Báo cáo khóa học: Some properties of human small heat shock protein Hsp20 (HspB6) potx

Eur J Biochem 271, 291–302 (2004) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03928.x Some properties of human small heat shock protein Hsp20 (HspB6) Olesya V Bukach1, Alim S Seit-Nebi1, Steven B Marston2 and Nikolai B Gusev1 Department of Biochemistry, School of Biology, Moscow State University, Moscow, Russia; 2Imperial College School of Medicine at National Heart and Lung Institute, London, UK Human heat shock protein of apparent molecular mass 20 kDa (Hsp20) and its mutant, S16D, mimicking phosphorylation by cyclic nucleotide-dependent protein kinases, were cloned and expressed in Escherichia coli The proteins were obtained in a homogeneous state without utilization of urea or detergents On size exclusion chromatography at neutral pH, Hsp20 and its S16D mutant were eluted as symmetrical peaks with an apparent molecular mass of 55–60 kDa Chemical crosslinking resulted in the formation of dimers with an apparent molecular mass of 42 kDa At pH 6.0, Hsp20 and its S16D mutant dissociated, and were eluted in the form of two peaks with apparent molecular mass values of 45–50 and 28–30 kDa At pH 7.0–7.5, the chaperone activity of Hsp20 (measured by its ability to prevent the reduction-induced aggregation of insulin or heat-induced aggregation of yeast alcohol dehydrogenase) was similar to or higher than that of commercial a-crystallin Under these conditions, the S16D mutant of Hsp20 possessed lower chaperone activity than the wild-type protein At pH 6.0, both a-crystallin and Hsp20 interacted with denatured alcohol dehydrogenase; however, a-crystallin prevented, whereas Hsp20 either did not affect or promoted, the heat-induced aggregation of alcohol dehydrogenase The mixing of wild-type human Hsp27 and Hsp20 resulted in a slow, temperaturedependent formation of hetero-oligomeric complexes, with apparent molecular mass values of 100 and 300 kDa, which contained approximately equal amounts of Hsp27 and Hsp20 subunits Phosphorylation of Hsp27 by mitogen activated protein kinase-activated protein kinase was mimicked by replacing Ser15, 78 and 82 with Asp A 3D mutant of Hsp27 mixed with Hsp20 rapidly formed a hetero-oligomeric complex with an apparent molecular mass of 100 kDa, containing approximately equal quantities of two small heat shock proteins Human small heat shock proteins (sHsp) form a large group of proteins, consisting of 10 members with a molecular mass in the range of 17–23 kDa [1] These proteins are grouped together because all contain an a-crystallin domain, of 80–100 amino acid residues, which is located in the C-terminal part of the protein [2,3] Some sHsp, such as aB-crystallin and Hsp27, are ubiquitous and expressed in practically all tissues [1,2,4,5], whereas other sHsp (such as HspB7 and HspB9) are expressed only in specific tissues [1,4,5] sHsp tend to form large oligomers that vary in structure and number of monomers [6,7] These complexes can be formed by identical or nonidentical subunits Subunits of a-crystallin, Hsp20, Hsp22, and Hsp27 seem to be involved in the formation of different heterooligomeric complexes [8–12] Hsp27 and aB-crystallin have been analyzed in detail [2–5,13,14], whereas other members of the large superfamily of sHsp are less well characterized Hsp20 was described by Kato et al [8] as a byproduct of purification of human aB-crystallin and Hsp27 Hsp20 is expressed in practically all tissues, reaching a maximal level of 1.3% of total proteins in skeletal, heart and smooth muscles [2,9,15] Since 1997, the laboratory of Colleen Brophy has performed detailed investigations of the role of Hsp20 in the regulation of smooth muscle contraction It has been shown that cAMP- and cGMP-dependent protein kinases phosphorylate Ser16 of Hsp20 and that phosphorylation of Hsp20 is associated with smooth muscle relaxation that is independent of the level of phosphorylation of the myosin light chain [16–20] These findings have been confirmed and extended [21–23] Insulin induces phosphorylation of rat Hsp20 at Ser157 [24] and Hsp20 phosphorylated at two different sites (Ser16 and Ser157) differently affects glucose transport [25,26] Recently, Hsp20 was detected in blood and it has been shown that Hsp20 binds to and inhibits platelet aggregation [27] Thus, significant progress has been achieved in revealing a possible physiological role of Hsp20 However, investigation of the biochemical properties of isolated Hsp20 lag behind Indeed, the biochemical properties of rat Hsp20 were only briefly characterized in the reports of Kato et al [8,9] and van de Klundert et al [15], whereas the corresponding properties of human Hsp20 remain practically uncharacterized Therefore, the present work was devoted to the cloning and purification of wild-type human Hsp20 and its Correspondence to N B Gusev, Department of Biochemistry, School of Biology, Moscow State University, Moscow 119992, Russia Fax:/Tel.: + 095 9392747, E-mail: NBGusev@mail.ru Abbreviations: ADH, yeast alcohol dehydrogenase; DMS, dimethylsuberimidate; EDC, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; Hsp, heat shock protein; 3D mutant, human Hsp27 with replacement of Ser15, 78 and 82 by Asp; NHS, N-hydroxysuccinimide; S16D, mutant of human Hsp20 with replacement of Ser16 by Asp; sHsp, small heat shock proteins (Received 21 October 2003, accepted 17 November 2003) Keywords: small heat shock proteins; phosphorylation; chaperone activity Ó FEBS 2003 292 O V Bukach et al (Eur J Biochem 271) mutant mimicking phosphorylation of Ser16, analysis of their oligomeric state, chaperone activity and their ability to interact with human Hsp27 Materials and methods Proteins Cloning and mutagenesis of human Hsp20 and Hsp27 The full-length cDNA encoding human Hsp20 (GenBank accession no.: AK056951) was amplified from Marathon-Ready cDNA, Heart (Clontech) using the following forward 5¢-GAGATATACATATGGAGATCC CTGTGC-3¢ (NdeI restriction site underlined) and reverse 5¢-GTGCTCGAGTTACTTGGCTGCGGCTGGCGG-3¢ (XhoI restriction site underlined) primers, and Pwo DNA polymerase (Roche) The 480 bp PCR product was purified after electrophoresis in an agarose gel, then digested with the restriction endonucleases NdeI and XhoI and inserted into the plasmid vector pET23b (which had been predigested with the same endonucleases) The resulting construct was verified by DNA sequencing and used for expression and mutagenesis A two step PCR-based ÔmegaprimerÕ method [28,29] was used for the replacement of Ser16 of Hsp20 with Asp In this case, the primer S16D (5¢-GCCGCGCCGACGCCCCG TTGC-3¢) was used for site-directed mutagenesis The human Hsp27 full-length cDNA (GenBank accession no.: NM001540) was amplified from Marathon-Ready cDNA, Lung (Clontech) using the following forward 5¢-GAGATATACATATGGCCGAGCGC-3 and reverse 5¢-CCGGATCCCTACTTCTTGGCTGG-3¢ primers containing, respectively, NdeI and BamHI restriction sites (underlined) The PCR product was purified and inserted into the plasmid vector, pET11c (Novagen) The resulting construct was verified by DNA sequencing and used for expression and site-directed mutagenesis Three serine residues of Hsp27 (Ser15, Ser78 and Ser82) were replaced with Asp This was achieved by using the following primers: 5¢-CGGGGCCCCGACTG GGACCCC-3¢ for S15D and 5¢-GACCCCGCTGTC GAGTTGCCGGTCGAGCGCGC-3¢ for the S78D and S82D mutants The two step PCR-based ÔmegaprimerÕ method [28,29] permits creation of the so-called 3D mutant of Hsp27 with replacements of Ser15, Ser78 and Ser82 by Asp This type of mutation mimics phosphorylation of Hsp27 by mitogen activated protein kinaseactivated protein kinase [30,31] Expression and purification of human Hsp20 and Hsp27 Expression was performed in Escherichia coli BL21(DE3) pUBS520 E coli was cultured with aeration, on Luria–Bertani (LB) media containing ampicillin (150 lgỈmL)1) and kanamycin (40 lgỈmL)1), to an attenuance (D600) of 0.5 Isopropyl thio-b-D-thiogalactoside (IPTG) was added to a final concentration of 0.5 mM and culture was continued for a further h at 30 °C The cells were harvested, frozen and used for isolation of recombinant human wild-type Hsp20, its S16D mutant, recombinant human wild-type Hsp27 and its 3D mutant The initial stages of purification of Hsp20 and its S16D mutant were performed as described previously [32] Briefly, the crude extract of Hsp20 in lysis buffer (50 mM Tris/HCl, pH 8.0, 100 mM NaCl, mM EDTA, 0.5 mM phenylmethanesulfonyl fluoride, 14 mM b-mercaptoethanol) was fractionated with (NH4)2SO4 (0–30% saturation) and subjected to ion-exchange chromatography on a High-Trap Q column (Amersham-Pharmacia) equilibrated with buffer B (20 mM Tris/acetate, pH 7.6, 10 mM NaCl, 0.1 mM EDTA, 0.1 mM phenylmethanesulfonyl fluoride, 14 mM b-mercaptoethanol) and developed by a linear (10–410 mM) gradient of NaCl Further purification was achieved by hydrophobic chromatography on a phenylsuperose column (Amersham-Pharmacia) equilibrated with 20 mM phosphate buffer (pH 7.0), containing 0.3 M (NH4)2SO4, and developed by a decreasing (0.3–0.005 M) gradient of (NH4)2SO4 The final preparations of Hsp20, or its S16D mutant, were concentrated by ultrafiltration and stored frozen in buffer B The initial steps of purification of Hsp27 or its 3D mutant were similar to those described for Hsp20 Hsp27 and its 3D mutant were fractionated by (NH4)2SO4 (0–50% saturation) and subjected to ion-exchange chromatography on a High-Trap Q column (Amersham-Pharmacia), followed by gel filtration on a Sephacryl S300 High-Prep 16/60 column (Amersham-Pharmacia) If necessary, further purification was achieved by hydrophobic chromatography on phenylsuperose (Amersham-Pharmacia) Preparations of Hsp27 and its 3D mutant were concentrated by ultrafiltration and stored frozen in buffer B containing 10% glycerol Denaturation and renaturation of sHsp Denaturation and renaturation of Hsp20 and commercial a-crystallin (Sigma) was performed according to van de Klundert et al [15] Recombinant wild-type Hsp20 in buffer B was freezedried The samples of freeze-dried Hsp20 or commercial a-crystallin were dissolved in 50 mM phosphate (pH 7.5), containing 100 mM Na2SO4, 0.02% b-mercaptoethanol and M urea, up to a final protein concentration of mgỈmL)1, and then stored on ice for h After incubation, the samples were diluted sixfold in the same buffer, minus urea and b-mercaptoethanol, and dialyzed against two changes of the same buffer overnight IEF and electrophoresis Isoelectrofocusing (IEF) was performed, as described previously [29], in a 5.4% polyacrylamide gel containing 8.5 M urea, 2% Triton-X-100, 0.4% ampholine (pH 3–10) and 1.6% ampholine (pH 5–7) Phosphoric acid (10 mM) and sodium hydroxide (20 mM) were used as electrode buffers After fixation and removal of ampholine, the proteins were stained with Coomassie R-250 SDS gel electrophoresis was performed according to Laemmli [33] For quantitative measurements the gels were stained with Coomassie R-250 and evaluated using the program ONEDSCAN Size exclusion chromatography The oligomeric state of sHsp was determined by size exclusion chromatography on Superdex 200 HR 10/30 using the ACTA-FPLC system The column was usually equilibrated with buffer C (20 mM Tris/HCl, pH 7.5, containing 150 mM NaCl and 15 mM b-mercaptoethanol) Ó FEBS 2003 Human 20 kDa small heat shock protein (Eur J Biochem 271) 293 In the case of renaturation experiments, the same column was equilibrated and developed with 50 mM phosphate (pH 7.5) containing 100 mM Na2SO4 The column was calibrated using the following molecular mass markers: thyroglobulin (669 kDa), ferritin (440 kDa), catalase (240 kDa), aldolase (158 kDa), BSA (66 kDa) and chymotrypsinogen (25 kDa) For investigating the exchange of subunits between Hsp20 and Hsp27, equimolar quantities (0.4 mgỈmL)1 Hsp20 and 0.54 mgỈmL)1 Hsp27) of sHsp were mixed in buffer B The mixture obtained was either immediately loaded onto the column or incubated for h at 30 or 37 °C, or for 15 h at 18 °C, before chromatography at room temperature In control experiments, isolated Hsp20 or Hsp27 were incubated under exactly the same conditions and subjected to size exclusion chromatography The protein composition of the fractions obtained in the course of size exclusion chromatography was analyzed by means of SDS gel electrophoresis [33] The effect of pH on the oligomeric state of Hsp20 was also analyzed by size exclusion chromatography To achieve this, the Superdex 200 HR 10/30 column was equilibrated with buffer D (50 mM phosphate, 150 mM NaCl, mM EDTA, 15 mM b-mercaptoethanol), pH-adjusted to 5.5, 6.0, 6.5, 7.0 or 7.5 The protein sample (150 lL, 0.6 mgỈmL)1) was mixed with an equal volume of 2· buffer D at the test pH and incubated for h at 20 °C before chromatography at room temperature Chemical crosslinking Three different methods were used for crosslinking Hsp20 In the first, Hsp20 (0.2 mgỈmL)1) was dialyzed overnight against 50 mM phosphate buffer (pH 7.5), containing 100 mM Na2SO4 Before SDS gel electrophoresis, the samples were either treated with an excess of b-mercaptoethanol or loaded onto the gel in the absence of b-mercaptoethanol In the second method, Hsp20 (0.75 mgỈmL)1) in 0.2 M triethanolamine (pH 7.5) was incubated with dimethylsuberimidate (20 mM) for h at 20 °C The reaction was stopped by the addition of SDS sample buffer The protein composition of the samples thus obtained was analyzed by SDS gel electrophoresis In the third method, Hsp20 (1 mgỈmL)1), in 20 mM imidazole/HCl (pH 7.0) containing 150 mM NaCl, was incubated for h at 30 °C in the presence of 1-(3dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) (5 mM) and N-hydroxysuccinimide (NHS) (5 mM) The reaction was stopped by the addition of SDS sample buffer and subjected to SDS/PAGE (15% gel) [33] CD spectroscopy sHsp ( mgỈmL)1) were dialyzed overnight, against 50 mM phosphate buffer containing 150 mM NaCl, at three different pH values (6.0, 6.8 or 7.5) The samples thus obtained were subjected to centrifugation (12 000 g, 20 min) and the pellet was discarded Far UV CD spectra were recorded in 0.05 cm cells at room temperature on a Mark V Jobin Yvon autodichrograph All spectra presented represent the average of three accumulations Determination of chaperone activity The chaperone activity of Hsp20 and of bovine lens a-crystallin (Sigma) was determined by their ability to retard or to decrease aggregation of the insulin B-chain (Sigma) [15] All experiments were performed in buffer E (50 mM phosphate, pH 7.5, 100 mM Na2SO4) Insulin (6.5 mgỈmL)1), dissolved in 2.5% acetic acid, was added to the incubation mixture (270 lL) to a final concentration of 0.25 mgỈmL)1 The mixture was incubated at 40 °C and the reaction started by addition of a water solution of dithiothreitol up to a final concentration of 20 mM Reduction of the disulfide bonds of insulin was accompanied by aggregation of the B-chain and an increase of turbidity that was measured at 360 nm on an Ultraspec 3100 Pro spectrophotometer The chaperone activity of Hsp20 and of commercial a-crystallin was also determined by their ability to retard or to prevent the heat-induced aggregation and precipitation of yeast alcohol dehydrogenase (ADH) [29,34] The incubation mixture (280 lL) comprised equal volumes of buffer B and buffer F (100 mM phosphate, 300 mM NaCl) at pH 6.0 or 7.0 Yeast ADH (Sigma) was added to the incubation mixture to a final concentration of 0.15–0.26 mgỈmL)1 and the sample was incubated at 42 °C The reaction was started by the addition of dithiothreitol and EDTA up to final concentrations of 30 mM and mM, respectively Heating and removal of divalent cations induces the aggregation of ADH; this process was followed at 360 nm on an Ultraspec 3100 Pro spectrophotometer The optical measurement of aggregation was complemented by a centrifugation assay where the samples were withdrawn at different time-points of incubation and subjected to centrifugation (12 000 g, 10 min) The protein composition of the pellet and supernatant was determined by quantitative SDS gel electrophoresis [33] Results Isolation of human Hsp20 and its S16D mutant As described in the Materials and methods, we developed procedure for purification of recombinant wild-type human Hsp20 All steps of extraction and purification were performed in the absence of urea or detergents The method provided 5–7 mg of recombinant wild-type Hsp20 from L of the E coli culture When the S16D mutant of Hsp20 was expressed in E coli, most of the protein was insoluble in the lysis buffer and this buffer extracted less than 20% of the protein The S16D mutant that was extracted with lysis buffer was subjected to the same steps of purification as the wild-type protein and, according to the SDS gel electrophoresis, had the same apparent molecular mass as the wild-type protein (Fig 1A) Most of the S16D mutant that was not soluble in the lysis buffer could be dissolved in the same buffer containing M urea and was subjected to ion-exchange chromatography on a High-Trap Q column in buffer B, containing M urea, at pH 8.5 According to SDS gel electrophoresis, the apparent molecular mass of the protein thus obtained was 2–3 kDa less than the corresponding molecular mass of the wild-type Hsp20 or water-soluble 294 O V Bukach et al (Eur J Biochem 271) Ó FEBS 2003 Fig Characterization of recombinant human wild-type Hsp20 and its S16D mutant SDS gel electrophoresis (A) and IEF (B) of wild-type Hsp20 (1), and of its S16D mutant that is soluble in the absence (2) and in the presence (3) of urea Arrows indicate the position of molecular mass markers (14 and 25 kDa) and direction of pH gradient (C) Primary structure of wild-type Hsp20, and of its S16D mutant soluble in the absence and in the presence of urea, as determined by HPLC/ tandem MS The experimentally determined sequence is shown in bold; shadowed residues were not detected in the experiment S16D mutant of Hsp20 (Fig 1A) Tandem MS analysis performed by Dr R Wait (Kennedy Institute of Rheumatology Division, Faculty of Medicine, Imperial College, London) was unable to detect peptides beyond residue 102 in the urea-soluble S16D mutant (Fig 1C), whereas both N- and C-terminal peptides were clearly detected in the wild-type Hsp20 and water soluble S16D preparations (Fig 1C) IEF, under denaturing conditions, indicated that the pI value of the urea-soluble S16D mutant was higher than that of the intact wild-type Hsp20 (Fig 1B) By analyzing the distribution of the charge residues in the C-terminal region of Hsp20, and by calculating the theoretical pI values of differently truncated species of Hsp20, we found that cleavage of the polypeptide chain only between residues 122 and 127 resulted in the formation of a protein species with a theoretical pI higher than that of intact Hsp20 Thus, we propose that during expression or purification, the S16D mutant tends to undergo proteolysis of the C-terminal region All experiments described in this report were performed with an S16D mutant that was soluble in the absence of urea The pI value of this soluble S16D mutant was 0.2 units lower than that of the wild-type Hsp20 A similar shift of pI was observed previously for the point mutants of Hsp25 with replacement of Ser with Asp [29] The method developed for purification of recombinant human Hsp27 and its 3D mutant was similar to that described previously [32,36] and yields 5–7 mg of homogeneous protein from L of E coli culture As in the case with Hsp20, all stages of Hsp27 purification were performed in the absence of urea or detergents Fig Size-exclusion chromatography of recombinant human wild-type Hsp20 (A) Lack of effect of dilution or sample volume on the apparent molecular mass of Hsp20 Equal volumes (240 lL) containing 624 lg (1) or 72 lg (2) of Hsp20, or equal quantities (72 lg) of Hsp20 dissolved in 240 lL (2) or 30 lL (3) volumes, were subjected to chromatography on a Superdex 200 HR 10/30 column (B) Effect of urea-induced denaturation followed by renaturation on the chromatographic behavior of small heat shock proteins a-Crystallin (1 and 2) and wild-type Hsp20 (3 and 4) were subjected to size-exclusion chromatography before (1 and 3) or after (2 and 4) urea-induced denaturation, followed by renaturation Oligomeric state of recombinant human Hsp20 Recombinant human wild-type Hsp20 was subjected to size exclusion chromatography, at neutral pH, on a Superdex 200 column under three different experimental conditions In the first we loaded the column with 240 lL of a 2.6 mgỈmL)1 concentration of protein (curve on Fig 2A) In the second, the column was loaded with the same volume of 0.3 mgỈmL)1 protein (curve on Fig 2A) In the third, the column was loaded with 30 lL of protein at a concentration of 2.6 mg mL)1 (curve on Fig 2A) Under these experimental conditions, the apparent molecular mass of recombinant human wild-type Hsp20 was 58, 54 and 56 kDa for the first, second and third experimental conditions, respectively We also analyzed the effect of urea induced denaturation, followed by renaturation, on the oligomeric state of Hsp20 As shown in Fig 2B (curves and 4) denaturation–renaturation showed practically no effect on the oligomeric state of Hsp20, and both intact and Ó FEBS 2003 Human 20 kDa small heat shock protein (Eur J Biochem 271) 295 Fig Crosslinking of Hsp20 (A) Formation of disulfide crosslinked Hsp20 dimers A sample of oxidized Hsp20 treated with an excess of b-mercaptoethanol (2), or loaded onto the gel without the addition of reducing agents (3) (B) Crosslinking of Hsp20 with dimethylsuberimidate Hsp20 before (2) or after (3) incubation with 20 mM dimethylsuberimidate (C) Zero-length crosslinking of Hsp20 Hsp20 before (2) and after (3) incubation with 1-(3-dimethylaminopropyl)3-ethylcarbodiimide hydrochloride (5 mM) and N-hydroxysuccinimide (5 mM) In all cases the mixture of standards containing proteins with molecular masses 94, 67, 43, 30, 20 and 14 kDa was loaded on the first track renatured proteins had an apparent molecular mass of 54–56 kDa However, denaturation–renaturation of commercial a-crystallin was accompanied by a significant decrease of molecular mass The molecular mass of a-crystallin that was not subjected to urea treatment was > 900 kDa, whereas after urea treatment and renaturation its molecular mass was 570 kDa (Fig 2B) As size-exclusion chromatography was insufficient for the exact estimation of oligomeric forms of Hsp20, and the apparent molecular mass of 54–56 kDa determined by this method may correspond to dimers or trimers of Hsp20, we performed additional crosslinking experiments The removal of b-mercaptoethanol was accompanied by the appearance of an additional band of molecular mass 40 kDa, as shown by SDS/PAGE (Fig 3A) This band disappeared if, prior to electrophoresis, the sample was treated with an excess of b-mercaptoethanol Therefore, we suggest that the 40 kDa band corresponds to Hsp20 dimer crosslinked via single Cys46 Crosslinking of Hsp20 with dimethylsuberimidate was also accompanied by the formation of an additional band with molecular mass 40 kDa (Fig 3B), that probably also corresponds to Hsp20 dimer Similar results were obtained if Hsp20 was subjected to zero-length crosslinking by EDC and NHS (Fig 3C) In this case we observed two or three closely separated bands with apparent molecular mass 38–40 kDa that probably correspond to isomers of Hsp20 dimers Thus, under the experimental conditions used, Hsp20 predominantly forms dimers of 40 kDa molecular mass, as judged by SDS gel electrophoresis, and 54–58 kDa by size-exclusion chromatography We considered that changes in pH might somehow affect the quaternary structure of Hsp20 At pH 7.5–7.0 Hsp20 was eluted as a more or less symmetrical peak with apparent molecular mass 54–58 kDa (Fig 4) At pH 6.5, both wildtype protein and its S16D mutant were eluted as broader Fig Effect of pH on the oligomeric state of recombinant human wildtype Hsp20 and its S16D mutant Three-hundred microliter samples containing 90 lg of wild-type Hsp20 (solid lines) or its S16D mutant (dotted lines) were loaded onto the column of Superdex 200 HR 10/30 equilibrated with buffer D (50 mM phosphate, 150 mM NaCl, mM EDTA, 15 mM b-mercaptoethanol) with a pH of 7.5, 7.0, 6.5, 6.0 or 5.5 For clarity, the pairs of elution profiles obtained at different pH values are shifted from each other by 10 mAu peaks with a slightly smaller apparent molecular mass (46–47 kDa) (Fig 4) When the pH was decreased to 6.0, two peaks with apparent molecular masses of 47–50 and 28–30 kDa were observed on the chromatogram (Fig 4) At pH 5.5, the high molecular mass peak completely disappeared and the small molecular mass peak became broader and more asymmetric (Fig 4) A decrease in pH from 7.5 to 5.5 was accompanied not only by a decrease of the apparent molecular mass of Hsp20, but also by a decrease in the area under the protein peaks on the chromatogram Acidification probably results in the dissociation of small oligomers of Hsp20 and its S16D mutant to monomers that tend to unfold and aggregate These aggregates are retarded on the top of the column and therefore not detected on the chromatogram The data presented indicates that acidic pH induced unfolding of Hsp20 In order to confirm this, we analyzed far UV CD spectra of Hsp20 and a-crystallin at different pH values At a high concentration of wild-type Hsp20 ( 1.0 mgỈmL)1), dialysis against pH 6.0 buffer was accompanied by partial protein precipitation The molar ellipticity of Hsp20 remaining in the supernatant ( 0.5 mgỈmL)1) had a negative maximum at 220 nm (Fig 5A) After dialysis at pH 6.8, wild-type Hsp20 ( 1.0 mgỈmL)1) was predominant in the supernatant and the maximum peak of molar ellipticity was shifted to 218 nm (Fig 5A) Dialysis of wild-type Hsp20 ( 1.0 mgỈmL)1) at pH 7.5 was not accompanied by any precipitation and the molar ellipticity at pH 7.5 was lower than that at acidic pH values with a shift in the maximum to 216 nm The data presented confirm that acidification leads to partial unfolding and precipitation of Hsp20 and indicate that the secondary (or tertiary) structure of Hsp20 remaining in the supernatant at acidic pH is different from that at neutral pH values Similar results were obtained with the S16D mutant of Hsp20 (data 296 O V Bukach et al (Eur J Biochem 271) Fig Far UV CD spectra of the wild-type Hsp20 (A) and commercial a-crystallin (B) The spectra were recorded at pH 6.0 (1), 6.8 (2) or 7.5 (3) not shown) Analogous experiments were performed with commercial a-crystallin In this case, independently of pH, a-crystallin was not precipitated and remained in the supernatant The changes of pH in the range of 6.0–7.5 weakly affect both amplitude and the position of maximum on the far UV CD spectra of a-crystallin (Fig 5B) Thus, acidification induced small changes in the secondary (or tertiary) structure of a-crystallin and these changes were not accompanied by protein aggregation As already mentioned, acidification induces substantial changes in the secondary structure of Hsp20 These structural changes probably result in the dissociation of Hsp20 dimers and aggregation of partially unfolded monomers Chaperone activity of human Hsp20 The reduction of disulfide bonds induces dissociation and aggregation of the insulin B-chain that is accompanied by a substantial increase in the optical density (Fig 6, curve 1) At pH 7.5, the addition of increasing quantities of intact wild-type Hsp20 results in an increase of the lag period and a decrease in the amplitude of light scattering Significant retardation of the insulin B-chain aggregation was observed at an insulin/Hsp20 ratio of : At a mass ratio of : 1, the sHsp almost completely prevented the aggregation of Ó FEBS 2003 reduced insulin (Fig 6A) Denaturation by M urea followed by renaturation had no effect on the chaperone activity of the wild-type Hsp20, and complete prevention of insulin aggregation was achieved at the same Hsp20/insulin ratio as for intact protein (Fig 6B) The S16D mutant of Hsp20 also decreased the aggregation of insulin (Fig 6C); however, it was less effective than the wild-type protein Denaturation–renaturation of the S16D mutant only weakly affected its chaperone properties (Fig 6D) Commercial a-crystallin that was not subjected to urea treatment was very ineffective in preventing reduction-induced aggregation of insulin Even at a ratio of : 1, a-crystallin only slightly decreased the aggregation of insulin (Fig 6E) Ureainduced denaturation followed by renaturation significantly improved the chaperone activity of a-crystallin (Fig 6F) This was probably caused by a change in the aggregation state of a-crystallin that was induced by urea treatment and identified by size-exclusion chromatography (see Fig 2B) However, even after treatment with urea, the chaperone activity of a-crystallin was similar to that of the wild-type Hsp20 Thus, at pH 7.5 and with reduced insulin as a model substrate, the chaperone activity of the wild-type Hsp20 was comparable to or greater than that of commercial a-crystallin At pH 7.0, the heating of isolated ADH in the absence of divalent cations was accompanied by aggregation and a large increase in the light scattering (Fig 7, curve 1) Addition of increasing quantities of the wild-type Hsp20 resulted in retardation of the onset of aggregation and a decrease in the amplitude of light scattering (Fig 7A, curves 2–5) At the ADH/Hsp20 ratio of : (wt/wt), aggregation of ADH was completely prevented Similar results were obtained with the S16D mutant of Hsp20 (Fig 7B) and a-crystallin (Fig 7C) However, at a lower concentration, when the ratio of ADH/sHsp was : (wt/wt), the efficiency of three sHsp decreased in the following order: wild-type Hsp20 > S16D mutant > a-crystallin (Fig 7D) Thus, phosphorylation (or a mutation mimicking phosphorylation) decreased the chaperone activity of Hsp20 measured both with insulin and ADH (Figs and 7) It is worthwhile to note that at the same time, phosphorylation of Ser16 of Hsp20 significantly enhanced the relaxation effect of Hsp20 on the smooth muscle contraction [16–23] The optical method used for measuring the chaperone activity of Hsp20 was complemented by the centrifugation assay Upon heating, isolated ADH formed aggregates that were easily precipitated and, after only 20 of incubation, more than 75% of the ADH was detected in the pellet (Fig 7E, curve 1) After 60 of incubation, isolated ADH was completely aggregated and precipitated a-Crystallin was rather ineffective in preventing the aggregation of ADH (Fig 7E, curve 2) This fact seems to contradict with the results obtained by light scattering where a-crystallin at least partially inhibited the aggregation of ADH (Fig 7C) However, this apparent contradiction can be explained by the suggestion that small complexes are effectively precipitated during centrifugation but contribute only slightly to light scattering These complexes can be formed either by denatured ADH or by denatured ADH being bound to sHsp Indeed, we found that at the end of incubation more than 30% of a-crystallin was coprecipitated with denatured ADH (Fig 7F) Hsp20 was more effective in preventing Ó FEBS 2003 Human 20 kDa small heat shock protein (Eur J Biochem 271) 297 Fig Influence of recombinant human Hsp20 (A and B), the S16D mutant of Hsp20 (C and D) or commercial a-crystallin (E and F) before (A, C and E) or after (B, D and F) urea treatment followed by renaturation on the reduction induced aggregation of insulin The chaperone activity was measured by the prevention of dithiothreitol-induced aggregation of insulin (0.25 mgỈmL)1) at 40 °C under conditions described in the Materials and methods Insulin alone (1), or insulin in the presence of 0.06 mgỈmL)1 (2), 0.12 mgỈmL)1 (3) or 0.25 mgỈmL)1 (4) small heat shock proteins precipitation of denatured ADH (Fig 7E, curve 3) Much smaller quantities of Hsp20 were coprecipitated with denatured ADH (Fig 7F, curve 2) It is worthwhile mentioning that isolated sHsp were not precipitated in the absence of ADH, even after 60 of incubation (Fig 7F, curve 3) Similar results were obtained with the S16D mutant of Hsp20 (data not shown) Thus, at pH 7.0, Hsp20 is a more potent chaperone than a-crystallin, probably because complexes formed by Hsp20 with denatured ADH are smaller or more soluble than the corresponding complexes formed by denatured ADH and a-crystallin As discussed above, a decrease in the pH to pH 6.0 may induce partial unfolding and dissociation of small oligomers formed by Hsp20 or its S16D mutant (Figs and 5) As unfolding and dissociation may affect the chaperone activity of Hsp20, we analyzed the effect of different sHsps on the aggregation of ADH at pH 6.0 Under these conditions, heating also induced the aggregation of ADH (Fig 8, curve 1) Addition of increasing quantities of wild-type Hsp20 increased the rate and amplitude of light scattering (Fig 8A, curves 2–5) Thus, the wild-type Hsp20, instead of preventing, promotes the aggregation of ADH This is probably a result of the formation of insoluble complexes of Hsp20 and denatured ADH Indeed, as shown in Figs 8E,F, incubation of ADH with the wild-type Hsp20 resulted in the formation of a pellet containing both proteins At pH 6.0 and at the low concentrations used in the experiment, isolated Hsp20 itself is completely soluble and does not precipitate (Fig 8A, curve 6, Fig 8F, curve 3) However, complexes formed by partially unfolded Hsp20 and denatured ADH tend to aggregate and precipitate Qualitatively similar results were obtained with the S16D mutant of Hsp20 (Fig 8B) However, in this case addition of increasing quantities of the S16D mutant resulted in a small retardation of the onset of ADH aggregation and either did not affect the amplitude of light scattering or slightly increased it Using the centrifugation assay we found that after a short incubation, the S16D mutant predominantly remained in the supernatant, whereas after a long incubation a large proportion of the S16D mutant coprecipitated with ADH (data not shown) At pH 6.0, a low concentration of a-crystallin either did not affect or slightly increased the thermal aggregation of ADH (Fig 8C) At an ADH/crystallin ratio of : 1, a significant decrease in the extent of ADH aggregation was observed (Fig 8C, curve 5) a-Crystallin was more effective than Hsp20 in preventing the precipitation of ADH (Fig 8E) and smaller quantities of a-crystallin were coprecipitated with denatured protein (Fig 8F) Therefore, at pH 6.0, a-crystallin possessed higher chaperone activity than Hsp20 or its S16D mutant Formation of mixed oligomer complexes between recombinant human Hsp20 and Hsp27 In tissue extracts, Hsp20 forms high molecular weight complexes [9,19] and is copurified with aB-crystallin and Hsp27 [8,9] Indirect data also indicate that Hsp20 may interact with Hsp27 and aB-crystallin [10] However, to our knowledge, the hetero-oligomeric complexes formed by Hsp20 with other sHsp have not been characterized and reported in the literature Therefore, we investigated the 298 O V Bukach et al (Eur J Biochem 271) Ó FEBS 2003 Fig Effect of Hsp20, its S16D mutant and a-crystallin on the heat-induced aggregation of yeast alcohol dehydrogenase (ADH) at pH 7.0 Aggregation of ADH (0.26 mgỈmL)1) was induced by the addition of EDTA and dithiothreitol and incubation at 42 °C, and was measured either by light scattering (A–D) or by centrifugation (E–F) Panels A–C, ADH alone (1), or ADH in the presence of 0.026 mgỈmL)1 (2), 0.052 mgỈmL)1 (3), 0.13 mgỈmL)1 (4) or 0.26 mgỈmL)1 (5) of the wild-type Hsp20 (A), the S16D mutant of Hsp20 (B), or a-crystallin (C) (D) Comparison of the effect of different small heat shock proteins (0.13 mgỈmL)1) on the aggregation of ADH (0.26 mgỈmL)1) ADH alone (1), or ADH in the presence of the wild-type Hsp20 (2), the S16D mutant of Hsp20 (3) or a-crystallin (4) (E) Heat-induced precipitation of isolated ADH (0.26 mgỈmL)1) (1), or ADH in the presence of either a-crystallin (0.13 mgỈmL)1) (2) or wild-type Hsp20 (0.13 mgỈmL)1) (3) The percentage of ADH in the pellet is plotted against the time of incubation (F) Co-precipitation of a-crystallin (1) or wild-type Hsp20 (2) with heat denatured ADH The percentage of small heat shock protein in the pellet is plotted against the time of incubation Lack of precipitation of isolated small heat shock proteins is shown on curve interaction of Hsp20 and its mutant mimicking phosphorylation with Hsp27 Interaction of the wild-type Hsp20 with the wild-type Hsp27 was analyzed by means of size-exclusion chromatography Hsp20 and Hsp27 were eluted from the Superdex 200 column as single peaks with molecular masses of 56 and 560 kDa, respectively The chromatographic behavior of isolated Hsp20 and Hsp27 was not altered if, prior to loading on the column, these proteins were preincubated for h at 30 or 37 °C or for 15 h at 18 °C If equimolar quantities of these two proteins were mixed and immediately subjected to size-exclusion chromatography, two well separated peaks with apparent molecular masses 560 and 56 kDa, corresponding to isolated Hsp27 and Hsp20, were detected on the chromatogram (Fig 9A, curve 1) According to SDS/PAGE, the high molecular mass peak contained exclusively Hsp27, whereas the small molecular mass peak contained only Hsp20 The elution profile was not changed upon preincubation of this mixture of proteins for 15 h at 18 °C (data not shown) If, prior to loading on the column, the mixture of the wild-type Hsp27 and Hsp20 was incubated for h at 30 °C, the elution profile was significantly changed The amplitude of the high molecular mass peak decreased and its apparent molecular mass was 470 kDa This peak was asymmetric with a prominent trailing edge In addition, a new peak, with an apparent molecular mass of 91 kDa, appeared on the chromatogram and the peak corresponding to isolated Hsp20 (56 kDa) decreased in size (Fig 9A, curve 2) Even more prominent changes were observed if the mixture of two wild-type proteins was incubated for h at 37 °C (Fig 9A, curve 3) In this case we observed two protein peaks with apparent molecular masses of 300 and 100 kDa, and each of these peaks, according to SDS/PAGE, contained almost identical quantities of Hsp27 and Hsp20 (insert on Fig 9A) Similar results were obtained if the wild-type Hsp27 was mixed with the S16D mutant of Hsp20 Thus, after mixing at 30–37 °C, homo-oligomers of wild-type Hsp27 and Hsp20 (or the S16D mutant of Hsp20) may rearrange, forming mixed hetero-oligomers that contain similar quantities of these two sHsp The isolated 3D mutant of Hsp27 produces a broad peak with apparent molecular mass 96–106 kDa A significant decrease in molecular mass compared with the wild-type Hsp27 is a result of the fact that mutations mimicking phosphorylation induce dissociation of large oligomers of Hsp27 [29–31] As already mentioned, the wild-type Hsp20 and its S16D mutant are eluted as a single peak with an apparent molecular mass of 56 kDa A mathematical summation of elution profiles obtained for the 3D mutant of Hsp27 and the wild-type Hsp20 is presented on curve of Fig 9B Only one broad asymmetric peak, with an apparent molecular mass of 100 kDa, was observed if, immediately after mixing, the two proteins were loaded onto the column (Fig 9B, curve 2) The position and shape of this peak were different from the sum of the two elution profiles obtained for the isolated 3D mutant of Hsp27 and wild-type Hsp20 (compare curves and on Fig 9B) If the mixture of the Ó FEBS 2003 Human 20 kDa small heat shock protein (Eur J Biochem 271) 299 Fig Influence of Hsp20, its S16D mutant and a-crystallin on the heat-induced aggregation of yeast alcohol dehydrogenase (ADH) at pH 6.0 Aggregation of ADH (0.15 mgỈmL)1) was measured either by light scattering (A–D) or by centrifugation (E–F) A–C, ADH alone (1), or ADH in the presence of 0.015 mgỈmL)1 (2), 0.03 mgỈmL)1 (3), 0.075 mgỈmL)1 (4) or 0.15 mgỈmL)1 (5) of the wild-type Hsp20 (A), the S16D mutant of Hsp20 (B) or a-crystallin (C) Lack of aggregation of isolated small heat shock proteins (0.15 mgỈmL)1) is shown on curve (D) Comparison of the effect of different small heat shock proteins (0.15 mgỈmL)1) on the aggregation of ADH (0.15 mgỈmL)1) ADH alone (1), or ADH in the presence of wild-type Hsp20 (2), the S16D mutant of Hsp20 (3) or a-crystallin (4) (E) Heat-induced precipitation of isolated ADH (0.15 mgỈmL)1) (1), or ADH in the presence of either wild-type Hsp20 (0.15 mgỈmL)1) (2) or a-crystallin (0.15 mgỈmL)1) (3) The percentage of ADH in the pellet is plotted against the time of incubation (F) Co-precipitation of wild-type Hsp20 (1) or a-crystallin (2) with heat denatured ADH The percentage of small heat shock proteins in the pellet is plotted against the time of incubation Lack of precipitation of isolated small heat shock proteins is shown on curve 3D mutant of Hsp27 and wild-type Hsp20 (or S16D mutant of Hsp20) were incubated for h at 30 °C, only one peak with an apparent molecular mass 95 kDa was detected on the chromatogram Thus, homo-oligomers formed by the 3D mutant of Hsp27 and wild-type Hsp20 (or its S16D mutant) rapidly rearrange, forming hetero-oligomeric complexes Discussion To our knowledge there are only two publications that report a detailed investigation of the biochemical properties of isolated Hsp20 Kato et al [9] reported that Hsp20 is presented in so-called aggregated and dissociated forms with apparent molecular masses of 200–300 and 67 kDa, respectively Using size-exclusion chromatography, van de Klundert et al [15] also detected two forms of Hsp20, with apparent molecular masses of 470 and 43 kDa, that, depending on the protein concentration may convert to each other In our case, size-exclusion chromatography revealed only an oligomer of Hsp20 with an apparent molecular mass of  54–58 kDa (Fig 2) According to our crosslinking experiments, Hsp20 predominantly forms dimers with an apparent molecular mass of 40 kDa, as judged by SDS/PAGE (Fig 3) Therefore, the question arises as to why we did not observe the high molecular mass oligomers of Hsp20 detected previously by Kato et al [9] and van de Klundert et al [15] We presumed that the exposure of Hsp20 to a high concentration of urea [9,15], or to urea and detergents [35], as used in the previously published reports, might affect the quaternary structure of Hsp20 In order to verify this, we denatured Hsp20 (purified by our method) by M urea and renatured it under the conditions described by van de Klundert et al [15] This treatment had no effect either on the apparent molecular mass, as determined by sizeexclusion chromatography, or on the chaperone activity measured by the prevention of insulin aggregation Thus, treatment with urea cannot explain the difference in molecular mass identified in our experiments and in data published previously [9,15] Another explanation was based on the fact that practically all previously published results were obtained using rat Hsp20 [9,15,35], whereas in the present study human Hsp20 was used Although rat and human Hsp20 are highly homologous ( 90% identity of the primary structure), the rat Hsp20 consists of 162 residues, whereas the human protein consists of 160 residues and the dipeptide deletion is located at the very C-terminal end (residues 154–155 of rat Hsp20) It is known that the C-terminal extension affects the oligomerization and chaperone action of Hsp27 [37] Therefore, we propose that the difference in the C-terminal extension of human and rat Hsp20 results in a different oligomeric state of these two proteins However, this suggestion is speculative and needs experimental verification Finally, as previously mentioned, when expressing the S16D mutant we found that truncation of 30–50 C-terminal amino acid residues results in the formation of protein aggregates that were soluble only in the presence of a high concentration of urea Previously it has been shown that the truncation of a short C-terminal 300 O V Bukach et al (Eur J Biochem 271) Fig Formation of hetero-oligomeric complexes between Hsp27 and Hsp20 (A) Rearrangement of the complexes formed by the wild-type Hsp27 and Hsp20 The wild-type Hsp27 and Hsp20 were loaded onto the column immediately after mixing (1) or were incubated at 30 °C (2) or at 37 °C (3) for h For clarity the profiles are shifted from each other by 40 mAu The protein composition of profile fractions 25–33 is shown on the insert The positions of Hsp27 and Hsp20 are marked by arrows (B) Rearrangement of the complexes formed by the 3D mutant of Hsp27 and the wild-type Hsp20 A mathematical summation of the elution profiles of the isolated 3D mutant of Hsp27 and isolated wild-type Hsp20 is presented on curve Experimental elution profiles of the mixture of the 3D mutant of Hsp27 and Hsp20 loaded onto the column immediately after mixing (2), or after incubation for h at 30 °C (3) For clarity the profiles are shifted from each other by 40 mAu peptide increases the hydrophobicity of Hsp27 and decreases its chaperone effect [37] There were no signs of proteolytic degradation in the samples of Hsp20 purified by Kato et al [9] and van de Klundert et al [15]; however, truncation of a short (2–4 kDa) fragment can be easily overlooked Therefore, we suggest that during expression and/or purification, Hsp20 can undergo limited proteolysis, and deletion of a short C-terminal fragment may result in the formation of a mixture of small and large aggregates that were reported in the previous publications Ó FEBS 2003 Van de Klundert et al [15] claimed that Hsp20 is a poor chaperone In our investigation we found that at neutral or slightly alkaline pH, Hsp20 has comparable or even higher chaperone activity than commercial a-crystallin (Figs and 7) sHsp protect the cell against unfavorable conditions, among them acidosis For instance, the data of Wang [38] indicate that a-crystallin prevents acidification-induced aggregation of creatine kinase and luciferase In our study, at pH 6.0, a-crystallin partially prevented the aggregation of yeast ADH, whereas the wild-type Hsp20 retained its ability to interact with denatured substrates, but, instead of preventing, promoted the aggregation of denatured ADH (Fig 8) This was caused by the fact that at low pH Hsp20 tends to unfold, and dimers of Hsp20 dissociate to monomers Under these conditions, partially unfolded monomers of Hsp20 interact with denatured ADH and form poorly soluble complexes Similar effects have been observed for the truncated form of Hsp27 [37] and for the alternative splicing product of aA-crystallin [39] Thus, although Hsp20 and a-crystallin are closely related, they have different properties Acidification induced a small decrease of the chaperone activity of a-crystallin, but significantly decreased the chaperone activity of Hsp20 Similar conclusions were reached by van de Klundert et al [40], who postulated that Hsp20 and a-crystallin might be involved in distinct protective activities in living cells It is worthwhile of note that the measurement of chaperone activity and analysis of the quaternary structure of Hsp20 was performed in buffers with compositions that are not completely physiological This was implemented in order to compare our results with data in the published literature However, limitations of biochemical experiments should be taken into account when interpreting our results at a physiological level The data obtained with the help of a yeast two-hybrid system indicate that different sHsps may interact with each other [10] Moreover, Bova et al [11], using the method of fluorescence energy transfer, have directly shown that Hsp27 and a-crystallin may form mixed oligomers If crude extracts of skeletal muscle or heart were subjected to size exclusion chromatography, Hsp20 was eluted in one or two high molecular mass peaks Kato et al [9] detected two peaks with apparent molecular masses 200–300 and 68 kDa, whereas Pipkin et al [19] detected only one peak with an apparent molecular mass of 230 kDa Brophy et al [17] postulated that cAMP-dependent phosphorylation results in the change of macromolecular associations of Hsp20 Finally, Hsp20 is usually copurified with Hsp27 and a-crystallin [9,19] Thus, all these data indirectly indicate the formation of mixed oligomer complexes between Hsp20 and a-crystallin or Hsp27 To verify this, we analyzed the chromatographic behavior of the mixture of Hsp20 and Hsp27 In good agreement with Bova et al [41], we found that at low temperature the rate of subunit exchange between the wild-type Hsp20 and Hsp27 was very slow However, at 30 or 37 °C the rate of exchange was significantly increased and we detected two hetero-oligomeric complexes with apparent molecular masses 100 and 300 kDa that contained similar quantities of Hsp20 and Hsp27 subunits (Fig 9A) Mutation S16D, imitating phosphorylation of the Ser16 of Hsp20, had no significant effect on the rate of subunits exchange or on the composition or Ó FEBS 2003 Human 20 kDa small heat shock protein (Eur J Biochem 271) 301 structure of the hetero-oligomeric complexes formed by Hsp20 and Hsp27 Mixing of the 3D mutant of Hsp27 with the wild-type Hsp20 or its S16D mutant was accompanied by a very rapid exchange of subunits and formation of a mixed oligomer with an apparent molecular mass of 95 kDa (Fig 9B) Thus, Hsp20 and Hsp27 may form two types of heterooligomeric complexes – one with an apparent molecular mass of  100 kDa and another of  300 kDa The low molecular mass oligomer is formed by both the wild-type and pseudophosphorylated sHsp, whereas only the wildtype sHsp formed the large molecular mass complex The 3D mutant of Hsp27 forms hetero-oligomeric complexes more readily than the wild-type protein Previously, a similar conclusion was reached concerning the interaction of Hsp22 with the wild-type Hsp27 and its 3D mutant [12] Summing up, we conclude that Hsp20 and Hsp27 readily form two types of hetero-oligomeric complexes with molecular masses 100 and 300 kDa The rate of exchange of sHsp subunits depends on a mutation mimicking the phosphorylation of Hsp27 The experimental conditions are not directly comparable with those in living cells However, taking into account a high efficiency of complex formation and a high concentration of Hsp20 and Hsp27 in certain tissues [8,9], we may suppose that hetero-oligomeric complexes are also formed in vivo This will lead to a decrease in the concentration of homo-oligomeric sHsp, affect oligomer structure and result in the accumulation of hetero-oligomeric complexes with properties that might be different from homo-oligomers Isolation and detailed characterization of hetero-oligomeric complexes will provide new, important information on the functioning of sHsp in 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