the AHSP^V56G apparently does not bind long enough (0.5 s versus 2 s for the WT) to complete the structural modifications. The overall replacement reaction (AHSP·α-Hb + β-Hb → AHSP + αβ) can be quite long, especially if there is an excess of AHSP relative to β-Hb monomers.     

α-Hemoglobin-stabilizing Protein (AHSP) Perturbs the Proximal Heme Pocket of Oxy-α-hemoglobin and Weakens the Iron-Oxygen Bond

α-Hemoglobin (αHb)-stabilizing protein (AHSP) is a molecular chaperone that assists hemoglobin assembly. AHSP induces changes in αHb heme coordination, but how these changes are facilitated by interactions at the αHb·AHSP interface is not well understood. To address this question we have used NMR, x-ray absorption spectroscopy, and ligand binding measurements to probe αHb conformational changes induced by AHSP binding. NMR chemical shift analyses of free CO-αHb and CO-αHb·AHSP indicated that the seven helical elements of the native αHb structure are retained and that the heme Fe(II) remains coordinated to the proximal His-87 side chain. However, chemical shift differences revealed alterations of the F, G, and H helices and the heme pocket of CO-αHb bound to AHSP. Comparisons of iron-ligand geometry using extended x-ray absorption fine structure spectroscopy showed that AHSP binding induces a small 0.03 Å lengthening of the Fe-O₂ bond, explaining previous reports that AHSP decreases αHb O₂ affinity roughly 4-fold and promotes autooxidation due primarily to a 3–4-fold increase in the rate of O₂ dissociation. Pro-30 mutations diminished NMR chemical shift changes in the proximal heme pocket, restored normal O₂ dissociation rate and equilibrium constants, and reduced O₂-αHb autooxidation rates. Thus, the contacts mediated by Pro-30 in wild-type AHSP promote αHb autooxidation by introducing strain into the proximal heme pocket. As a chaperone, AHSP facilitates rapid assembly of αHb into Hb when βHb is abundant but diverts αHb to a redox resistant holding state when βHb is limiting.     

Production of Functional Soluble Dectin-1 Glycoprotein Using an IRES-Linked Destabilized-Dihydrofolate Reductase Expression Vector

Dectin-1 (CLEC7A) is a C-type lectin receptor that binds to β-glucans found in fungal cell walls to act as a major pattern recognition receptor (PRR). Since β-glucans epitope is not present in human cells, we are of the opinion that Dectin-1 can have therapeutic functions against fungal infections. We thus set out to produce a soluble extracellular domain of murine Dectin-1 (called sDectin-1) in sufficient titers to facilitate such studies in mouse models. Since sDectin-1 has previously been shown to be glycosylated, we chose to produce this protein using Chinese Hamster Ovary (CHO) cells, a mammalian host cell line suitable for the high-titer production of recombinant glycoproteins. To ensure a high titer production of sDectin-1 and minimize the effects of gene fragmentation, we constructed a mammalian expression vector with a PEST-destabilized dhfr amplifiable marker downstream of an attenuated IRES element, which was in turn downstream of the sDectin-1 gene and a CMV IE promoter. Stably transfected and MTX-amplified cell pools were generated using this vector, and maximum sDectin-1 titers of 246 mg/l and 598 mg/l were obtained in shake flask batch culture and bioreactor fed-batch culture respectively. The purified recombinant sDectin-1 was shown to be glycosylated. Protein functionality was also demonstrated by its ability to bind to zymosan particles and to the cell wall of Saccharomyces cerevisiae. We describe for the first time the use of an attenuated IRES-linked PEST-destabilized dhfr amplifiable marker for the production of recombinant proteins with stably amplified cell pools. With our process, we reached the highest reported titer for producing recombinant proteins smaller than 50 kDa in cell pools. sDectin-1 protein produced is glycosylated and functional. This vector design can thus be used efficiently for the high-titer production of functional recombinant proteins.     

Thermal Stress Induced Aggregation of Aquaporin 0 (AQP0) and Protection by α-Crystallin via Its Chaperone Function

Aquaporin 0 (AQP0) formerly known as membrane intrinsic protein (MIP), is expressed exclusively in the lens during terminal differentiation of fiber cells. AQP0 plays an important role not only in the regulation of water content but also in cell-to-cell adhesion of the lens fiber cells. We have investigated the thermal stress-induced structural alterations of detergent (octyl glucoside)-solubilized calf lens AQP0. The results show an increase in the amount of AQP0 that aggregated as the temperature increased from 40°C to 65°C. α-Crystallin, molecular chaperone abundantly present in the eye lens, completely prevented the AQP0 aggregation at a 1∶1 (weight/weight) ratio. Since α-crystallin consists of two gene products namely αA- and αB-crystallins, we have tested the recombinant proteins on their ability to prevent thermal-stress induced AQP0 aggregation. In contrast to the general observation made with other target proteins, αA-crystallin exhibited better chaperone-like activity towards AQP0 compared to αB-crystallin. Neither post-translational modifications (glycation) nor C-terminus truncation of AQP0 have any appreciable effect on its thermal aggregation properties. α-Crystallin offers similar protection against thermal aggregation as in the case of the unmodified AQP0, suggesting that αcrystallin may bind to either intracellular loops or other residues of AQP0 that become exposed during thermal stress. Far-UV circular dichroism studies indicated a loss of αhelical structures when AQP0 was subjected to temperatures above 45°C, and the presence of α-crystallin stabilized these secondary structures. We report here, for the first time, that α-crystallin protects AQP0 from thermal aggregation. Since stress-induced structural perturbations of AQP0 may affect the integrity of the lens, presence of the molecular chaperone, α-crystallin (particularly αA-crystallin) in close proximity to the lens membrane is physiologically relevant.