Characterization of novel sequence motifs within N- and
C-terminal extensions of p26, a small heat shock protein
from Artemia franciscana
Yu Sun and Thomas H. MacRae
Department of Biology, Dalhousie University, Halifax, Canada
The small heat shock proteins (sHSPs), characterized
by a conserved a-crystallin domain of approximately
90 residues and the ability to reversibly oligomerize,
constitute a distinctive molecular chaperone family
composed of monomers ranging in mass from 12 to
43 kDa [1–4]. The a-crystallin domain [5–7] is bor-
dered on one end by a variable N-terminal extension
involved in substrate interaction, oligomerization and
subunit dynamics [8–14], and on the other by a poorly
conserved, charged, highly flexible, C-terminal exten-
sion active in oligomer formation, promotion of solu-
bility and chaperoning [11,14–16]. Functions assigned
to N- and C-terminal extensions vary, reflecting envi-
ronmental demands on organisms in addition to the
types of molecular tasks that different sHSPs must per-
form. Generally speaking, sHSPs constitute the first
line of defense in stressed cells, binding denatured pro-
teins in a process requiring oligomer disassembly and
Keywords
molecular chaperone; p26 structure ⁄
function; small heat shock protein; stress
resistance; Artemia franciscana
Correspondence
T. H. MacRae, Department of Biology,
Dalhousie University, Halifax, N.S. B3H 4J1,
Canada
Fax: +1 902 494 3736
Tel: +1 902 494 6525
E-mail: tmacrae@dal.ca
(Received 9 June 2005, revised 11 August
2005, accepted 16 August 2005)
doi:10.1111/j.1742-4658.2005.04920.x
The small heat shock proteins function as molecular chaperones, an activ-
ity often requiring reversible oligomerization and which protects against
irreversible protein denaturation. An abundantly produced small heat
shock protein termed p26 is thought to contribute to the remarkable stress
resistance exhibited by encysted embryos of the crustacean, Artemia francis-
cana. Three novel sequence motifs termed G, R and TS were individually
deleted from p26 by site-directed mutagenesis. G encompasses residues
G8–G29, a glycine-enriched region, and R includes residues R36–R45, an
arginine-enhanced sequence, both in the amino terminus. TS, composed of
residues T169–T186, resides in the carboxy-extension and is augmented in
threonine and serine. Deletion of R had more influence than removal of G
on p26 oligomerization and chaperoning, the latter determined by thermo-
tolerance induction in Escherichia coli, protection of insulin and citrate syn-
thase from dithiothreitol- and heat-induced aggregation, respectively, and
preservation of citrate synthase activity upon heating. Oligomerization of
the TS and R variants was similar, but the TS deletion was slightly more
effective than R as a chaperone. The extent of p26 structural perturbation
introduced by internal deletions, including modification of intrinsic fluores-
cence, 1-anilino-8-naphthalene-sulphonate binding and secondary structure,
paralleled reductions in oligomerization and chaperoning. Three-dimen-
sional modeling of p26 based on wheat Hsp16.9 crystal structure indicated
many similarities between the two proteins, including peptide loops associ-
ated with secondary structure elements. Loop 1 of p26 was deleted in the
G variant with minimal effect on oligomerization and chaperoning,
whereas loop 3, containing b-strand 6 was smaller than the corresponding
loop in Hsp16.9, which may influence p26 function.
Abbreviations
ANS, 1-anilino-8-naphthalene-sulphonate; aTc, anhydrotetracycline; CD, circular dichroism; sHSP, small heat shock protein; WT, wild type.
5230 FEBS Journal 272 (2005) 5230–5243 ª2005 FEBS
where substrates are held in a folding-competent state
[7,10,14,17–21]. Subunit dynamics and chaperone
activity are closely related in sHSPs from yeast, plants
and bacteria, but less so in human aA-crystallin [22].
Substrate release from sHSPs and subsequent refolding
depend on ATP-requiring chaperones such as HSP70
[18,23,24]. The sHSPs confer stress tolerance on living
organisms [25], modulate apoptosis [26–28] and inter-
act with cell components such as membranes [29,30],
the cytoskeleton [31–34], and intranuclear elements
[35,36]. When perturbed by mutation or post-transla-
tional modification the sHSPs contribute to cataract
and desmin-related myopathy, among other diseases
[37–40].
The extremophile crustacean, Artemia franciscana,
populates aquatic environments of high salinity, where
they are subject to several stressors [41,42]. One adaptive
strategy exhibited by Artemia in response to its habitat
is to undertake different developmental pathways. Ovo-
viviparous development yields swimming embryos ready
to take advantage of favorable growth conditions. In
contrast, during oviparous development, embryos arrest
as gastrulae, encyst and enter diapause [42,43], a condi-
tion characterized by profound reduction in metabolic
activity and extreme stress resistance including anoxia
tolerance for several years [44–46]. Diapause-destined
embryos synthesize large amounts of a developmentally
regulated but stress-indifferent sHSP termed p26, which
peaks in encysted embryos and remains at high levels
until larvae emerge from cysts [42,47,48]. Composed of
20.8 kDa monomers, p26 forms oligomers as large as 34
subunits with a molecular mass approximating 700 kDa
[11,49]. p26 is thought to contribute to stress resistance
in encysted Artemia embryos by acting as a molecular
chaperone. In support of this proposal, the protein pro-
tects citrate synthase against heat-induced aggregation
and inactivation of its enzymatic activity and shields
insulin from dithiothreitol-induced denaturation in vitro
[11]. p26 also guards tubulin against heat-induced dena-
turation [32] and confers thermotolerance on trans-
formed bacteria [11,25]. That p26 functions in more
than one major cell compartment is indicated by reversi-
ble cytoplasmic to nuclear translocation in Artemia
embryos during development, upon exposure to stress
and by pH modulation in vitro [50–53].
As shown in this paper, the p26 a-crystallin domain
consists predominantly of b-strands arranged as a
b-sheet sandwich. The N-terminal extension is 60 resi-
dues in length and the C-terminal is 40, both with lim-
ited similarity to corresponding regions in other sHSPs
(Fig. 1). The extensions may determine distinct sHSP
properties and in this context the p26 N-terminus
possesses a novel peptide, 8-GGFGGMTDPWSDP
FGFGGFGGG-29 containing 10 glycines, as com-
pared to three or four glycines in similar locations of
other sHSPs. Additionally, six arginines occur in the
sequence 36-RPFRRRMMRR-45. The p26 C-terminal
extension encompasses 12 serine ⁄threonine residues
in the peptide 169-TTGTTTGSTASSTPARTT-186.
These unusual regions were deleted by site-directed
mutagenesis in order to examine their contribution to
p26 structure and function and ascertain their role in
Artemia stress resistance.
Results
Mutagenesis and purification of p26 produced
in E. coli
Alignment of sHSPs from several species, a selection
of which is shown (Fig. 1), demonstrated two novel
sequence motifs in the p26 N-terminal extension and
another in the C-terminus. The deletion of these
motifs, termed G (multiple glycine), R (multiple argin-
ine), and TS (multiple threonine ⁄serine), was confirmed
by sequencing and the modified cDNAs were cloned in
expression vectors. In addition to the p26 sequence,
each bacterial expression vector contained DNA from
the original p26-3-6-3 template clone that encoded a
short N-terminal peptide (PRAAGIRHELVLK) and
the His-tag. Bands corresponding in size to p26 were
just visible in Coomassie blue stained SDS ⁄polyacryl-
amide gels containing protein extracts from anhydro-
tetracycline (aTc)-induced bacteria transformed with the
G and R constructs, but not the TS construct, however,
all extracts contained polypeptides that reacted with
anti-p26 antibody (Fig. 2A,B). Upon purification, single
bands of the expected size were observed in stained gels
and these polypeptides were recognized on western blots
by antibody to p26 (Fig. 2C,D).
p26 synthesis and localization in mammalian
cells
In order to examine oligomerization and cell localiza-
tion, both interesting in the context of Artemia embryo
development and sHSP function, mammalian cells were
transfected with p26 cDNA. Immunoprobing of western
blots revealed p26 in protein extracts from transiently
transfected COS-1 cells, with the yield of TS somewhat
lower than for the other variants (Fig. 3A,B). The trans-
fected cells stained strongly with anti-21 antibody
(Fig. 3C). Wild-type (WT) p26 localized exclusively to
the cytoplasm of transfected cells, whereas all modified
versions of p26 occurred in both the cytosol and nuclei.
The p26 variants G and TS were found in only some
Y. Sun and T. H MacRae Small heat shock protein sequence motifs
FEBS Journal 272 (2005) 5230–5243 ª2005 FEBS 5231
nuclei whereas p26 lacking the R motif was in the nuclei
of all transfected cells (Fig. 3C).
Oligomerization of p26
As revealed by sucrose density gradient centrifugation,
oligomers with the largest mass and greatest number of
monomers were produced in bacteria expressing WT
p26 (Fig. 4A,B; Table 1). Oligomers formed in bacteria
with G variants of p26 were somewhat smaller than WT
p26, followed by R and TS which were very similar.
Purification of p26 from bacterial extracts had no effect
on oligomer mass. Except for WT p26, the maximum
monomer number was greater for oligomers assembled
in mammalian cells than bacteria (Fig. 4; Table 1).
Additionally, in contrast to the situation with bacteria,
the maximum monomer number for oligomers produced
by G and WT p26 in mammalian cells was the same.
Maximum monomer numbers for oligomers of R and
TS p26 produced in mammalian cells were identical and
somewhat smaller than for wild type.
Fig. 1. Multiple sequence alignment of
representative sHSPs. The amino acid
sequences of selected sHSPs were ana-
lyzed by CLUSTAL W (1.82). Ap26, A. francis-
cana p26, AAB87967; HCRYAA, Homo
sapiens aA-crystallin, P04289; HCRYAB,
H. sapiens aB-crystallin, P02511; HHSP27,
H. sapiens Hsp27, NP_001532; MHSP25,
Mus musculus Hsp25, JN0679; DHSP26,
Drosophila melanogaster Hsp26, P02517;
CHSP16-1, Caenorhabditis elegans Hsp16–
1, P34696; YHSP26, Saccharomyces cere-
visiae Hsp26, NP_009628. sHSP domains
are indicated above the alignment and
regions corresponding to the deleted resi-
dues are boxed. Residue number is indica-
ted on the right. No residue (–), identical
residues (*), conserved substitutions (:)
and semiconserved substitutions (.) are
indicated.
A
MGRTSWTV MG RTS WT
C
BD
Fig. 2. Purification of bacterially produced p26. Cell-free extracts
from transformed E. coli BL21PRO induced with aTc were electro-
phoresed in SDS polyacrylamide gels and either stained with Coo-
massie blue (A) or blotted to nitrocellulose and reacted with
antibody to p26 (B). Proteins purified by affinity chromatography
were electrophoresed in SDS polyacrylamide gels and either
stained with Coomassie blue (C), or blotted to nitrocellulose and
reacted with antibody to p26 (D). All lanes received 10 lL of sam-
ple. Lane V, vector lacking p26 cDNA; lane M, molecular mass
markers of 97, 66, 45, 31, 21 and 14 kDa; other lanes received
wild-type or modified p26 as indicated. Arrow, p26.
Small heat shock protein sequence motifs Y. Sun and T. H MacRae
5232 FEBS Journal 272 (2005) 5230–5243 ª2005 FEBS
p26 confers thermotolerance on transformed
bacteria
E. coli expressing WT p26 were more resistant to heat
stress than bacteria expressing modified versions of the
protein, and all transformed bacteria were significantly
more thermotolerant than those containing only the
pPROTet.E233 vector which failed to survive the
60 min heat shock (Fig. 5A). Thermotolerance levels
induced by expression of G and TS were similar to
each other (P> 0.05) and significantly higher than the
thermotolerance conferred by variant R (P<0.05).
However, because the amount of TS p26 in trans-
formed bacteria was low (Fig. 2A,B), this protein is
superior to the other modified p26 versions in confer-
ring thermotolerance.
p26 exhibits chaperone activity in vitro
Purified WT p26 effectively prevented dithiothreitol-
induced denaturation of insulin (Fig. 5B). For example,
A
B
C
MGRTSWTV
Fig. 3. p26 synthesis and localization in transfected COS-1 cells.
Equal volumes of cell-free extracts were obtained from COS-1 cells
transiently transfected with the vector pcDNA ⁄4⁄TO ⁄myc-His.A
containing p26 cDNA inserts, electrophoresed in SDS ⁄polyacryl-
amide gels and either stained with Coomassie blue (A) or blotted to
nitrocellulose and stained with antibody to p26 (B). Lane V, vector
lacking p26 cDNA; lane M, molecular mass markers of 97, 66, 45,
31, 21 and 14 kDa; other lanes received wild-type or modified p26
as indicated. (C) Transiently transfected COS-1 cells were incubated
with antibody to p26 followed by FITC-conjugated goat antirabbit
IgG antibody (green). Nuclei were stained with propidium iodide
(red). p26 variants are indicated in the figure. The bar represents
100 lm and all 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 in COS-1 cell extracts
(C) were centrifuged at 200 000 gfor 12 h at 4 C in 10–50%
continuous sucrose gradients. Samples from gradient fractions
were electrophoresed in SDS ⁄polyacrylamide gels, blotted to
nitrocellulose and reacted with antibody to p26 followed by HRP-
conjugated goat antirabbit IgG. The top of each gradient is to the
right and fractions are numbered across the top. The molecular
mass markers, a-lactalbumin, 14.2 kDa; carbonic anhydrase,
29 kDa; bovine serum albumin, 66 kDa; alcohol dehydrogenase,
150 kDa; apoferritin, 443 kDa; and thyroglobulin, 669 kDa are indi-
cated by numbered arrows.
Y. Sun and T. H MacRae Small heat shock protein sequence motifs
FEBS Journal 272 (2005) 5230–5243 ª2005 FEBS 5233
WT p26 inhibited insulin aggregation by 39% after
30 min at 0.1 lm, and almost completely at 1.6 lm,a
0.4 : 1 monomer to monomer molar ratio of chaperone
to substrate. Mutant R was the least effective, whereas
G and TS provided an intermediate level of protection,
with G moderately more effective at higher concentra-
tions. Bovine serum albumin (BSA) and IgG at 1.6 lm
failed to inhibit insulin aggregation (not shown). Puri-
fied, bacterially produced WT p26 also exhibited the
greatest ability to shield citrate synthase from heat-
induced denaturation while mutant R had the least,
although all mutants provided protection (Fig. 5C). At
600 nmWT p26, representing a chaperone to target
molar ratio of 4 : 1 (p26 monomer to citrate synthase
dimer), citrate synthase aggregation was inhibited
almost completely for 1 h at 43 C (Fig. 5C), a result
similar to that obtained with p26 purified from
Artemia (not shown). At 37.5 nm, where the molar
ratio of WT p26 to citrate synthase was 1 : 4, heat-
induced turbidity was reduced by 46% after 1 h at
Table 1. Oligomerization of p26. The molecular mass of p26 oligo-
mers was determined by sucrose density gradient centrifugation.
Monomer mass refers to the molecular mass of p26 polypeptides.
Oligomer mass range represents the smallest to largest oligomers
observed while oligomer mass maximum refers to the mass of the
largest oligomer. Maximum monomer number refers to monomer
number in the largest oligomer.
p26 mutant
Monomer
mass (kDa)
Oligomer mass
Maximum
monomer
number
Range
(kDa)
Maximum
(kDa)
E. coli
G 23.7 14.2–443 443 19
R 24.1 14.2–300 300 12
TS 23.8 14.2–300 300 13
WT 25.5 29.0–669 669 26
COS-1 cells
G 18.7 14.2–443 443 24
R 19.4 14.2–300 300 16
TS 19.1 14.2–300 300 16
WT 20.8 14.2–500 500 24
015
8
0
0.01
0.02
0.03
0.04
0.05
0.06
AB
CD
7
6
5
4
3
2
1
0
70
60
50
40
30
20
10
0
30 45
Time (min)
E. coli thermotolerance Insulin aggregation
Citrate synthase inactivation
CS activity (umole citrate/mg protein/min
Citrate synthase aggregation
Log10 of CFU/ml
A400
A360
Time (min) p26 (nm)
p26 (µM)
60
No p26
No p26
WT
G
TS
R
WT
G
TS
R
G
TS
WT
No p26
R
G
TS
WT
No p26
R
0 4 8 12 16 20 24 28 32 36 40 44 48 52 56 60 1200
0.1
0.09
0.08
0.07
0.06
0.05
0.04
0.03
0.02
0.01
0
600 300 150 75 37.5
1.6 0.8 0.4 0.2 0.1
Fig. 5. Chaperone activity of p26. (A) Transformed E. coli was incubated at 54 C, diluted, plated in duplicate on LB agar and colonies were
counted after incubation overnight at 37 C. (B) Purified, bacterially produced p26 was incubated with insulin for 30 min in the presence of
dithiothreitol and solution turbidity was measured at 400 nm. The p26 variants are in the same order in each histogram group. (C) Purified,
bacterially produced p26 at 600 nMwas heated at 43 C for 1 h with 150 nMcitrate synthase, and solution turbidity was measured at
360 nm. The A
360
values were multiplied by 1000. (D) Citrate synthase at 150 nMwas heated at 43 C for 1 h in either the absence or the
presence of p26 and enzyme activity was determined. The p26 variants are in the same order in each histogram group. Results in all experi-
ments are averaged from three independent experiments.
Small heat shock protein sequence motifs Y. Sun and T. H MacRae
5234 FEBS Journal 272 (2005) 5230–5243 ª2005 FEBS