Oligomerization, Chaperone Activity and Nuclear Localization of p26, a Small Heat Shock Protein from Artemia franciscana

: Artemia franciscana embryos undergo encystment, developmental arrest and diapause, the latter characterized by profound metabolic dormancy and extreme stress resistance. Encysted embryos contain an abundant small heat shock protein termed p26, a molecular chaperone that undoubtedly has an important role in development. To better understand the role of p26 in Artemia embryos, the structural and functional characteristics of full length and truncated p26 expressed in Escherichia coli and COS-1 cells were determined. p26 chaperone activity declined with increasing truncation of the protein, and those deletions with the greatest adverse effect on protection of citrate synthase during thermal stress had the most influence on oligomerization. When produced in either prokaryotic or eukaryotic cells the p26 a -crystallin domain consisting of amino acid residues 61-152 existed predominately as monomers, and p26 variants lacking the amino terminal domain but with intact carboxy-terminal extensions were mainly monomers and dimers. The amino-terminus was, therefore, required for efficient dimer formation. Assembly of higher order oligomers was enhanced by the carboxy-terminal extension, although removing the ten carboxy-terminal residues had relatively little effect on oligomerization and chaperoning. Full length and carboxy-terminal truncated p26 resided in the cytoplasm of transfected COS-1 cells, however variants missing the complete amino-terminal domain and existing predominantly as monomers/dimers entered nuclei. A mechanism whereby oligomer disassembly assisted entry of p26 into nuclei was suggested, this of importance because p26 translocates into Artemia embryo nuclei during development and stress. However, when examined in Artemia , p26 oligomer size was unchanged under conditions that allowed movement into nuclei, suggesting a process more complex than just oligomer The The cells were three then for at (FITC)-conjugated goat anti-rabbit IgG ImmunoResearch) diluted in PBS-Triton. After washing three times with PBS-Triton, the cells were incubated with RNaseA at 5 mg/ml for 10 min and stained with propridium iodide at 0.4 mg/ml for 2 min. Cover-slips were rinsed with and placed on (cid:212) Cells expressing His-tagged p26 fusion proteins were also stained with diluted in PBS-Triton, followed by FITC-conjugated goat anti-mouse IgG antibody Immunofluorescently stained cells were examined with a Zeiss 410 confocal laser scanning microscope.

movement into nuclei, suggesting a process more complex than just oligomer dissociation.

INTRODUCTION
The small heat shock proteins (sHSPs), characterized by a conserved α-crystallin domain exhibiting an immunoglobulin-like fold bordered by variable amino-and carboxy-terminal extensions (1-7), function as molecular chaperones. The molecular mass of sHSPs ranges from 12-43 kDa and most oligomerize by a multi-step process often with dimers as stable suboligomeric units (8,9), although for bovine αB-crystallin monomers may be basic building blocks (10). For sHSPs such as HSP20 (11) and HSP22 (12), dimers are the predominant complex and they exhibit chaperone activity. Of medical significance, αA-crystallin truncations occur in mammalian lens, suggesting a relationship between carboxy-terminal modification and cataract (13,14). Also, dropping either thirteen or twenty-five carboxy-terminal residues from human αB-crystallin causes myofibrillar myopathy, with modified proteins exerting dominant negative effects (15). The truncated and normal αB-crystallins appear to interact, yielding oligomers slightly smaller than those generated by wild type protein.
Embryos of the brine shrimp, Artemia franciscana, undergo ovoviviparous and oviparous development, the former yielding nauplii and the latter encysted gastrulae or cysts (31). Cysts enter diapause (32,33), characterized by deep reduction in metabolic activity (34) and resistance to extreme environmental stress such as long term anoxia, desiccation and heat shock (35,36).
p26, an abundant sHSP in Artemia cysts, forms oligomers, functions as a molecular chaperone in vitro and confers thermotolerance on transformed bacteria (29,(37)(38)(39)(40)(41), undoubtedly contributing to embryo stress tolerance. p26 enters nuclei upon synthesis in developing embryos (38), and migrates reversibly by a poorly understood mechanism into Artemia nuclei during anoxia and heat shock (42)(43)(44). Upon exposure to anoxia the internal pH of post-diapause cysts drops to about 6.6, and reversible movement of p26 into nuclei occurs in vitro upon pH reduction.
Domain-specific effects on p26 oligomerization, chaperone activity and nuclear localization were examined in this study and the data are related to the structural/functional properties of p26 within oviparously developing Artemia embryos.

EXPERIMENTAL PROCEDURES
Cloning of p26 cDNAs-Truncated p26 cDNAs were generated by site-directed mutagenesis using the QuikChange™ Site-directed Mutagenesis kit (Stratagene, La Jolla, CA), p26-3-6-3 as template (38), and designated primers ( Table 1). The truncated p26-3-6-3 cDNAs were recovered from agarose gels with the GFX™ PCR DNA and Gel Band purification kit (Amersham Biosciences, Piscataway, NJ), inserted into pRSET.C, a polyhistidine-tagged (Histagged) prokaryotic expression vector linearized by digestion with BamH1 and Xho1 prior to purification from agarose gels, and cloned in Escherichia coli strain BL21(DE3)PLysS (Invitrogen, Carlsbad, CA). Full-length and truncated p26 cDNAs were also generated previously by PCR and inserted into the T/A vector, pCRII (37). The p26 cDNA-containing pCRII vectors and the mammalian expression vector pSecCMV (Invitrogen) were digested with BamH1 and Xba1, followed by electrophoresis in 1% agarose gels. p26 cDNAs were recovered with the GFX™ PCR DNA and Gel Band purification kit, inserted into linearized pSecCMV, and cloned in E. coli DH5α™ (Invitrogen). Full length p26 cDNA (p26-full) and the cDNA fragments p26-N∆60 and p26-alpha were excised from pSecCMV with BamH1 and Xba1, inserted into pcDNA4/His.A (Invitrogen), a His-tag-containing mammalian expression vector, and cloned in E. coli DH5α. All inserts were sized by restriction digestion followed by electrophoresis in agarose, and PCR fidelity was verified by DNA sequencing (DNA Sequencing Facility, Centre for Applied Genomics, Hospital for Sick Children, Toronto, ON).
Purification of p26-Expression plasmids containing either full length or truncated p26 cDNAs transformed into E. coli strain BL21(DE3)PLysS were induced by addition of 1 mM isopropyl thio-β-D-galactoside (IPTG) (Sigma, St. Louis, MO) for 5 h. The cultures were cooled on ice and centrifuged at 5,000 × g for 5 min at 4 o C. The cell pellets were washed once with Extraction/Wash buffer (50 mM Na 3 PO 4 , 300 mM NaCl, pH 7.5) and resuspended in 4 ml of the same buffer prior to addition of 100 µg/ml lysozyme (Sigma), 1 mM phenylmethylsulfonyl fluoride (PMSF) (Sigma), 1 µg/ml pepstatin A (Sigma) and 1 µg/ml leupeptin (Sigma). The mixtures were incubated at 30 o C for 15 min, cooled on ice and sonicated three times for 10 sec using a Branson Sonifier™ 150 (Branson Ultrasonics, Danbury, CT) at medium setting with intermittent cooling on ice for 30 sec. The sonicated cells were centrifuged at 12,000 × g for 20 min at 4 o C, the supernatants recovered and protein concentrations ascertained with the Bio-Rad Protein determination kit (Bio-Rad, Hercules, CA). His-tagged p26 was purified from bacterial extracts using 2 ml BD TALON™ affinity columns as described by the manufacturer (BD Biosciences, Mississauga, ON). The samples were desalted by dialysis against 10 mM NaH 2 PO 4 , pH 7.1 and concentrated with Centriprep YM-10 centrifugal devices (Amicon Bioseparations, Billerica, MA).
Bacterial extracts and purified p26 were electrophoresed in 12.5% SDS polyacrylamide gels followed either by staining with Coomassie blue or transfer to nitrocellulose. Blots were stained with 2% Ponceau S (Sigma) in 3% trichloroacetic acid (TCA) to verify protein transfer, washed  with TBS-Tween (10 mM Tris-HCl, 140   Fifteen µl from each fraction was electrophoresed in 12.5% SDS polyacrylamide gels, blotted to nitrocellulose, and probed with either anti-p26 antibody or Omni-probe as described previously.

RESULTS
Cloning of p26 cDNAs-Full length and truncated p26 cDNAs cloned in the prokaryotic expression vector pRSET.C included p26-full-His, the full length polypeptide; p26-N∆36-His, residues 1-36 deleted; p26-N∆60-His, residues 1-60 removed; p26-C∆40-His, final forty residues eliminated; p26-C∆10-His, lacked the last ten residues; p26-alpha-His, missing residues 1-60 and 153-192, thereby consisting of the α-crystallin domain (Fig. 1). All p26 cDNAs generated by site-directed mutagenesis contained, in addition to the p26 sequence, an amino terminal peptide of 13 residues encoded by p26-3-6-3 and the His-tag. The presence of the His-tag is indicated by "-His" in the name of the p26 cDNA or protein. The p26 cDNAs recovered from the storage vector pCRII (37) were cloned in pSecCMV for expression in COS-1 cells. p26-full, p26-N∆60 and p26-alpha were also prepared in pcDNA4/His.A for expression as His-tagged fusion proteins. All cloned p26 cDNA fragments were sized by electrophoresis in agarose after restriction digestion of recombinant plasmids and PCR fidelity was confirmed by sequencing all cloned inserts (not shown).
p26 oligomer formation and chaperone activity-IPTG induction of transformed E. coli yielded polypeptides on western blots of the appropriate size that reacted strongly with Omni-probe, although bands corresponding to p26 were not readily visible in Coomassie blue stained gels (Figs. 2a, b). With the exception of p26-N∆60-His and p26-alpha-His, all of the polypeptides were recognized by antibody to p26 (not shown). Subsequent to purification on TALON™ affinity columns single bands of the expected size appeared on Coomassie blue stained gels (Fig.   2c). These polypeptides reacted strongly with Omni-probe (Fig. 2d) indicating they were Histagged p26, although purified p26-N∆60-His and p26-alpha-His were again not recognized by anti-p26 antibody. Protein bands were not detected on blots containing extract from E. coli transformed with vector lacking p26 cDNA (Figs. 2a, b, lane 7).
The molecular mass of oligomers assembled from purified p26-full-His synthesized in E. coli ranged from 26 kDa to 600 kDa, with the peak at about 150 kDa, or 6 monomers per oligomer (Fig. 3, Table 2). The oligomers assembled with full length p26 were larger than those obtained with truncated proteins, although oligomers of p26-C∆10-His were similar in mass. At the other extreme, p26-alpha-His migrated predominately as monomers and p26-N∆60-His existed as monomers and dimers, although larger complexes occurred. Oligomers assembled from other p26 variants were intermediate in mass to those just described.
Purified p26-full-His possessed the greatest chaperone activity and p26-alpha-His the least, as determined by heat-induced denaturation of citrate synthase, although all variants provided some protection (Fig. 4). At a chaperone to target molar ratio of 4:1 (p26 monomer to citrate synthase dimer), or 1:1.5 if the peak oligomer size of p26-full-His is used for comparison, citrate synthase protection was almost complete after 1 h at 43 o C (Fig. 4a). At a 1:4 molar ratio of p26-full-His to citrate synthase, or 1:24 (oligomer to dimer), purified p26 reduced the heat-induced turbidity increase by approximately 25% (Fig. 4e). In contrast, p26-alpha-His was almost devoid of chaperone activity even at molar ratios of 4:1 (Fig. 4). p26-N∆60-His was marginally better than p26-alpha-His as a chaperone, followed by p26-N∆36-His and p26-C∆40-His which were similar to one another. p26-C∆10-His approached p26-full-His in chaperone potency especially at high concentrations. BSA and IgG at 600 nM provided almost no protection upon heating of citrate synthase (Fig. 4f).

Synthesis and oligomerization of p26 in transfected mammalian cells-Except for p26-N∆60
and p26-alpha, which reacted with Omni-probe, protein extracts from COS-1 cells transfected with p26 cDNA-containing expression vectors yielded polypeptides of the expected size on western blots with anti-p26 antibody (Fig. 5). Neither primary antibody gave protein bands on blots with extracts from COS-1 cells transfected with vectors lacking p26 cDNA (not shown).
Full length p26 synthesized in COS-1 cells produced oligomers as large as 512 kDa and composed of up to 21 monomers, with monomer number essentially the same in the presence and absence of His-tag (Figs. 6a, b). Oligomers assembled with p26-C∆10 were only slightly smaller than those produced from full length p26 (Fig. 6c), but removal of the complete carboxy-terminal extension gave oligomers of a narrower size range and smaller mass (Fig. 6d).
Oligomer mass became progressively smaller upon sequential removal of the amino-terminus (Figs. 6e, f) with polypeptides encoded by p26-N∆60-His present as monomers and dimers, as was true for p26-alpha-His (Fig. 6g), although the latter was more enriched in monomers. The properties of p26 oligomers produced in COS-1 cells are summarized in Table 3.
Truncated p26 localizes to COS-1 nuclei-In order to monitor p26 synthesis transiently transfected COS-1 cells were stained with anti-p26 antibody, Omni-probe and propridium iodide, revealing that polypeptides encoded by p26-full and p26-full-His (Figs. 7a, b) and the carboxy-terminal truncations, p26-C∆40 (Fig. 7c) and p26-C∆10 (Fig. 7d), localized exclusively to the cytoplasm. In contrast, upon amino-terminal modification p26 occurred in cytoplasm and nuclei, the latter shown by yellow staining. p26-N∆36 encoded polypeptides were in the nuclei of only some cells (Figs. 7e, f), but polypeptides p26-N∆60/p26-N∆60-His lacking the amino-terminus (Figs. 7g, h), or p26-alpha/p26-alpha-His composed of the αcrystallin domain (Figs. 7i, j), resided in the nuclei of all transfected cells, indicating that the His-tag had no effect on p26 localization. The results suggested that disassembly of oligomers is responsible for p26 movement into nuclei of COS-1 cells and by extrapolation, the nuclei of encysted Artemia embryos.
Oligomer size of p26 from Artemia embryos is unaffected by pH and heat shock-p26 moves into the nuclei of Artemia embryos upon exposure to reduced pH in vitro and upon heat shock in vivo by an unknown mechanism that, as just suggested by the previous results, entails oligomer mass reduction. However, the p26 oligomers obtained from Artemia cysts homogenized at either pH 6.5 or 7.0 were identical in molecular mass, whether or not they were incubated 30 min at room temperature before gradient centrifugation (Fig. 8a-d). p26 migrated into the nuclei of heat shocked Artemia embryos under the conditions used in this work (Figs. 9a, b), but the size of p26 oligomers in extracts from cysts (Figs. 9c, d) and nuclei (Figs. 9e, f) remained constant.
The p26 in nuclear extracts tended to smear upon electrophoresis and blotting, probably due to the presence of DNA.

DISCUSSION
Full length p26 produced in either bacteria or mammalian cells yielded oligomers that were somewhat smaller than those from encysted Artemia embryos, but which represented effective oligomerization nonetheless. In contrast, the α-crystallin domain existed mainly as monomers and dimers, as was true for p26 lacking the entire amino-terminus. These data indicate a role for the amino-terminus in formation of dimers and thus higher oligomers, a conclusion strengthened by the comparatively greater oligomerization of p26 lacking thirty-six rather than sixty residues.
The α-crystallin domain and amino-terminal truncated p26 variants were poor chaperones in relation to full length p26, with chaperone activity decreasing as truncation increased. Reduced chaperone activity and oligomerization were likely caused by the loss of residues involved in oligomer formation and interaction with substrates. As one example, the p26 motif 17WSDPF21 corresponds to 15SWEPF19 in Chinese hamster HSP27, a sequence important for oligomer formation and chaperoning (45). p26 structural organization and function may also depend on other hydrophobic elements of the amino-terminal region with the deleted residues 1-36 and 1-60 possessing 72.2% and 61.7% hydrophobicity, respectively, both significantly higher than the 51% hydrophobicity of the full length protein.
In comparison to p26, yeast HSP26 lacking amino-terminal sequences formed dimers devoid of chaperone activity even though dissociation of oligomers into dimers is a functional prerequisite (8). Human αB-crystallin missing amino acid residues 1-56 produced dimers with significant chaperone activity (46), while removing either 50 or 56 residues from the aminoterminus of αA-crystallin decreased oligomer mass from 550 to 60 kDa, the latter composed of tetramers and dimers (47). The results indicate that sHSP dimerization occurs independent of the amino-terminal domain, but formation of larger oligomers requires this region, conclusions in partial agreement with the findings for p26. HSP16.5 from M. jannaschii (19) and HSP16.9 from T. aestivum (18) assemble well defined (monodisperse) oligomers and for HSP16.5 the amino terminus facilitates oligomerization, but does not necessarily determine the final architectural structure (48). Deletion of 42 amino-terminal residues from rice HSP16.9 decreased chaperone activity but not oligomer size (49), and in the bacterium Bradyrhizobium japonicum, where the amino-terminus is required for oligomer assembly, at least a portion of the region drives dimer formation (17), as appears to be true for p26.
Removal of ten carboxy-terminal residues had little effect on p26, however deleting the entire carboxy-terminus, including the conserved motif I/V-X-I/V (as VPI) (17), reduced oligomerization and chaperoning. The carboxy-terminus of p26 was not required for dimer formation, but the region contributed to oligomer assembly, although less so than the aminoterminus. Deletion of the entire p26 carboxy-terminal extension reduced protection of citrate synthase upon heating, indicating a role in chaperoning which may depend upon oligomerization, recognition of substrate proteins, chaperone/substrate solubility, or a combination of these.
Precipitation of truncated p26 was not observed even with complete removal of the carboxyterminus which contains twenty polar and four charged amino acids in the final thirty residues. In contrast, loss of the last sixteen amino acids from C. elegans HSP16-2 was without effect on oligomer size and chaperone activity, but the modified protein precipitated upon freeze/thawing (50), suggesting reduced solubility.
The carboxy-terminus promotes sHSP/substrate solubility and chaperoning to varying degrees (17,(50)(51)(52)(53). Eliminating the last eighteen amino acid residues from mouse HSP25, which excludes the conserved I/V-X-I/V motif (17), had little affect on oligomerization and chaperoning of citrate synthase at 43 o C, but protection of α-lactalbumin against dithiothreitolinduced denaturation was lost (52). Deletion of 10 carboxy-terminal residues has minimal impact on αA-crystallin, and sometimes even enhanced substrate protection, albeit modestly (14,47), but this result was never obtained with p26. In contrast, removal of 11 or more Cterminal residues from αA-crystallin, including the I/V-X-I/V motif drastically reduced oligomer size and chaperoning, with Arg-163 particularly important. Additionally, the carboxyterminus of the B. japonicum sHSP, and especially the conserved motif I/V-X-I/V, plays a role in oligomer assembly (17), as is true for Pfu-sHSP from the hyperthermophilic microorganism Pyrococcus furiosus (54), all results in agreement with those obtained with p26.
The role of sHSPs in nuclei has received limited attention. αB-crystallin and HSP27 associate with nuclear speckles and nucleoli of various human cell lines in non-stress conditions and may exert regulatory roles in these locations (55, 56). Human HSP27 translocates into nuclei of transfected A549 cells occurs stress, although protection occurs independent of nuclear localization (57). p26 migrates into nuclei early in oviparous development (38), during physiological stress and upon exposure to acidic conditions in vitro (42)(43)(44). A nuclear localization signal as occurs in tomato HSP16.1-CIII (58) is not apparent, but residues 36-45 of p26 include six arginines (39), a potential nuclear localization signal.
In this study, full length and carboxy truncated p26 resided exclusively in transfected COS-1 cell cytoplasm. In contrast, complete removal of the amino-terminal domain, including the putative nuclear localization signal, resulted in nuclear translocation. One interpretation of this finding is that decreased oligomer disassembly permits p26 movement through nuclear pores by simple diffusion, and nuclear translocation of rat HSP20 and HSP25 upon heat shock, where they may play protective roles, is accompanied by stress-induced decrease in oligomer mass, perhaps to dimers for HSP20 (59). However, p26 reduced in oligomer size due to carboxy-terminal truncation remained in the cytoplasm, suggesting that simple diffusion is not occurring. In support of this, modification of a single p26 residue by site-directed mutagenesis had little effect on oligomerization, but led to nuclear localization (manuscript in preparation). Additionally, p26 oligomers in heat stressed and control cysts, including those from nuclei, were similar and their mass was maintained in reduced pH, a condition that promotes p26 translocation into nuclei in vivo and in vitro. The most direct conclusion is that p26 migration into cyst nuclei depends on a mechanism other than oligomer mass reduction, although the transient formation of monomers or dimers followed by reassembly in the nucleus is possible. Clearly, however, movement of p26 into the nuclei occurs independent of the arginine-enriched amino-terminal sequence.
To conclude, the amino-terminal domain of p26, a sHSP occurring in embryos that exhibit extreme stress resistance, promotes α-crystallin dimerization and in concert with the carboxyterminal extension enhances protein oligomerization. As measured by heat-induced denaturation of citrate synthase, the α-crystallin domain of p26 lacks effective chaperone activity, depending on amino-and carboxy-terminal regions for full function. The nuclear translocation of p26 in COS-1 cells occurs independent of oligomer size and a putative nuclear localization in the amino-terminal region, suggesting a complex mechanism for movement into the nuclei of encysted Artemia embryos. Additional work to support these conclusions and to define domainspecific p26 amino acid residues with structural/functional implications is underway. cDNA-containing vector pSecCMV were incubated with antibody to p26 followed by FITCconjugated goat anti-rabbit IgG antibody (green) (a, c, d, e, f). Cells transfected with the p26 cDNA-containing vector pcDNA4/His.A were exposed to anti-p26 antibody followed by FITCconjugated goat anti-rabbit IgG (green) (g, i) or to Omni-probe followed by FITC-conjugated goat anti-mouse IgG (green) (b, h, j). Nuclei were stained with propridium iodide (red) and samples were examined by confocal microscopy. a, p26-full; b, p26-full-His; c, p26-C∆40; d, p26-C∆10; e and f, p26-N∆36; g, p26-N∆60; h, p26-N∆60-His; i, p26-alpha; j, p26-alpha-His. The scale bar represents 50 µm and all figures are the same magnification.