Purification, Structure and In vitro Molecular-Chaperone Activity of Artemia P26, a Small Heat-Shockh/α-Crystallin Protein

Eur. J. Biochem. 243, 225-232 (1997) 0 FEBS 1997

Purification, structure and in vitro molecular-chaperone activity of Artemia p26, a small heat-shockh-crystallin protein Ping LIANG’, Reinout AMONS’, Thomas H. MACRAE‘ and James S. CLEGG’ ’ Department of Biology, Dalhonsie University, Halifax, Nova Scotia, Canada

Department of Medical Biochemistry, University of Leiden, The Netherlands ’ Bodega Marine Laboratory, University of California, Davis, Bodega Bay, USA

(Received 14 August 1996) - EJB 96 1215/1

Encysted brine-shrimp gastrulae bring their metabolism to a reversible standstill during diapause and quiescence, demonstrating a remarkable resistance to unfavourable environmental conditions. For example, mortality of Artemia embryos under normal temperature and hydration is very low, even after two years of anoxia, and embryos commonly experience complete desiccation as part of their developmental program. Previous evidence from our laboratories indicated that p26, an abundant low-molecular-mass cyst-specific protein capable of translocation into the nucleus, may have a protective function in Artemia cysts. p26 was purified to apparent homogeneity and a continuous sequence of 141 of its amino acids was determined by peptide sequencing, revealing that it is a member of the small-heat-shock/u-crystallin family of proteins. As determined by molecular-sieve chromatography and sucrose-density-gradient centrifugation, native p26 is a multimer of about 27 monomers with a molecular mass of approximately 700 kDa. Inactivation of citrate synthase was less when the enzyme was heated in the presence rather than the absence of p26. Additionally, the renaturation of heat-inactivated citrate synthase was promoted by p26. These results indicated that p26 possesses molecular-chaperone activity, a property of other small heat-shock/u-crystallin proteins. Our findings demonstrate that p26 has the potential to protect the macromolecular components of Artemia embryos, either as they encyst or upon exposure to environmental extremes. Protection may depend upon the ability of p26 to function as a molecular chaperone.

Keywords: chaperone ; heat-shocWu-crystallin protein; diapause ; Arternia

The crustacean, Artemia franciscana reproduces via two alternative pathways. In the ovoviviparous mode, embryos develop in the ovisac and are released from the female as free- swimming nauplii. In contrast, for the oviparous route, development is arrested at the late gastrula stage, the embryos encyst and they are released from the female as encysted gastrulae, hence the term cysts [1]. The cysts, composed of about 4000 cells and enclosed in a shell that is impermeable to non-volatile molecules, are in a dormant state known as diapause [l-71. They are resistant to severe insults, including exposure to several organic solvents, y-irradiation, temperature extremes, desiccation and anoxia [1-3, 81. Moreover, the embryos withstand re- peated cycles of hydration and desiccation, losing this ability at about the time they emerge from the cyst as larvae [X, 91. The period of post-gastrula development within the cyst occurs in the absence of DNA synthesis and mitosis [4, 101, implying extensive morphological rearrangement and differentiation of preexisting cells. Because cysts are readily available, their development has been investigated extensively at the molecularhio- chemical level [11-141. Diapause in Artemia has been studied to a Iesser extent [2, 31, but it is known that its occurrence is ‘anticipated’, and embryos following ovoviviparous and oviparous routes display differences early in development [3, 151. Diapause embryos do not resume development, even under fa- vourable environmental conditions, until they receive the appro- priate environmental signals, at which time they are said to be

Correspondence to T. H. MacRae, Department of Biology, Dalhousie University, Halifax NS, B3H 4J1, Canada

Abbreviation. HSP, heat-shock protein.

activated. Metabolism then resumes if permissive conditions prevail. Activated (post-diapause) embryos are particularly resistant to anoxia, and they survive while fully hydrated in the absence of oxygen, in a condition of anaerobic dormancy, for at least two years [16-191. The metabolism of these embryos is reduced to the point where it cannot be detected [20-231. Such observations are interesting because they imply the embryos exhibit activities contrary to the generality that, under normal hydration and temperature, cell maintenance requires a constant and substantial free-energy flow.

Artemia embryos must possess a means to survive these treatments and be able to resume development while confined within a shell that is largely impermeable to internal and external molecules. That is, because only volatile molecules can enter and leave, the embryo is obliged to maintain a complement of functional macromolecules and cell organelles even when desic- cated or deprived of oxygen. There is no convincing evidence that the structural components and enzymes of the embryo are unusually stable. Alternatively, the embryo may possess protective mechanisms that either prevent damage or assist in the re- pair of disrupted macromolecules and organelles. One strong candidate for this role is p26, a low-molecular-mass protein pro- duced by embryos that encyst but not by those that develop di- rectly into nauplii [15, 18, 191. p26 constitutes approximately IS % of the non-yolk protein in Artemia cysts and it is lost when the embryos become sensitive to anoxia, namely upon emergence of the nauplius.

In previous work we found that p26 consists of several iso- forms, exhibits a molecular mass of approximately 500 kDa when determined by analysis of cell-free supernatants, and is

226 Liang et al. (EuI: J . Biochem. 243)

reversibly translocated into the nucleus upon exposure of activated embryos to anoxia [18, 191. These features of p26 are characteristics of the small heat-shocWu-crystallin proteins, which share sequence similarities and are either synthesized constitutively or induced if cells are stressed [reviewed in 24-30]. For example, when challenged by increased temperature, the enhanced production of these proteins is reported to protect cells from thermal damage 131 -351, although there is not complete agreement on this point 136, 371. The a-crystallins, composed of subunits termed a A and aB, have at least one established function; they are major structural proteins of the vertebrate eye with a role in lens transparency 127, 281. Both subunits also occur in cells other than those of the eye. and their synthesis is stress induced 134, 38-40]. Moreover, the a-crystallins and the small heat-shock proteins are molecular chaperones [24-26, 41 -481. As such, they prevent denaturation and aggregation of proteins, and assist in their folding. It is undoubtedly the chaperone activity that protects cells under stress and this is likely to be one of the most important functions of the small heat-shocWcc-crystallin proteins. On the basis of its demonstrated properties, we asked if p26 is a member of this family of proteins, and if it has chaperone activity. The answer obtained for each question is consistent with a protective role for p26 during encystment, diapause and anaerobic dormancy in the encysted embryos.

MATERIALS AND METHODS

Purification of p26. Encysted Artetnin embryos used for the purification of p26 were obtained from Sanders Brine Shrimp Co. The cysts were hydrated at 0°C for 6 h, washed under suc- tion on a Buchner funnel with cold 100 mM Pipes, 1 mM EGTA, 1 mM MgCI,. pH 6.5 (buffer A), and homogenized in the same buffer in 50-100-g amounts with a Retsch motorized mortar and pestle (Brinkman Instruments Canada). The homogenate was centrifuged at 16000Xg for 1 0 min and the supernatant, after passage through two layers of Miracloth (Calbiochem) was centrifuged at 40000Xg for 30 min at 4°C. The upper 70% of the supernatant was transferred to a fresh tube, centrifuged under the same conditions for 20 min, and used immediately or frozen at -70°C. This cell-free extract was termed S, .

20-ml samples of S , were applied to a column (2.5 cmX7.0 cm) of DEAE-cellulose (DE-52, Whatman) washed previously in buffer A containing 1 M NaCl and equilibrated in buffer A. Protein in the flow-through fractions was pooled and applied to a phosphocellulose (PI 1, Whatman) column (2.5 cmX7.0 cm) prepared as described for the DE-52 column. The P11 column was washed with buffer A, then sequen- tially with buffer A containing 0.2 M and 0.4 M NaCI. Fractions from the final wash were pooled, and protein was precipitated by addition of (NH,),SO, to 40% (6.3 g in 28 ml at 4°C). The precipitate was collected by centrifugation at 10000Xg for 10 min and dissolved in 3.0 ml buffer A. 2 ml of the (NH,),SO, fraction was stored at -70°C for future use. The remaining 1 ml was adjusted to 0.4 M NaCl and mixed with 1 .O ml of an affinity matrix, constructed as described by Harlow and Lane [491 from Protein-A-Sepharose CL-4B (Sigma Chem. Co.) and the IgG fraction of antibody raised in rabbits to p26. After overnight incubation with gentle agitation at room temperature, the slurry was poured into a column and washed with 10 vol. 0.1 M Tris/ glycine, pH 7.4. The protein was eluted with 0.1 M glycine, pH 2.3, into tubes containing 0.1 vol. 1 .O M Tris, pH 8.0. Protein fractions from each stage of purification were electrophoresed in 12.5 5% SDS/polyacrylamide gels, which were either stained with Coomassie blue or the proteins were transferred to nitrocellulose and immunostained with antibody to p26.

Preparation of antibody to p26. To prepare antibodies to p26, before the purification procedure described herein was de- veloped, cell-free Artemin extracts were incubated at 37" for 30 min, causing the protein to aggregate into particles larger than those described later. I-ml samples of the extract were centrifuged for 30 min at 40000Xg through 10-ml 15% sucrose cushions in buffer A at 20°C. The pellets, rinsed with warm buffer A, were suspended in 150 pl buffer A, incubated on ice for 30 min with occasional vortexing, and centrifuged as above through 2.5-ml cushions of 15% or 30% sucrose. The pellets were rinsed, suspended in buffer A, diluted twofold with 0.063 M Tris, 2% SDS, 10% glycerol, 5% 2-mercaptoethanol, pH 6.8 and electrophoresed in 10 % SDS/polyacrylamide gels. The gels were processed and p26, a major low-molecular-mass protein in the gel, was recovered for use as antigen as described previously [50]. The suspended, lyophilized protein sample was diluted twofold (by vol.) with Freund's complete adjuvant and rabbits were injected subcutaneously. Three additional injections at 2-week intervals consisted of lyophilized p26 in acrylamide mixed 1 : 1 (by vol.) with Freund's incomplete adjuvant and administered subcutaneously. Specificity was determined by in- cubating the antibody with western blots containing Artetnin cell-free extract proteins that had been resolved in 10% SDS/ polyacrylamide gels before transfer.

Sequencing of p26. Attempts to sequence the entire protein did not yield data, indicating that the amino terminus of p26 was blocked. To circumvent this problem, purified p26 and peptides derived therefrom, were digested under various conditions. Typi- cally, enzymatic digestions were performed on 50-200 pg p26 or 5-50 pg p26-derived peptides in 50-100 p1 at 37°C. Trypsin at 0.01 -0.10 pg/p1 was used in 0.2 M ammonium carbonate, pH 8.5, for 3-16 h. Endoproteinase Lys-C was at 0.5-1 ng/pl in 0.2 M Tris/HCl, pH 8.6, for 48 h and endoproteinase Glu-C was at 0.01 -0.02 pg/pl in 0.2 M ammonium carbonate, pH 8.5, for 24 h. All enzymes were purchased as sequence grade from Boehringer Mannheim. Exposure to BrCN (Merck) was in 200 p1 6 M guanidinium chloride, 0.2 M HCl at 10 pg/pl p26 for 16-20 h at ambient temperature.

The peptides were separated by reversed-phase HPLC on a column (4 mmX1OO mm) of Merck Lichrospher 100 RP18e, at a flow rate of 0.6 ml/min, with a I-h gradient from 0.1 % (mass/ vol.) tritluoroacetic acid to 0.08 % (by vol.) trifluoroacetic acid, 75 % (by vol.) acetonitrile. When larger peptides were expected, a Vydac C, protein column (4 mmX250 mm) was used at a flow rate of 1 .0 ml/min under the chromatographic conditions just described. Prior to HPLC separation, excess BrCN was removed by exposing solutions to a stream of nitrogen. Sequence determinations, using 20-200 pmol peptide, were performed in a Model 475A Applied Biosystems pulse liquid sequencer, connected on line to a Model 120A PTH-amino acid analyzer.

Evaluation of possible molecular-chaperone activity of p26 in vitro. To test the ability of p26 to influence renaturation of a heat-inactivated enzyme, pig-heart citrate synthase from Sigma at 15 pM in buffer C (40 mM Hepes, 20 mM KOH, S O mM KCl, 2 mM potassium acetate, pH 7.8) was heated at 50°C for 20 min, quickly cooled on ice and rapidly diluted 100- fold with 0.1 M Tridglycine, pH 7.5. These reaction mixtures were incubated at 23°C for 30 min in the absence or presence of p26 at 35 nM and assayed for citrate synthase activity [51]. When present, either GTP or ATP was at 3 mM. In some experiments, after analysis of citrate synthase activity, the remainder of each heated reaction mixture was frozen at -20°C. To deter- mine if the heat denaturation of citrate synthase was affected by p26, the frozen reaction mixtures were thawed, analyzed for citrate synthase activity, then heated at 50°C for 5 min, and analyzed for enzyme activity. The analysis was performed on two

Liang et al. ( E M J. Biochem. 243) 227

independent preparations of p26. To examine the influence of p26 concentration on reactivation of heat-denatured citrate synthase, the enzyme was heated as described previously, then incubated in the presence of increasing concentrations of p26. These results were corrected by subtraction of values obtained for the spontaneous reactivation of citrate synthase in the absence of p26.

Multimer formation by p26. The association of p26 into particles of higher-order structure, a characteristic of low-molecular-mass heat-shock proteins, and an important consideration in relation to chaperone activity, was analyzed by two methods. Size determination by gel-filtration chromatography was on a Sepharose CL-6B column (1.0 cmX48 cm) equilibrated with 0.1 M Tridglycine, pH 7.4, and standardized with molecular markers from Sigma, including carbonic anhydrase (29 kDa), albumin (66 kDa), /I-amylase (200 kDa), apoferritin (443 kDa) and thyroglobulin (699 kDa). The void volume was determined by use of dextran blue (2000 kDa). 500 pg purified p26 was applied to the standardized column and eluted with Tridglycine, pH7.4, at 20ml/h at 4°C. 1-ml fractions were collected and from each of these 75 pI was electrophoresed on 15% SDS/ polyacrylamide gels followed by transfer to nitrocellulose. The blots were reacted with antibody to p26 by means of the enhanced-chemiluminescence procedure (Amersham) as recom- mended by the manufacturer. Relative amounts of p26 in the column fractions were determined by scanning blots with a Hewlett Packard ScanJet 1 1 cx at 400 dots per inch and measuring the density of bands with the Scan Analysis (Biosoft) program.

The size of multimers was also examined by applying 200 pg purified p26 to 10-ml continuous sucrose gradients (from 17% to 40%) in 0.1 M Tris/glycine, pH 7.4, and centrifuging at 40000Xg for 21 h in a Beckman SW41Ti rotor. The gradients were standardized under these centrifugation conditions by means of the size markers described above. After centrifugation, the gradients were divided into I-ml fractions and a sample was prepared from the bottom of the tube to test the possibility that p26 had pelleted. The positions of size markers in the gradients were determined by measuring the absorbance of each fraction at 280nm, while the position of p26 was revealed by western blotting, as for samples from the Sepharose columns.

RESULTS

Purification of p26 to apparent homogeneity. Previous data suggested that p26 is a stress protein with a role in brine-shrimp encystment and that it protects the embryo upon exposure to unfavourable environmental conditions such as anoxia (see the introduction). To test these possibilities, p26 was purified as a prerequisite for subsequent characterization (Fig. 1). p26 in cell- free extracts from hydrated cysts was recovered in the flow- through fraction from DE-52 cellulose, eluted from phosphocellulose PI 1 with 0.4 M NaCl in buffer A and precipitated in the 0 to 40% (NH,),SO, fraction. Although greatly enriched at this stage, the sample of p26 was contaminated by several minor proteins that were subsequently eliminated by affinity chromatography. When 20 pg of the affinity-purified protein was electrophoresed in an SDS/polyacrylamide gel and staincd with Coo- massie blue, only one band was visible (Fig. l A). A low-molecular-mass polypeptide of uncertain origin, which reacted with the antibody to p26, was observed in the phosphocellulose PI 1 fractions, but was removed by (NH,),SO, fractionation. The protein recoveries during purification arc summarized in Table 1, and reveal that 3.6 mg p26 were obtained from 900 mg starting material.

1 2 3 4 5 6 1 2 3 4 5 Fig. 1. Purification of p26 to apparent homogeneity. p26 was purified from hydrated brine-shrimp cysts as described in Materials and Methods. The fractions obtained during the different stages of purification were electrophoresed in 12.5 % SDS/polyacrylamide gels and either visualized with Coomassie blue (A) or blotted to nitrocellulose and inimunostained by the alkaline-phosphatase procedure (B). Lane 1, 220 pg cell-free protein extract: lane 2, 150 pg (protein) flow-through fraction from DE-52; lane 3, 70 pg (protein) of the 0.4-M-NaCl fraction from phosphocellulose P11: lane 4, 100 pg (protein) of the 0 to 40% (NH,),S04 fraction; lane 5 , 20 pg affinity-purified p26: lane 6, molecular-mass markers of 97, 66, 43, 31, 22 and 14 kna. The location of p26 is shown in A.

Table 1. Protein recovery during purification of p26. The yield of protein at each stage in the purification of p26 from 20 g (wet mass) of cysts is rcported. The values shown represent the average of three preparations and include the standard deviations. The preparation of each fraction is described in Materials and Methods.

Fraction Vol- Protein Total Protein ume concentration protein recovery

ml mg/ml mg %

s , 20 45 2 3 900 -t60 100 DE-52 36 16 ? I 576 2 3 6 64 2 4

64 i- 5 7.1 5 0 . 5 2.3 +- 0.2 P11 28 2.7 t 0.1 24 5 1 (NH4)2SO, 3.0 8.020.3

Affinity column 4.5 0.8 t 0.1 3.6-t 0.5 0.450.1

Sequence similarity between p26 and the small-heat-shock/ a-crystallin family of proteins. Purified p26 was digested by enzymatic and chemical methods, and peptides were recovered by HPLC chromatography, sequenced and arranged. These peptides, representing a continuous polypeptide stretch of 141 amino acid residues, are depicted in Fig. 2. Comparison to sequences in the NCBI data base demonstrated that p26 contains a conserved sequence characteristically found toward the carboxy terminus of proteins in the small-heat-shock/a-crystallin family (Fig. 3 ) . Comparisons of p26 with the proteins shown in Fig. 3 revealed that within the similar regions, the numbers of identical residues were as high as S O % , while those of conserved residues reached 73 %. p26 is therefore a small heat-shock/a-crystallin protein.

Does p26 exhibit molecular-chaperone activity in vitro ? Some small heat-shock proteins are molecular chaperones in vi- tm 124-26, 41 -481 and previous work had indicated that p26 from Arteinia exhibits several properties that are characteristic of molecular chaperones [IS, 191. To explore this possibility,

228 Liang et al. ( E m J. Biochem. 243)

tr RR* A L K * DTADEFQVQLDVGHFLPNEI>> TTDDDILVH>>HDER* EFR* tr RGPDTSR* ELATPGSLR* SDEYGHVQR* tr eLATPGSLRDtAd>> tr -TADEFQVQLDVGhFLPNEItVKt-d--il>>

ELATPGSLRDTADE>> TTDDDILVHGKHDER-DEYGHVq--F--

VGH FLPNE * FRR BrCN RRGPDTSRALKELATPg-lR>> p26 RRGPDTSRALKELATPGSLRDTADEFQVQLDVGHFLPNEITVKTTDDDILVHGKDERSDEYGHVQREFRR

ITVKTTDDDILVHGK>> YGHVQR>> l Y S glu glu

LATPGSLRDT>> FQVQLd>>

IEGGTTGTTTGSTASSTPAr tr RYR* TALS S PT E R* tr YRLPEHVKPESVsst>> IVPITPAPAVg>> tr LPEHVKPESVSSTL>> lys -y-LP>> TALSSPTERIVPITPAPAVGRIEGGTT>> glu HVKp-* glu RYRLPe* SVSSTLSSDGVLTIHAPKtaL->> p26 RYRLPEHVKPESVSSTLSSDGVLTIHAPKTALSSPTERIVPITPAPAVGRIEGGTTGTTTGSTASSTPAR

Fig. 2. Sequencing protocol and partial primary structure of p26. Purified p26 and peptides derived therefrom were digested with trypsin (tr), endoproteinase Lys-C (lys), endoproteinase Glu-C (glu) and cyanogen bromide (BrCN). The peptides were separated by reversed-phase HPLC and sequence determinations, with 20-200 pmol peptide. were performed in a Model 475A Applied Biosystem pulse liquid sequencer, connected to a Model 120 A PTH-amino acid analyzer. Peptides were positioned in a linear series by analysis of their sequence overlap, yielding the partial sequence of p26 used in subsequent comparisons to other proteins. -, amino acid not determined; *, end of run; 9, run interrupted. Lower-case letters indicate that the identity of residues was uncertain

P26 C-A-A-H C-A-B-H EL-F OV2 5- 2 -N HSP27-H

P2 6 C-A-A-H C-A-B-H EL-F OV25-2-N HSP27-H

P2 6 C -A-A- H C-A-B-H EL-F OV2 5-2-N HSP27 -H

~2 6 C-A-A-H C-A-B-H EL-F OV2 5-2 - N HSP27-H

~2 6 C-A-A-H C-A-B-H EL-F OV2 5- 2 - N HSP27-H

MD-------VTIQHPWFKRTLGPFY-PSRLFDQFFGEGLFYDLLPFL-- MD-------IAIHHPWIRRPFFPFHSPSRLFDQFFGEHLLESDLFP-T-- MSWPLMF-----RDWWDELDF?M-RTSRLLDQHFGQGLKRDDLMSSVWN

MTERRVPFSI,L,RGPSW--DPFRDWYPHSRLFDQAPGLPRLPEEWSQWL-- QTSPMERFIV------------------NLLDSTFDD-----------RS

RRGPDTSRAL--KELATPGSLRDTADE --SSTISPYY--R-------------QSLFRTV---LDSGISEVRSDRDK --STSLSPFYLRP-------------PSFLRAPSW-FDTGLSEMRLEKDR SRPTVLRSGYLRPWHTNSLQKQESG----------------STLNIDSEK SRP-------LHSVAPYWLHQPELNEC--------NIGNSLGEVINEKDK --GGSSWPGYVRPLPPAAIESPAVAAPAYSRALSRQLSSGVSEIRHTADR

__________-----____----

FQVQLDVGHFLPNEITVKTTDDDILVHGKHDERSD--E--YGQREFRRRY FVIFLDVKHFSPEDLTVKVQDDmIHGKHNERQD--DHGYISREFHRRY FSVNLNVKHFSPEELKVKVLGDVIEVHGKHEERQD--EHGFISREFHRKY FEVILDVQQFSPSEITVKVADKFVIVEGKHEEKQD--EHGWSRQFSRRY FAVRADVSHFHPKELSVSVRDRELVIEGHHEERTDPAGHGSIERHFIRKY WRVSLDVNHFAPDELTVKTKDGVVEITGKHEERQD--EHGYISRCFTRKY . . * .* * . . * . . . * * . * . * * * . *

RLPEHVKPESVSSTLSSDGVLTIHAPKTALS--SPTERIVPITPAPAVGR RLPSNVDQSALSCSLSADGl-iLTFCGPKIQTGLDAHTERAIPVSRE----- RIPADVDPLAITSSLSSDGVLTVNGPRKQVS---GPERTIPITRE----- QLPSDVNPDTVTSSLSSDGLLTIKAPMKALP-PPQTERLVQIT------- VLPEEVQPDTIESHLSDKGVLTISANKTAIGTTAS--RNIPIRASP---- TLPPGVDPTQVSSSLSPEGTLTVEAPMPKLATQSN-EITIPVTFE---- . . * t t t . . * . IEGGTTGTTTGSTASSTPAR

EKPTS--APSS-- EKPAVTAAPKK--

-QTG?SSKEDNAKKVETSTA _--_- KEPEANQKSAINDAK

SRAQLGGRSCKIR

- - - - - - - - - - - - - -

- - - - - - -

Fig. 3. Multiple-sequence alignment of p26 with small heat-shock proteins and a-crystallins. The sequence of p26 was compared, by use of the CLUSTAL V sequence-alignment program, to the sequences of other small heat-shock proteins and a-crystallins available in the NCBI database. C-A-A-H, human a-crystallin A chain; C-A-B-H, human a- crystallin B chain; EL-F, fruit fly embryo lethal (2) 13-1; OV25-2-N, nematode small heat-shock protein OV25-2 : HSP27-H, human 27-kDa heat-shock protein. -, no residue; *. identical residue; 0, conserved residue.

we examined the ability of native p26 to reactivate thermally- inactivated citrate synthase, an enLyme that has been used by others to study chaperone activity in vitro [42, 521. Citrate synthase was heated as described in Materials and Methods, rapidly diluted and incubated in the absence or presence of native p26, prior to measuring enzyme activity. Table 2 summarizes the results for two preparations of p26. Some spontaneous renatur-

Table 2. The effect of p26 on recovery of citrate synthase activity after heating. Citrate synthase was heated at 50°C for 20 min, diluted and incubated at 23 "C for 30 min before assay as described in Materials and Methods. The values are means ? standard errors (n = 3). When present, p26 was at 35 nM, citrate synthase was at 150 nM (both as monomer concentrations), and BSA was at 35 nM. The final concentration of GTP was 3 mM. n.d., not determined.

Conditions Citrate synthase activity of

preparation 1 preparation 2

nmol coenzyme A/min

Control (no heating) 10.9 ? 0.9 11.1 2 0 . 3 Control (heating) 3.8 2 0.3 3.2 t- 0.3 P26 6.1 ? 0.4 6.5 2 0.2 p26 + GTP 6.8 ? 0.6 7.1 t- 0.2 BSA 4.2 2 0.2 n. d.

ation of heat-treated citrate synthase was observed in the absence of p26, amounting to about 30% of the unheated control. Incubation of heat-treated citrate synthase with native p26 resulted in a twofold increase over the spontaneous level. The results of a one-way analysis of variance revealed that the recovery of enzyme activity in the presence of p26 alone is significantly greater than in its absence (P<O.OS) for both preparations of p26. Since Artemia embryos contain very large concentrations of guanine nucleotides [53], the effects of adding GTP were examined (Table 2). A modest, but repeatable increase in the recovery of heat-treated citrate synthase was observed, which was about the same when ATP was used (data not shown). Although the addition of GTP in individual experiments always resulted in a small increase in the recovery of enzyme activity following heat treatment, the pooled results indicate no statisti- cally significant differences (P > 0.05) due to GTP addition. The recovery of enzyme activity in the presence of BSA was not significantly different ( P > 0.05) from recovery in the absence of p26, but it was significantly lower (P<O.OS) than when p26 or p26 and GTP were present. The conditions used in these studies have not been manipulated to maximize the activity of p26, so the results we have obtained thus far can probably be improved.

Liang et al. ( E m J. Biochem. 243)

25

.- 2 20

2 15

u) c 0)

a

m al

'0

- a

10

5 --

229

-- 600 --

-- 500 -- h m 0 443

-- 400 2. u) -- u)

I" -- 300 ;6

r -- 200 I

- =I

0 --

-- 100

o - ; : : : : 0

Fig. 4. Effect of p26 on citrate synthase activity after thermal treatments. Citrate synthase at 15 pM was heated at 50°C for 20 min, cooled and rapidly diluted to 150 nM. The levels of citrate synthase activity (nmol coenzyme Nmin) in the absence and presence of either p26, or p26 and GTP, were then determined (open bars). These reaction mixtures were frozen at -20"C, thawed and the activity of citrate synthase determined (cross-hatched bars) before heating at 50°C for 5 min and once again measuring citrate synthase activity (solid bars).

[p26 (monomer)] (nM)

Fig.5. Effect of p26 concentration on recovery of citrate synthase activity. Citrate synthase was heat inactivated and incubated in the presence of increasing concentrations of p26 as described in the legend to Fig. 4. The plotted values for citrate synthase activity (nmol coenzyme Nmin) were corrected for spontaneous renaturation of citrate synthase measured in the absence of p26.

Further study involved a sequential treatment, in which citrate synthase was heated in the absence of p26, then assayed as described in Table 2. These reaction mixtures were subjected to a freezelthaw cycle followed by a second heat treatment. The results of one such experiment are shown in Fig. 4. As expected from the results shown in Table 2, the addition of p26 after the first heat treatment resulted in a substantial increase in the recovery of citrate synthase. When these reaction mixtures were frozen at -20°C for a week, thawed and assayed for enzyme activity, a substantial protectionlreactivation was observed. A second heating (5OoC, 5 min) was carried out on these freezelthawed preparations (Fig. 4). The results showed that, in the absence of p26, very little citrate synthase survived the sequential treatment. In contrast, the addition of p26 enhanced substantially the recovery of activity, about 25-fold compared with the recovery in its absence. The presence of GTP seemed to enhance the effect of

B "7 0 699

T 700

1 15 21 23 25 27 30 34 38

Elution Volume (mi)

Fraction# 1 2 3 4 5 6 7 8 S 10 D

C

D 60-

50 --

40 -- r u) c al

> m

c .- 30 --

.- c -

20 --

l o 1 0 200

0 699

1 2 3 4 5 6 7 8 9 1 0 1

Fraction Number

Fig. 6. Molecular mass of p26 multimers. Purified p26 was applied to a Sepharose CL-6B column previously standardized with the following markers: carbonic anhrydrase, 29 kDa; albumin, 66 kDa; P-amylase, 200 kDa; apoferritin, 443 kDa; thyroglobulin, 699 kDa. The markers are labelled in the figure with their conesponding molecular mass (kDa). Samples of equal volume were taken from each column fraction, electrophoresed in 15 % SDS/polyacrylamide gels, blotted to nitrocellulose and stained with an antibody to p26 (A). Some of the samples, which failed to yield a band after reaction with antibody are not shown. The blots were scanned with a Hewlett Packard ScanJet l l cx at 400 dots pcr inch and the density was determined with the Scan Analysis (Biosoft) program. The density determinations, in arbitrary units (closed circles) were plotted in concert with the elution profile peaks for the molecular mass markers (open circles) (B). The molecular mass of p26 was determined by densitometric analysis of samples obtained by centrifugation on continuous sucrose gradients and stained with antibody after blotting to nitrocellulose (C, D). Standardization of gradients was accomplished with the size markers used for the Sepharose CL-6B columns. Vo, void volume; Ve, eluate volume; p, pellet.

700

600

500 3 2. UJ

400 0 I - ;6

300 g I

200

100

0

230 Liang et al. (Eur: J. Binchenz. 243)

p26 to a modest level. Because p26 is present in substantial amounts and may simply be acting through some sort of colliga- tive property, the ability of BSA to perform roles similar to those observed for p26 was tested. With BSA at the same mass/vol. as p26, no reactivation of heat-denatured citrate synthase was observed (Table 2).

The effect of p26 concentration on the renaturation of heat- inactivated citrate synthase was examined under the assay conditions described except that plotted values were corrected for spontaneous reactivation of citrate synthase in the absence of p26 (Fig. 5). Maximal recovery occurred at a monomer ratio of 10 citrate synthase: 1 p26 (Fig. 5) . However. using molecular masses of 100 kDa for native citrate synthase [51 I and 700 kDa for native p26, the molar ratio at maximal activity was about 130 citrate synthase: 1 p26.

Multimer formation by p26. Analysis by molecular-sieve chromatography on Sepharose CL-6B (Fig. 6A, B) and sucrose-density-gradient centrifugation (Fig. 6C, D) demonstrated that purified p26 exists as a multimer. Although its size varied, the largest number of multimers had a mass of about 700 kDa. As- suming a monomeric molecular mass for p26 of 26 kDa, there are about 27 monomers in a multinier of 700 kDa.

DISCUSSION

Arternia embryos are among the most stable of all eukaryotes when exposed to environmental insults, including prolonged anoxia, severe desiccation. extremes of temperature, and various forms of radiation [ 1 . 3, 11 - 131. Desiccation is a normal part of the life history of this species, and the embryos tolerate continuous anoxia for at least two years when fully hydrated at ordinary temperatures 1161. They achieve this by bringing their metabolism to a reversible standstill [15, 20-231, apparently con- tradicting the widely held concept that cells are unstable under ordinary conditions of temperature, pressure and water content. Given this, how does the anoxic Artemia embryo maintain its integrity'? For example, how are globular proteins, which are generally agreed to be inherently unstable at physiological temperatures [54-561, prevented from unfolding and aggregating over several years '?

Previous work on p26 has shown that it is present in very large amounts, making up 10-15% of the total non-yolk embryo protein, and that it is restricted to the encysted embryo stage [15, 191. Upon exposure to various kinds of stress, a large fraction of the total p26 is reversibly translocated into the nucleus and probably to other compartments and locations [18, 191. In view of these results and the literature on stress proteins [24- 261, it seemed that p26 belonged to the small-heat-shockla-crystallin family of proteins. Thus, purification and sequencing of the protein were undertaken to resolve this issue. Moreover, the native form of p26 was required to further evaluate its role as a molecular chaperone.

One unusual observation during purification of p26 to apparent homogeneity was the appearance of an immunoreactive polypeptide smaller than p26 in samples from phosphocellulose PI 1. This polypeptide was subsequently removed by ammonium-sulphate fractionation. This result suggests proteolytic di- gestion of p26, perhaps by an enzyme normally sequestered in an inactivated form. but which is activated during purification. Such an enzyme may be required for destruction of p26 as nauplii emerge and hatch.

The 141-amino-acid stretch of p26 reported here contains a region toward the carboxy terminus with significant similarity to a conserved region, the cn-crystallin domain, of the small-heat-

shockla-crystallin family of proteins [27-301. For example, in the conserved region, p26 and the human a-crystallin B chain are 51 % identical over a 91-amino-acid residue stretch, and this increases to 67% similarity when conserved residues are in- cluded. p26 is equally similar to small heat-shock proteins from Drosophila and Caenorhuhditis elegans. As is common to other proteins of this family, the conserved region is preceded by a variable amino-terminal domain. The sequence analysis demon- strates that p26 is a member of the small-heat-shock/a-crystallin family of proteins.

Purified p26 exists as multimers of 700 kDa composed of about 27 monomers. Previous work [19] indicated that p26 forms multimers, but because these experiments were done with cell-free extracts, the contribution of other proteins to particle mass, and thus to the quantitation of p26 monomers, was uncertain. Multimer formation is a characteristic of the small heat- shockla-crystallin proteins, and different representatives of the family may coaggregate [47]. The particles range from 10 nm to 18 nm, from about 200 kDa to 800 kDa, and from about 12 to 40 monomers [31, 41, 57-59]. Thus, the multimer size of p26 falls within the range expected for a small heat-shock/a-crystallin protein.

p26 exhibits molecular-chaperone activity in vitro. As determined by measuring enzyme activity, p26 assists in the renaturation of heat-inactivated citrate synthase, and prevents its denaturation. Chaperone activity does not require nucleotide hydroly- sis, a finding also true of other small heat-shock/a-crystallin proteins [41, 421 and it occurs at a molar ratio of citrate synthase (molecular mass of 100 kDa)/p26 (molecular mass of 700 kDa) of about 130: 1. However, no attempt has been made to optimize the conditions for chaperone assay.

The effectiveness of p26 as a molecular chaperone can be compared with that of other small heat-shockla-crystallin proteins although the experiments were carried out differently and the parameters used to measure activity varied. Horwitz [4X] demonstrated that a-crystallin suppressed heat-induced or guanidine-hydrochloride-induced aggregation of y-crystallin, alcohol dehydrogenase and rx-glucosidase at molar ratios of test protein to a-crystallin of between 1 : 1 and 12: 1. Delays in aggregation were noted at molar ratios as high as 120: 1. a-crystallin did not prevent heat-induced inactivation of these enzymes, nor of [-crystallin/quinone-reductase activity [46] and carbonic anhydrase [60]. Aggregation of the latter two enzymes was, however, prevented. In another study 1471, bovine a-crystallin and murine heat-shock protein HSP25 delayed the heat-induced aggregation of P-crystallin at a molar ratio of target enzyme/chaperone of 40: I , with the calculation based on a molecular mass of 600 kDa for HSP25. rx-crystallin prevented aggregation of rhodanese [61] at a molar ratio of 1.2: 1, representing a ratio of 33 enzyme molecules/molecule of a-crystallin. However, a-crystallin did not assist in the refolding of rhodanese, &crystallin or y-crystallin upon dilution of these proteins from 6 M guanidinium chloride [61]. In contrast, Jakob et al. [42] showed that rx-crystallin, HSP27 and HSP25 enhance protein refolding. Citrate synthase recoveries were 8% and 25% in the absence and presence, re- spectively, of small heat-shockla-cry stallin proteins, while a-glucosidase recovery increased from 20% to 45 9%. Refolding was optimal at a chaperonekitrate synthase ratio of 1 : 3. As a final example, folding yields of citrate synthase and lactate dehydrogenase after denaturation in 6 M guanidine hydrochloride, were enhanced by HSP18.1 and HSP17.7 from pea [41]. The citrate synthase activity was increased greater than 40% in the presence of the small heat-shock proteins, but only 17% in their absence. The results for lactate dehydrogenase were similar. Refolding was saturated at a 1 : 1 molar ratio of small-heat-shock protein

Liang et al. (ELM J. Biochem. 243) 23 1

complex (1 2 monomers)/target protein, but significant results were achieved at lower ratios.

The observation that the small heat-shockla-crystallin proteins form complexes with denaturing proteins and that they prevent their aggregation has particular significance in the consideration of p26 activity. As proposed by Jacob et al. [42] for other small heat-shock proteins, p26 may keep the number of folding intermediates with the potential to aggregate at a low level. It therefore acts as a primary protective mechanism within the cell, but does not necessarily have an enzymatic effect on the folding of proteins. Thus, p26 may recognize non-native substrates and bind reversibly in the absence of ATP, exhibiting a high capacity to prevent aggregation, as shown by a-crystallin in the eye [61]. This would limit the damage in stressed cells, such as those in diapause or anoxia, by preventing complete denaturation and aggregation of cellular proteins. Additionally, in a system where anoxia can be prolonged and cellular energy reserves quite low, a stabilizing chaperone that is ATP-independent represents an adaptive process. Upon return to permissive conditions, the proteins assume their native conformation, but the mechanism may not be overly efficient because it is passive in nature. Such a proposal has been made for HSP18.1 and HSP17.7 from pea [41]. An entirely different mechanism of action for p26 is suggested by the finding that a-crystallin and HSP25 inhibit elastase [47]. If p26 has a similar property, it may prevent serine-protease activity in the absence of protein turnover, as occurs in Artemia during diapause and anoxia. In this context, it was difficult to obtain peptides for sequence analysis from some preparations of p26, suggesting resistance to proteolytic action in these samples.

In summary, the findings presented in this paper, in concert with other information from our laboratories [15, 18, 191 are consistent with the conclusion that p26 functions as a molecular chaperone during diapause and anaerobic dormancy, indicating the molecular basis for at least one mechanism whereby encysted Arternia embryos resist stress. Moreover, the results broaden the range of physiological activities in which the small heat-shock/a-crystallin proteins are thought to have a role.

The expert technical contributions of Susan A. Jackson are gratefully acknowledged. The work was supported by a Natural Sciences and Engi- neering Research Council of Canada research grant to THM and an Izaak Walton Killam Memorial Scholarship to P. L.

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Documents

Purification, Structure and In vitro Molecular-Chaperone Activity of Artemia P26, a Small Heat-Shockh/α-Crystallin Protein