Báo cáo khoa học: Structural and functional roles for b-strand 7 in the a-crystallin domain of p26, a polydisperse small heat shock protein from Artemia franciscana pdf

a-crystallin domain of p26, a polydisperse small heat shock protein from Artemia franciscana Yu Sun, Svetla Bojikova-Fournier and Thomas H.. Keywords a-crystallin domain; Artemia francis

a-crystallin domain of p26, a polydisperse small heat shock protein from Artemia franciscana

Yu Sun, Svetla Bojikova-Fournier and Thomas H MacRae

Department of Biology, Dalhousie University, Halifax, NS, Canada

Protein folding and maintenance of an appropriate 3D

structure occur with the assistance of molecular

chap-erones, including Hsp60 (chaperonins), Hsp70, Hsp90,

Hsp104⁄ ClpB, Hsp110 and the small heat shock

pro-teins (sHSPs) [1–6] Several chaperones are actively

involved in protein folding, whereas others, and in par-ticular the sHSPs, protect proteins during stresses such

as heat shock, oxidation and hypoxia⁄ anoxia Mole-cular chaperones also remove damaged proteins through the action of CHIP, a ubiquitin ligase [6]

Keywords

a-crystallin domain; Artemia franciscana;

molecular chaperone; p26 structure ⁄

function; small heat shock protein

Correspondence

T H MacRae, Department of Biology,

Dalhousie University, Halifax, NS,

Canada B3H 4J1

Fax: +1 902 4943736

Tel: +1 902 4946525

E-mail: tmacrae@dal.ca

(Received 5 July 2005, revised 26 December

2005, accepted 5 January 2006)

doi:10.1111/j.1742-4658.2006.05129.x

Oviparous development in the extremophile crustacean, Artemia franciscana, generates encysted embryos which enter a profound state of dormancy, termed diapause Encystment is marked by the synthesis of p26, a polydis-perse small heat shock protein thought to protect embryos from stress In order to elucidate structural⁄ functional relationships within p26 and other polydisperse small heat shock proteins, and to better define the protein’s role during diapause, amino acid substitutions R110G, F112R, R114A and Y116D were generated within the p26 a-crystallin domain by site-directed mutagenesis These residues were chosen because they are highly conserved across species boundaries, and molecular modelling indicates that they are part of a key structural interface between dimers The F112R mutation, which had the greatest impact on oligomerization, placed two charged resi-dues at the p26 dimer–dimer interface, demonstrating the importance of b-strand 7 in tetramer formation All mutated versions of p26 were less able than wild-type p26 to confer thermotolerance on transformed bacteria and they exhibited diminished chaperone action in three in vitro assays; however, all variants retained protective activity This apparent stability of p26 may, by prolonging effective chaperone life in vivo, enhance embryo stress resistance All substitutions modified p26 intrinsic fluorescence, sur-face hydrophobicity and secondary structure, and the pronounced changes

in variant R114A, as indicated by these physical measurements, correlated with the greatest loss of function Although mutation R114A had the greatest effect on p26 chaperoning, it had the least on oligomerization These results demonstrate that in contrast to many other small heat shock proteins, p26 effectiveness as a chaperone is independent of oligomeriza-tion The results also reinforce the idea, occasioned by modelling, that R114 is removed slightly from dimer–dimer interfaces Moreover, b-strand

7 is shown to have an important role in oligomerization of p26, a function first proposed for this structural element upon crystallization of wheat Hsp16.9, a small heat shock protein with different quaternary structure

Abbreviations

ANS, 1-anilino-8-naphthalene-sulphonate; sHSP, small heat shock protein.

sHSPs usually occur as oligomers composed of

sub-units ranging in molecular mass from 12 to 43 kDa,

and they protect proteins from irreversible

denatura-tion independent of ATP [3,7–13] The conserved

a-crystallin domain of  90 amino acid residues,

located towards the C terminus, is important for

oligo-mer formation and chaperoning [14–16] The

a-crystal-lin domain is preceded by a poorly conserved

N-terminal region proposed to function in oligomer

assembly, subunit exchange and substrate binding [17–

22], and is followed by a flexible, polar, C-terminal

extension of variable sequence that influences solubility

and oligomerization [17,21,23–25] sHSP secondary

structure is dominated by b-pleated sheet, but the

quaternary structure is variable [26,27] Hsp16.5 from

the archaeon, Methanococcus jannaschii, and Hsp16.9

from wheat, Triticum aestivum, assemble monodisperse

oligomers and they have been crystallized, revealing

important sHSP structural attributes [14,16] sHSPs,

most of which form polydisperse oligomers, interact

with several substrates and a reservoir of intermediates

accrues, a progression involving oligomer dissociation

and subunit exchange [19,28–30], but which may also

occur upon rearrangement of oligomer structure in the

absence of dissociation [31] When stress is relieved,

substrates are released and renatured, processes

occur-ring spontaneously or with assistance from other

molecular chaperones [32,33] The sHSPs influence

cytoskeleton organization [34–36], apoptosis [37–40]

and development [7,41], thereby playing important

roles in cell activities

Artemia females release offspring as swimming

lar-vae (ovoviviparous development) or encysted gastrulae

(oviparous development), termed cysts The cysts enter

diapause, a resting stage where metabolic activity is

extremely low, even under favourable conditions [41]

Activation of encysted embryos by desiccation

pre-cedes reinitiation of development in the presence of

appropriate hydration, temperature and aeration

Arte-mia cysts are exceptionally resistant to harsh

condi-tions, and when fully hydrated, either during diapause

or in a postdiapause state of metabolic arrest, termed

quiescence, they survive for several years without

oxy-gen This is arguably the ultimate indifference to

anoxia of any metazoan [42], and qualifies the

organ-ism, as do other of its characteristics, as an

extremo-phile Because activated Artemia embryos resume

development immediately upon return to favourable

circumstances, macromolecular components must be

preserved in the presence of limiting ATP, an activity

within sHSP functional capability Just such a protein,

named p26, is synthesized in large quantities by

ovi-parous, but not ovoviviovi-parous, embryos [17,41,43–46]

The p26 a-crystallin domain is similar in sequence to this region in other sHSPs, including wheat Hsp16.9; the protein confers thermotolerance on transformed Escherichia coliand it has chaperone activity in vitro The objectives of the work described here are to reveal structural and functional characteristics of poly-disperse sHSPs by introducing single-site mutations in p26, and to better define the relationship between p26 and stress resistance in A franciscana The amino acids selected for study are highly conserved from species

to species (Fig 1); at least one causes disease when mutated [47–52] and, as indicated by molecular model-ling, they reside in a key structural interface, suggest-ing that their modification will affect oligomerization and chaperoning The role of b-strand 7 in oligomeri-zation was demonstrated in this work Additionally, as for other sHSPs, changing the conserved p26 a-crystal-lin domain arginine (R114) reduced chaperone activity, but in this case with only a minor effect on oligomeri-zation This showed, in concert with analysis of the F112R mutation, that oligomerization and chapero-ning are not linked in p26 The resistance of p26 chap-eroning activity to single-site mutations suggests a stable protein and this, in concert with the large amount of p26 present during oviparous development, undoubtedly contributes to the remarkable stress resistance of encysted Artemia embryos

Results

Site-directed mutagenesis and purification

of bacterially produced p26 cDNAs encoding the amino acid substitutions R110G, F112R, R114A and Y116D in the a-crystallin domain

of p26 were cloned in the prokaryotic expression vector, pPROTet.E233, and used to transform E coli BL21PRO Sequencing demonstrated that each p26 cDNA contained only the introduced substitution p26 synthesized in bacteria possessed an N-terminal His-tag and an additional short N-terminal peptide (PRAAGIRHELVLK) encoded by the clone used for site-directed mutagenesis, but comparisons throughout the study to p26 from Artemia and transfected mam-malian cells lacking these residues indicated that they had almost no effect on structure and function Cell-free extracts prepared from transformed bacteria induced with anhydrotetracycline (aTc) exhibited lightly stained bands, corresponding in size to p26 when electrophoresed in SDS polyacrylamide gels and stained with Coomassie blue, and these polypeptides reacted with anti-p26 immunoglobulin (Fig 2A,B) Expression levels in bacteria were very similar for all

variants and there was no indication of protein degra-dation After purification on TALONtm

affinity col-umns, a major polypeptide of the expected size recognized by anti-p26 immunoglobulin was obtained for each variant (Fig 2C,D)

p26 in COS-1 cells COS-1 cells were transiently transfected with p26 cDNAs cloned in the eukaryotic expression vector, pcDNA4⁄ TO ⁄ myc-His.A, and p26 synthesis was veri-fied by immunofluorescent staining and confocal laser-scanning microscopy (Fig 3) Wild-type (WT) p26 was localized predominantly, if not exclusively, in the cyto-plasm of transfected cells In contrast, all COS-1 cells transfected with cDNA containing the R114A muta-tion had p26 in nuclei as well as in the cytoplasm (Fig 3) p26 R110G, F112R and Y116D occurred in the cytoplasm and nuclei of transfected cells, although some nuclei lacked the protein (not shown) p26 was subsequently prepared from transfected COS-1 cells for determination of oligomer size

Fig 1 Multiple sequence alignment of rep-resentative small heat shock proteins (sHSPs) The amino acid sequences of selected sHSPs were analyzed by CLUSTAL W

(1.82) HHSP27, Homo sapiens Hsp27, P04792; MHSP25, Mus musculus Hsp25, P14602; HCRYAA, H sapiens aA-crystallin, P02489; HCRYAB, H sapiens aB-crystallin, P02511; Ap26, Artemia franciscana p26, AF031367; and WHSP16.9, wheat Hsp16.9, 1GME sHSP domains based on the sequence of p26 are indicated above the alignment, secondary structure elements based on the sequence of wheat Hsp16.9 are below the alignment, and the conserved a-crystallin domain amino acid residues selected for mutational analysis are shaded Residue number is indicated on the right –, no residue; *, identical residues; :, con-served substitution; , semiconcon-served sub-stitution.

Fig 2 Purification of p26 synthesized in Escherichia coli BL21PRO.

Cell-free extracts from transformed E coli BL21PRO induced with

anhydrotetracycline (aTc) were electrophoresed through SDS

poly-acrylamide gels and either stained with Coomassie blue (A) or

blot-ted to nitrocellulose and reacblot-ted with antibody to p26 (B) Proteins

purified by affinity chromatography were electrophoresed through

SDS polyacrylamide gels and either stained with Coomassie blue

(C), or blotted to nitrocellulose and reacted with antibody to p26

(D) Lane 1, R110G; lane 2, F112R; lane 3, R114A; lane 4, Y116D;

lane 5, wild-type (WT) p26; lane 6, vector lacking p26 cDNA Lanes

in panels A and B received 4.5–5.5 lg of protein and lanes in

pan-els C and D received 1 lg of protein Arrow, p26 M, molecular

mass markers of 97, 66, 45, 31, 21 and 14 kDa.

p26 oligomerization

WT p26 produced the largest oligomers, while, for

modified proteins, oligomer size was greatest for

R114A and smallest for F112R (Fig 4; Table 1) The

molecular mass of p26 variants synthesized in bacteria

was unaffected by purification (Fig 4A,B), indicating

that the methods employed had little effect on protein

structure, an important observation in relation to

ana-lysis of chaperone function Except for WT p26, the

maximum monomer number per oligomer was higher

for p26 synthesized in COS-1 cells than in bacteria,

but the variation, although observed consistently, was

minor (Fig 4, Table 1), indicating little difference

between the proteins from either source Of equal

sig-nificance, the F112R substitution greatly reduced p26

oligomer size upon synthesis in COS-1 cells,

demon-strating that results obtained upon synthesis in bacteria

were not specific to the organism or the recombinant

construct

Amino acid substitutions in the p26 a-crystallin

domain reduced chaperone activity

Although all p26 variants conferred thermotolerance

on bacteria, WT p26 was the most effective (Fig 5A)

Thermotolerance levels induced by R110G, F112R

and Y116D were similar to (P > 0.05) and significantly

higher than those conferred by R114A (P < 0.05),

which provided the least protection Bacteria lacking

p26 failed to survive the 60 min heat shock

WT p26 at 1.6 lm, representing a chaperone to

sub-strate molar ratio of 0.4 : 1 if monomers are compared,

almost completely prevented dithiothreitol-induced

insulin aggregation at 25
C, and even at 0.1 lm

p26 aggregation was inhibited by more than 40%

(Fig 5B) At all concentrations, WT p26 prevented insulin aggregation the most and R114A the least, fol-lowed by F112R, Y116D and R110G, with the latter two not significantly different from one another Chap-eroning of insulin by p26 purified from Artemia [45] and E coli was very similar, whereas BSA and IgG at 1.6 lm had no effect on dithiothreitol-induced insulin aggregation (not shown)

At 600 nm, a chaperone to target (monomer to dimer) molar ratio of 4 : 1, WT p26 inhibited citrate synthase aggregation almost completely after 1 h at

43
C (Fig 5C) R110G, Y116D and F112R were progressively less effective in protecting citrate

Fig 3 p26 synthesis in COS-1 cells COS-1 cells transiently

trans-fected with the p26 cDNA-containing vector pcDNA4/TO/myc-His.A

were incubated with antibody to p26 followed by fluorescein

iso-thiocyanate-conjugated goat anti-rabbit IgG (green) Nuclei were

stained with propidium iodide (red) The scale bar represents

100 lm and both figures are the same magnification.

A

B

C

Fig 4 p26 oligomer formation Bacterially produced p26 either before (A) or after (B) purification, and p26 synthesized in

transfect-ed COS-1 cells (C), were centrifugtransfect-ed at 200 000 g for 12 h at 4
C

in 10–50% (w ⁄ v) continuous sucrose gradients The gradients were fractionated and 15-lL samples from each fraction were electro-phoresed in SDS polyacrylamide gels, blotted to nitrocellulose and reacted with antibody to p26 followed by horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG The top of each gradient is

to the right and fractions are numbered across the top The posi-tions of the molecular mass markers alpha-lactalbumin, 14.2 kDa; carbonic anhydrase, 29 kDa; BSA, 66 kDa; alcohol dehydrogenase,

150 kDa; apoferritin, 443 kDa; and thyroglobulin, 669 kDa, are indi-cated by labeled arrows.

synthase, with the latter two not significantly

differ-ent from one another However, the modified p26

variants exhibited appreciable chaperone activity, and

at 1200 nm the variants were almost as good as WT

p26 (Supplementary Fig 1) R114A provided the

least protection, but was still about 60% as potent

as WT p26 at 600 nm WT p26 also shielded citrate

synthase enzyme activity against heat-induced

inacti-vation better than mutated p26, and at 1200 nm the

activity remaining was essentially the same as in

unheated preparations (Fig 5D) R114A was the

least effective of all p26 variants in protecting

enzyme activity, although differences disappeared at

lower concentrations Of the remaining p26 mutants,

chaperone activity decreased from R110G to Y116D,

which were similar, and then to F112R Chaperone

activities of p26 from Artemia [45] and E coli with

citrate synthase were similar, whereas BSA and IgG

at 1200 nm neither prevented citrate synthase

aggre-gation nor preserved enzyme activity (data not

shown)

To summarize, as determined by thermotolerance

induction in E coli, dithiothreitol induced insulin

aggregation at 25
C, heat-induced citrate synthase

aggregation at 43
C, and maintenance of citrate

syn-thase enzyme activity at 43
C, WT p26 possessed the

greatest chaperone activity and R114A the least It is

noteworthy, however, that all p26 variants protected

bacteria and substrate proteins in vitro

Modification of p26 structure by amino acid substitutions

Measurement of intrinsic fluorescence demonstrated that the maximum emission peak for each p26 mutant was less than for WT p26 (Fig 6A) Three variants (R110G, F112R and Y116D) had very similar emission spectra, whereas R114A possessed the lowest fluores-cence and the emission was red-shifted The results indicate altered microenvironments for aromatic amino acid residues, such as tryptophan, of which two reside

in the p26 N-terminal region at positions 6 and 17, with the greatest change caused by the R114A modi-fication All mutated versions of p26 exhibited less 1-anilino-8-naphthalene-sulphonate (ANS)-binding than

WT p26, an indication of reduced surface hydropho-bicity (Fig 6B), with R114A at the lowest level The enhancement of surface hydrophobicity by increasing the temperature from 25
C to 43
C was reduced for the p26 variants in comparison to WT p26 (Fig 6B) Additionally, as shown by far-UV CD, the spectra for p26 a-crystallin domain mutants possessed wider, more negative, shoulders ranging from 208 to 230 nm, and with one exception, a related positive shoulder peaking near 194 nm (Fig 7A,B) R114A exhibited the greatest variation from WT in CD spectra, this reflecting decreased b-structure content and an increase in a-heli-cal constituents (Table 2)

WT p26 from E coli and Artemia gave comparable fluorescence intensities in ANS-binding experiments at

25
C and 43
C (Fig 6B) The far-UV CD spectra of p26 from both sources were characteristic of b-sheet enrichment, with a negative shoulder near 214 nm and

a positive shoulder near 194 nm (Fig 7A) The only indication of a difference was the slight red-shifted intrinsic fluorescence of bacterial WT p26; however, the fluorescence intensities for p26 from bacteria and Artemia were similar (Fig 6A) This structural resem-blance, and the functional analysis mentioned previ-ously, indicate data obtained by examining bacterially produced p26 are indicative of the protein synthesized

in Artemia

Localization of amino acid substitutions within p26

The p26 tetramer, modeled on the 3D crystal structure

of wheat Hsp16.9, consists of two dimers, with mono-mers A and B in dimer 1 and C and D in dimer 2 (Fig 8) The a-crystallin domain of each monomer is composed of nine b-strands (labeled b2–b10), with the b6 strand situated in a large loop, L5⁄ 7, located between b-strands 5 and 7 b-strand 10 inhabits

Table 1 Characteristics of p26 oligomers The molecular mass of

p26 oligomers produced in transformed Escherichia coli BL21PRO

and transfected COS-1 cells was determined by sucrose density

gradient centrifugation Monomer mass refers to the mass of p26

monomers and was calculated using a p26 molecular mass of

20.8 kDa, as determined by GENERUNNER (version 3.05, Hastings

Software, Inc.) with corrections for protein modifications Oligomer

mass range represents the smallest to largest oligomers observed,

and maximum monomer number refers to the number of subunits

in oligomers of maximum mass.

Expression

system

p26

mutant

Monomer mass (kDa)

Oligomer mass range (kDa)

Maximum monomer number

C-terminal extensions which extend to neighboring

monomers, and the remaining b-strands, with the

exception of strand 6, are arranged in two antiparallel

beta sheets within the a-crystallin domain The

inter-face between monomers of a dimer involves interaction

between strands b2 and b6 of neighboring monomers

The p26 modifications examined in this study are not

located in either of these strands and they are not

con-sidered further

As the basic p26 oligomer building block, dimers

interact to form tetramers, the next level of

struc-ture Modeling indicates that tetramer formation

depends upon contact of b-strand 10 from the

C-ter-minal extensions of monomers A and D with

b-strands 4 and 8 in the a-crystallin domain of

monomers C and B, respectively (Fig 8) A more pro-minent dimer–dimer interface occurs with b-strand 7

of monomer B interacting with loop L5⁄ 7 of mono-mer C, and b-strand 7 of monomono-mer C reacting with L5⁄ 7 of monomer B, regions of high similarity between p26 and Hsp16.9 (Fig 8) The p26 residues examined in this study are situated in b-strand 7, with mutations R110G and F112R directly in the dimer–dimer interface As a result of the spatial dis-position of monomers and their b-strand elements in the a-crystallin domain, the amino acid substitution F112R introduces two changes at the dimer–dimer interface Although Y116 and R114 are in b-strand

7, neither is shown by the model to reside directly

in the dimer–dimer interface Modification of these

Fig 5 Chaperone activity of p26 (A) Transformed Escherichia coli BL21PRO cells were incubated at 54
C for 1 h with samples removed periodically, diluted, and plated in duplicate on Luria–Bertani (LB) agar followed by incubation at 37
C for 16 h The log 10 values of colony-forming units (CFU) per ml were plotted against heat shock in min Bacteria containing the pPROTet.E233 vector lacking p26 cDNA did not survive the entire 60 min Standard errors ranged from 3.3 to 7.1% (B) Bacterially produced p26 purified to apparent homogeneity was incu-bated with insulin for 30 min in the presence of dithiothreitol, and solution turbidity was measured at 400 nm The p26 variants tested are indicated in the figure and they appear in the same order in each histogram group The standard error ranged from 4.2 to 5.8% (C) Purified, bacterially produced p26 at 600 n M was heated at 43
C for 1 h with 150 n M citrate synthase and the solution turbidity was measured at

360 nm The A360values were multiplied by 1000 for construction of the curves The standard error ranged from 4.2 to 7.0% (D) Citrate synthase at 150 n M was heated at 43
C for 1 h in either the absence or presence of p26, and then enzyme activity was determined p26 concentrations are indicated and the p26 variants are in the same order in each histogram group The standard error ranged from 3.3 to 10.0%.

residues had little effect on oligomerization, although

the R114A substitution reduced chaperoning to the

greatest extent

Discussion

p26, an abundantly expressed, polydisperse sHSP thought to protect encysted Artemia embryos against physiological stress, was investigated by site-directed mutagenesis of a-crystallin domain residues and molecular modeling of protein structure The p26 a-crystallin domain contains nine b-strands arranged predominantly as paired b-sheets and possesses resi-dues conserved in many other sHSPs, including those

in the sequence 110REFRRRY116, where substitu-tions were generated Examination of mutasubstitu-tions within the selected sequence indicated that b-strand 7 is involved in dimer–dimer interactions, leading to higher-order oligomer structure In addition, it was concluded that p26 structural characteristics would promote Artemia survival during encystment, diapause and stress exposure

Fig 7 Secondary structure of p26 Far-UV CD spectra were

obtained for purified p26 dissolved in 10 m M NaH 2 PO 4 , pH 7.1, to

0.2 mgÆmL)1 The absorption data are expressed as molar ellipticity

in degrees cm 2 Ædmol)1(m deg), with each spectrum the average of

three scans.

Fig 6 Tertiary structure perturbation of p26 (A) The intrinsic fluorescence of purified p26 diluted in 10 m M NaH 2 PO 4 , pH 7.1, to 0.06 mgÆmL)1was determined The excitation wavelength was 280 nm, with a 2-nm band pass, and fluorescence emission was detected from 310 to 400 nm The standard error ranged from 3.5 to 7.5% (B) Surface hydrophobicity of purified p26 at 0.06 mgÆmL)1in 10 m M

NaH 2 PO 4 , pH 7.1, was determined by oversaturation with 1-anilino-8-naphthalene-sulphonate (ANS) Fluorescence was measured at an exci-tation wavelength of 388 nm and band pass of 8 nm, with emission wavelength at 473 nm and band pass of 8 nm Measurements were made at either 25
C (grey) or 43
C (black) Fluorescence generated by buffer containing ANS, but no p26, was subtracted The standard error ranged from 6.7 to 10%.

Table 2 Secondary structure elements of p26 The secondary ele-ment percentages were calculated using the CDNN v2.1 deconvolu-tion program for each p26 variant generated by site-directed mutagenesis and for purified wild-type (WT) p26 from transformed Escherichia coli and Artemia embryos.

Structural elements

R110G (%) F112R (%) R114A (%) Y116D (%)

WT (E coli) (%)

WT (Artemia) (%)

b-antiparallel 18.0 18.2 12.9 20.5 21.6 23.0 b-parallel 10.2 10.2 9.7 10.1 10.3 10.0

Random coil 34.8 34.8 34.9 33.3 34.0 32.5

Oligomers for each exogenously produced p26

vari-ant are composed of similar numbers of monomers

when synthesized in mammalian and bacterial cells,

and oligomerization is unaffected by protein

purifica-tion, observations important for subsequent analysis of

the protein in in vitro assays Single-site mutations to

the p26 a-crystallin domain generally decreased

oligo-mer size in comparison to WT p26, with mutation

F112R reducing oligomerization most dramatically A

tetramer model of p26 was constructed on the basis of

the crystallin structure of wheat Hsp16.9 [14], a

mono-disperse sHSP used for modeling of human aA- and

aB-crystallins [47], in order to position residues within

the a-crystallin domain, better understand the

conse-quences of amino acid substitutions, and identify

pro-tein regions involved in oligomer assembly The four

modified a-crystallin domain residues are spatially

close to one another in the p26 model, with R110 and

F112 occupying central positions in the dimer–dimer

interface The R110G mutation had relatively limited

effect on oligomer size, indicating that p26, and by

extrapolation, other polydisperse sHSPs tolerate charge reduction at the dimer–dimer interface The F112R modification, on the other hand, placed two positively charged residues in the dimer–dimer interface and the maximum oligomer mass dropped, as indicated by sucrose density gradient centrifugation, from 669 kDa, for WT p26, to150 kDa for the F112R variant Replacement of Y116 with negatively charged aspar-tic acid had limited effects on oligomerization, prob-ably as a result of the residue’s location at the edge of the dimer–dimer interface The maximum oligomer size obtained with p26 R114A was  500 kDa, closer to the mass of the WT oligomer than the other variants This compares to oligomers of 2–4 MDa for mutation R116C of aA-crystallin and 0.7–2 MDa and larger for R120G aB-crystallin [48–53], both significant increases

in mass when compared with oligomers of WT a-crys-tallins Modification of R114 in p26 obviously has less effect on oligomerization than equivalent substitutions

in aA and aB-crystallin In agreement with the limited effect on oligomer mass and the proposed importance

Fig 8 Structural model of a p26 tetramer (A) Sequence alignment of amino acid residues 59–158 of Artemia p26 (AAB87967) and residues 45–151 of wheat, Triticum aestivum, Hsp16.9 (1GME) used to generate the 3D structural model of the p26 tetramer The proteins share 25.9% sequence identity and overall similarity of 69.4% in the regions compared The boxed residues labeled b2–b10 indicate the Hsp16.9 b-strand positions [14] and the corresponding residues in p26 Residues highlighted in yellow were modified in p26 by site-directed mutagen-esis Residue numbers are given on the right (B) A structural model of the p26 tetramer generated by comparison to wheat Hsp16.9 is rep-resented in a ribbon diagram Mutated residues Arg110, Phe112, Arg114 and Tyr116 are shown in gray in ball-and-stick and are labeled along the dimer–dimer interface by using the three-letter amino acid code in the color of the parent monomer Monomers A (green) and B (yellow) form dimer 1, while monomers C (red) and D (blue) form dimer 2 L5⁄ 7, the loop between b-strands 5 and 7 which contains b-strand 6; N term, amino terminus of a p26 monomer; C term, carboxy terminus of a p26 monomer; the b-strands 2–10 are labeled in monomer A.

of tetramer formation in higher-order structure, the

p26 model predicts R114 to be positioned slightly

out-side the dimer–dimer interface Interestingly, in

Chi-nese hamster Hsp27, mutation R148G had a limited

effect on chaperone activity and reduced oligomers to

dimers [54], contrasting the results obtained with p26

R114A Whether this indicates fundamental differences

between the two proteins awaits further study

WT p26, purified from transformed bacteria, almost

completely prevented heat-induced citrate synthase

aggregation and loss of enzyme activity at a molar

ratio of 4 : 1 (monomer to dimer), a result obtained

previously [17] and which was similar to the activity of

p26 from Artemia embryos (data not shown)

Chemic-ally induced insulin aggregation at 25
C was inhibited

at a monomer to monomer ratio of 0.4 : 1, the first

measurement of p26 chaperone activity in vitro at a

temperature near the optimum for Artemia growth

Although it is difficult to compare chaperone activity

across species owing to variation in experimental

tech-niques, effective chaperone to substrate molar ratios

determined by heating citrate synthase in the presence

of other representative sHSPs are 2 : 1 for

Bradyrhizo-bium japonicum sHSPs [55],  3 : 1 for Caenorhabditis

elegans Hsp16–2 [56], 15 : 1 for Mycobacterium

tuber-culosisHsp16.3, and 5 : 1 for human aB-crystallin [57]

The bovine a-crystallin to substrate ratio for

protec-tion against dithiothreitol induced denaturaprotec-tion ranges

from 2 : 1 for insulin and a-lactalbumin, 8 : 1 for BSA

and 10 : 1 for ovotransferrin, with the ratio rising as

the molecular mass of the substrate increases [58] For

human aB-crystallin, the ratio is 1 : 1 [48] The p26

chaperone activity therefore approximates that of

other sHSPs and this, in concert with its abundance,

provides a large capacity for storage of partially

dena-tured proteins in oviparous Artemia embryos during

diapause and quiescence Upon return of embryos to

permissive conditions, proteins would be released from

p26 and renatured, permitting rapid resumption of

metabolism, cell growth and development, an

advant-age to the organism under most circumstances

In contrast to a marginal impact on oligomerization,

substitution R114A had the greatest detrimental effect

on p26 chaperone activity in all assays R114 is

prob-ably buried within the a-crystallin domain, stabilized

by a salt bridge with another charged residue(s) [59]

The R114A substitution would destroy ionic linkages

and expose negatively charged residues within

mono-mer interiors, with ensuing structural changes and

reduced chaperone activity In agreement with this

idea, modified intrinsic fluorescence spectra and

sur-face hydrophobicity – the latter an effector of sHSP

chaperone activity [60] – indicate that p26 structural

changes are greater for R114A than for other muta-tions Additionally, far-UV CD measurements showing decreased b-structure were most prominent for R114A, with similar observations reported for R120G in aB-crystallin [48–50] and R116C aA-crystallin at 37
C but not 25
C [51] Mutation R114A had the least effect on p26 oligomerization but the greatest conse-quence for function, demonstrating that chaperoning is independent of oligomerization

All a-crystallin domain mutants, including R114A, retain significant amounts of chaperone activity In comparison, loss of chaperone activity reported upon introduction of substitution R116C into aA-crystallin ranges from 40% to almost 100% [49,51,52] The aB-crystallin mutation R120G promotes protein aggrega-tion in in vitro turbidimetric assays, reduces in vitro chaperone activity [48–50], and decreases thermotoler-ance induction by 70% while promoting inclusion body formation [61], the latter not being observed for p26 R114A p26 chaperone activity appears to be more resistant to modification of this conserved a-crystallin domain arginine, suggesting that the residue is less crit-ical than in mammalian a-crystallins where modifica-tion leads to disease [62–64] The ramificamodifica-tions of these observations for p26 are worthy of note For example, a-crystallins function in the mammalian lens for a life-time, indicating, by comparison, that p26 is sufficiently stable to protect Artemia for long periods of time, as required in encysted embryos

p26 oligomers synthesized in mammalian and bacter-ial cells are similar in size to one another and to Arte-mia p26, indicating that characteristics derived by studying bacterially produced p26 are reflective of the protein from Artemia Moreover, p26 localization in transfected cells is interesting because the protein migrates into Artemia nuclei during diapause and stress [65] Other sHSPs, such as Hsp20, aB-crystallin and Hsp27, access nuclei where they may be associated with speckles and nucleoli [66,67] The R120G muta-tion disrupts aB-crystallin speckle localizamuta-tion, with lit-tle of the modified protein entering nuclei [67], and there is a tendency for R120G aB-crystallin to form inclusion bodies in the cytosol [68], but this was not observed with R114A p26 Human R116C aA-crystal-lin occurs mainly in the cytoplasm of epithelial cells [53] How p26 enters nuclei is unknown, as is true for most, but not all, sHSPs [69] Oligomers of p26 R114A enter all COS-1 nuclei in which the protein is expressed and they are equivalent in mass to WT oligomers, which, in contrast to Hsp27 and a-crystallin [67], reside only in the cytoplasm of unstressed cells How-ever, the much smaller F112R oligomers exhibit reduced translocation efficiency and they are not found

in the nuclei of all transfected COS-1 cells synthesizing

this p26 variant; these results are in agreement with

earlier work showing that p26 oligomers, reduced in

size by C-terminal truncation, remain in the cytoplasm

[17] p26 nuclear migration is apparently not

accom-plished by simple diffusion across the membrane upon

oligomer size reduction, and why a-crystallin domain

modifications promote translocation remains

uncer-tain

To summarize, analysis of individual amino acid

substitutions, coupled with molecular modeling of

protein structure, indicate that b-strand 7 of the

a-crystallin domain is an integral component of the

p26 dimer–dimer interface in polydisperse sHSPs p26

chaperoning is not dependent upon oligomerization,

and chaperone activity effectively tolerates structural

perturbation, this potentially contributing to stress

resistance in Artemia embryos The ability of p26 to

prevent aggregation and loss of enzyme activity, in

concert with its abundance, indicate a large protective

capacity during oviparous development Proteins

shiel-ded by p26 would be readily available upon

termin-ation of diapause to initiate development, conferring a

marked advantage on encysted Artemia embryos

Experimental procedures

Construction of p26 cDNAs

p26 amino acid substitutions were generated by

site-direc-ted mutagenesis by using the QuikChangetm

Site-directed Mutagenesis kit (Stratagene, La Jolla, CA, USA), using

pRSET.C-p26–3-6-3 as template [46] and designated

prim-ers (Table 3) PCR mixtures were incubated for 30 s at

95
C prior to 12 cycles of 30 s at 95
C, 1 min at 55
C

and 8 min at 68
C DNA products were digested with

DpnI at 37
C for 1 h and used to transform E coli

XL1-blue supercompetent cells (Stratagene) p26 cDNA inserts were recovered from pRSET.C plasmids by digestion with BamHI and XhoI, electrophoresis in agarose and purifica-tion with the GFXtm

PCR DNA and Gel Band purification kit (Amersham Biosciences, Piscataway, NJ, USA) before cloning in the eukaryotic expression vector, pcDNA4⁄ TO ⁄ myc-His.A (Invitrogen, San Diego, CA, USA) and transfor-mation of E coli DH5a (Invitrogen, Carlsbad, CA, USA) The p26 cDNAs were also cloned into pPROTet.E233 (Clontech Laboratories, Inc., Palo Alto, CA, USA), a His-tag-containing prokaryotic expression vector, using the BamHI and XbaI restriction sites Polypeptides encoded by pPROTet.E233 were longer than those encoded by pcDNA4⁄ TO ⁄ myc-His.A because the former employed a start codon upstream of the His-tag, while the latter initi-ated translation from the p26 start codon All p26 cDNA inserts were sequenced (DNA Sequencing Facility, Center for Applied Genomics, Hospital for Sick Children, Toronto, ON, Canada)

Bacterial synthesis and purification of p26 p26 was synthesized in transformed E coli BL21PRO (Clontech Laboratories, Inc., Mississauga, ON, Canada) induced with 100 ngÆmL)1anhydrotetracycline (aTc) (Clon-tech Laboratories) p26 was recovered from bacterial extracts using BD TALON resin (BD Biosciences Clontech, Mississauga, ON, Canada) and concentrated in Centrip-repYM-10 centrifugal filter devices (Amicon Bioseparations, Billerica, MA, USA) [17] Protein samples were electro-phoresed in 12.5% SDS polyacrylamide gels and either stained with Coomassie Brilliant Blue R-250 (Sigma) or blotted onto nitrocellulose (Bio-Rad, Hercules, CA, USA) for reaction with anti-p26 immunoglobulin [46] and Omni-probe (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), a monoclonal antibody recognizing the (His)6 tag Blots were then incubated with either horseradish peroxi-dase (HRP)-conjugated goat anti-rabbit IgG or HRP-conju-gated goat anti-mouse IgG (Jackson ImmunoResearch, Mississauga, ON, Canada) and immunoconjugates were detected with Western Lightning Enhanced Chemilumines-cence (ECL) Reagent Plus (PerkinElmer Life Sciences, Boston, MA, USA)

p26 synthesis and localization in transiently transfected COS-1 cells

Cloned p26 cDNA in SuperFecttm

(Qiagen, Mississauga,

ON, Canada) was employed to transiently transfect COS-1 cells [17] The cells were trypsinized 24 h after transfection for preparation of protein extract, centrifuged at 1500 g for

5 min, washed with 1 mL of phosphate-buffered saline (NaCl⁄ Pi) (140 mm NaCl, 2.7 mm KCl, 8.0 mm Na2HPO4, 1.5 mm KH2PO4, pH 7.4), and incubated on ice for 20 min

in lysis buffer consisting of 50 mm Tris⁄ HCl, pH 7.8,

Table 3 Primers for site-directed mutagenesis of p26 Single

amino acid substitutions were generated within the p26 a-crystallin

domain by site-directed mutagenesis using primers presented as

sense and antisense, respectively, for each mutation.

p26

mutation Primer

R110G 5¢-GGACACGTACAAGGAGAATTTCGACGACG-3¢

5¢-CGTCGTCGAAATTCTCCTTGTACGTGTCC-3¢

F112R 5¢-CACGTACAAAGAGAACGTCGACGACG-3¢

5¢-CGTCGTCGACGTTCTCTTTGTACGTG-3¢

R114A 5¢-GAGAATTTCGAGCACGATACAGACTCCC-3¢

5¢-GGGAGTCTGTATCGTGCTCGAAATTCTC-3¢

Y116D 5¢-CGACGACGAGACAGACTCCCAGAACATGTC-3¢

5¢-GACATGTTCTGGGAGTCTGTCTCGTCGTCG-3¢