MacRae Department of Biology, Dalhousie University, Halifax, Nova Scotia, Canada Oviparously developing embryos of the brine shrimp, Artemia franciscana, synthesize abundant quantities o
Trang 1Functional analysis of a small heat shock/a-crystallin protein
Oligomerization and thermotolerance
Julie A Crack, Marc Mansour, Yu Sun and Thomas H MacRae
Department of Biology, Dalhousie University, Halifax, Nova Scotia, Canada
Oviparously developing embryos of the brine shrimp,
Artemia franciscana, synthesize abundant quantities of a
small heat shock/a-crystallin protein, termed p26 Wild-type
p26 functions as a molecular chaperone in vitro and is
thought to help encysted Artemia embryos survive severe
physiological stress encountered during diapause and
anoxia Full-length and truncated p26 cDNA derivatives
were generated by PCR amplification of p26-3-6-3, then
cloned in either pET21(+) or pRSETC and expressed in
Escherichia coliBL21(DE3) All constructs gave a
polypep-tide detectable on Western blots with either p26 specific
antibody, or with antibody to the His6epitope tag encoded
by pRSETC Full-length p26 in cell-free extracts of E coli
was about equal in mass to that found in Artemia embryos,
but p26 lacking N- and C-terminal residues remained either
as monomers or small multimers All p26 constructs conferred thermotolerance on transformed E coli, although not all formed oligomers, and cells expressing N-terminal truncated derivatives of p26 were more heat resistant than bacteria expressing p26 with C-terminal deletions The C-terminal extension of p26 is seemingly more important for thermotolerance than is the N-terminus, and p26 protects
E coliagainst heat shock when oligomer size and protein concentration are low The findings have important impli-cations for understanding the functional mechanisms of small heat shock/a-crystallin proteins
Keywords: small heat shock/a-crystallin protein; oligomeri-zation; thermotolerance; diapause; Artemia franciscana
Cells respond to stress by the enhanced synthesis of heat
shock or stress proteins, which are also developmentally
regulated under normal physiological conditions Stress
proteins are divided into several families on the basis of size
and amino-acid sequence [1–5] Moreover, they function as
molecular chaperones, facilitating proper folding, transport
and multimerization of nascent proteins, as well as
preventing the irreversible aggregation of denaturing
proteins The small heat shock/a-crystallin proteins
consti-tute a structurally divergent, ubiquitous group within the
chaperone superfamily, ranging in molecular mass from 12
to 43 kDa [6] A conserved region, termed the a-crystallin
domain, distinguishes all small heat shock/a-crystallin
proteins, and a two or three domain structure is proposed
for these proteins [7,8] The a-crystallin domain, located
toward the C-terminus of the protein monomer, consists of
80–100 amino-acid residues and is important for oligomer
formation and chaperoning [9–13] Flexible C-terminal
extensions of small heat shock/a-crystallin proteins,
enriched in polar and charged amino-acid residues, vary
in length and sequence [8,14,15] Loss or modification of the
C-terminal extension has the potential to perturb function
and reduce solubility of these proteins and their complexes with target proteins [15–19] The N-terminus, which may be partly buried within the mature protein, promotes oligomer formation, subunit exchange, and capture of unfolding proteins [12,18,20–26]
Small heat shock/a-crystallin proteins confer thermotol-erance upon cells [27–33], protect against apoptotic death [34,35] and have chaperone activity in vitro, wherein the aggregation of client proteins is prevented [36–38] Chap-eroning is thought to depend upon formation of oligomers that reach 800 kDa in mass and possess quaternary structure modifiable by environmental parameters [8,18,20,22,39,40] Oligomers exhibit dynamic equilibrium with constituent subunits, which can affect chaperoning but
is not in itself sufficient to ensure chaperone activity [25,41,42] A small heat shock/a-crystallin protein from Methanococcus jannaschii, termed Mj hspl6.5, has been crystallized, revealing highly ordered oligomers of 24 subunits with a hollow center [9] Cryoelectron microscopy
of small heat shock/a-crystallin proteins from several sources has shown, however, that oligomer structure ranges from well defined to variable, leading to the idea that structural plasticity elicits low specificity and permits binding of different target proteins [10,24,42] Several molecules of denaturing proteins, present in an unstable molten globule state, interact with a single oligomer when chaperoning occurs The proteins are protected from irreversible aggregation under stress, their activity may be preserved, and they either refold spontaneously or with the assistance of other chaperones upon release [38,43–46] Embryos of the brine shrimp, Artemia franciscana, develop ovoviviparously, leading to release of swimming
Correspondence to T H MacRae, Department of Biology, Dalhousie
University, Halifax, Nova Scotia, B3H 4J1, Canada.
Fax: + 902 494 3736, Tel.: + 902 494 6525,
E-mail: tmacrae@is.dal.ca
Abbreviations: Gp4G, guanosine 5¢-tetraphospho-5¢-guanosine;
IPTG, isopropyl thio-b- D -galactoside; HRP, horseradish peroxidase.
(Received 12 October 2001, revised 3 December 2001, accepted 5
December 2001)
Trang 2nauplii from females Alternatively, oviparous development
occurs, embryos encyst as gastrulae composed of about
4000 cells and are discharged from females enclosed in a
shell permeable only to volatile molecules [47–49]
Subse-quent to release, encysted embryos enter a dormant state
known as diapause [50], wherein metabolic activity is
difficult to detect [51,52] Diapause continues, even under
favourable growth conditions, until the appropriate
activa-tion signal The embryos are very tolerant of physical
and chemical insults such as exposure to organic solvents,
a-irradiation, temperature extremes and desiccation, the
latter probably a cue that terminates diapause [53] As one
remarkable example of stress resistance, fully hydrated cysts
survive several years at physiological temperature in the
complete absence of oxygen [48,51,52,54], an unusual degree
of tolerance for any animal These observations contradict
the general belief that under ordinary hydration and
temperature, cell maintenance entails a constant and
substantial free energy flow [51,52] Anoxic cysts may
acquire sufficient energy to survive by utilization of
guanosine 5¢-tetraphospho-5¢-guanosine (Gp4G), an
abun-dant nucleotide at this developmental stage [55]
Previous work has revealed p26, a small heat shock/
a-crystallin protein found only in Artemia undergoing
oviparous development [47–49,56–59] p26 has chaperone
activity in vitro and imparts thermotolerance to
trans-formed bacteria [49,57] Although chaperoning and
ther-motolerance are not necessarily equivalent activities, the
results indicate that p26 prevents irreversible denaturation
of proteins in diapause/encysted Artemia embryos This
permits spontaneous and/or assisted refolding of proteins,
the former allowing rapid resumption of development
under limiting energy reserves, perhaps to exploit the
transient occurrence of favourable environmental
condi-tions encountered by Artemia In the current study,
functions of p26 N- and C-terminal regions were explored
through deletion mutagenesis Specifically, protein
solubil-ity, oligomerization, and the thermotolerance of
trans-formed bacteria were examined Such information may
illuminate the mechanism by which p26 protects Artemia
from physiological stress experienced during diapause and
anoxia, thereby enhancing our appreciation of small heat
shock/a-crystallin proteins
E X P E R I M E N T A L P R O C E D U R E S
Cloning of full-length and truncated p26 cDNAs Full-length and truncated p26 cDNAs were generated by PCR using p26-3-6-3 cDNA (GenBank accession no AF031367) [58] as template, and custom primers possessing BamHI and XhoI restriction sites on the sense and antisense oligoneucleotides, respectively (CyberSyn, Inc., Lenni, PA, USA) (Table 1) Fifty microliter PCR mixtures included
38 ng of template DNA, 0.01 lgmL)1each of sense and antisense primers, 5 lL of PCR buffer (ID Laboratories, London, Ontario, Canada), 1 mM dNTP, 50 mM Mg2+,
40 lL of H2O and 0.01 U of proof-reading Taq polymerase (ID Laboratories) Reaction mixtures, covered with mineral oil, were incubated for 2 min at 94°C prior to five cycles of
30 s at 94°C, 45 s at 40 °C, 30 s at 72 °C, then 30 cycles of
30 s at 94°C, 30 s at 55 °C and 30 s at 72 °C, followed by
10 min at 72°C PCR products were analyzed in 1.0% agarose gels in TAE buffer (0.04MTris HC1, 0.02Mglacial acetic acid, 0.001M EDTA) using 100-bp standards (Amersham-Pharmacia Biotech or BioRad) DNA frag-ments of appropriate length were ligated into the T/A vector, pCRII (Invitrogen, San Diego, CA, USA), using T4 DNA ligase overnight at 14°C, and E coli DH5a made competent by the calcium chloride procedure were trans-formed with the recombinant DNA [60] Putative p26 cDNA containing clones were selected by blue/white screening using the LacZ system, propagated in LB broth, and examined by restriction analysis for plasmids incorpo-rating inserts of the appropriate size, which were subcloned into the prokaryotic expression vector pET21(+) (Nov-agen, Inc., Madison, WI, USA) Briefly, pCRII constructs and pET21(+) were digested with BamHI and XhoI before electrophoresis in 1% agarose gels Linearized pET21(+) and p26 cDNAs were excised and purified with the GFXTM PCR DNA and Gel Band Purification Kit (Amersham-Pharmacia Biotech) Each p26 cDNA was ligated into pET21(+) using T4 DNA ligase, and competent E coli DH5a were transformed with the constructs [60] Bacteria containing p26 cDNA of the correct length were identified
by restriction digestion of constituent plasmids followed by electrophoresis in 1% agarose gels The p26 cDNAs were
Table 1 Full-length and truncated p26 cDNAs generated by PCR The primers are listed in the 5¢ fi 3¢ direction and restriction sites are underlined ATG, start codon; TTA, termination codon; bp, base pair.
p26 cDNAs
Amino acid
residues deleted Designations Primer sequences
Length (bp/amino acids) p26-full None (p26-1Bam-s) GCGCGGATCCACCATGGCACTTAACCCATG 576/192
(p26-192Xho-as) CGCGCCTCGAGTTAAGCTGCACCTCCTGATCT p26-ND36 1–36 (p26-36Bam-s) GCGCGGATCCACCATGCCCTTCCGGAGAAGA 468/156
(p26-192Xho-as) CGCGCCTCGAGTTAAGCTGCACCTCCTGATCT p26-ND60 1–60 (p26-60Bam-s) GCGCGGATCCACCATGTCCTTGAGGGACACA 396/132
(p26-192Xho-as) CGCGCCTCGAGTTAAGCTGCACCTCCTGATCT p26-CD40 153–192 (p26-1Bam-s) GCGCGGATCCACCATGGCACTTAACCCATG 459/153
(p26-153Xho-as) CGCGCCTCGAGTTAACGTTCTGTTGGTGAGCT p26-CD10 183–192 (p26-1Bam-s) GCGCGGATCCACCATGGCACTTAACCCATG 546/182
(P26-182 Xho-as) CGCGCCTCGAGTTATGGAGTTGAACTAGCTGT p26-alpha 1–60 and (p26-60Bam-s) GCGCGGATCCACCATGTCCTTGAGGGACACA 297/93
153–192 (p26-153Xho-as) CGCGCCTCGAGTTAACGTTCTGTTGGTGAGCT
Trang 3sequenced in both directions, either at the Hospital for Sick
Children (Toronto, Ontario, Canada) or the National
Research Council Laboratory (Halifax, Nova Scotia,
Canada) Sequence similarity amongst the p26 constructs
was analyzed byCLUSTAL W, with files viewed and edited
in Microsoft WORD Selected p26 cDNA fragments
recovered from pET21(+) constructs were also cloned into
pRSETC (Invitrogen) Full-length p26 cDNA cloned in
pRSETC, termed pRSET-p26-3-6-3, was prepared
previously [49]
Expression of p26 inE coli BL21(DE3)
Two ml of LB medium supplemented with 50 lgmL)1
ampicillin was inoculated with a single colony consisting of
transformed bacteria possessing either full-length or
trun-cated p26 cDNA, and incubated with shaking at 37°C until
the D600 reached 0.6–1.0 Cultures were stored at 4°C
overnight before incubation with shaking in 50 mL of fresh
LB medium containing 50 lgmL)1 ampicillin at either
30°C or 37 °C Isopropyl thio-b-D-galactoside (IPTG) was
added when the culture reached a D600 of 0.6–1.0 and
incubation continued for 5 h, followed by 5 min on ice and
centrifugation at 5000 g for 5 min at 4°C Growth rates of
E colitransformed with wild-type and mutated p26 were
not determined, but the D600increases for all cultures were
similar indicating that expressed p26 had no effect on cell
division The pelleted cells were washed twice with cold
buffer (50 mM Tris/HC1, 2 mM EDTA, pH 8.0) and
resuspended in 5 mL of the same buffer, before adding
lysozyme and Triton X-100 to final concentrations of
100 lgmL)1 and 0.01%, respectively Triton X-100 was
omitted from some preparations to learn if detergent
influenced p26 oligomerization Mixtures were incubated
at 30°C for 15 min, sonicated twice for 10 s at the high
output setting with a Branson Sonifier cell disruptor 200
fitted with a microtip, and centrifuged at 12 000 g for
15 min at 4°C Supernatants were either used immediately
or frozen at )70 °C until required The pellets, when
retained, were resuspended in 500 lL of SDS/PAGE
treatment buffer, placed in a boiling water bath for 3 min
and either electrophoresed immediately or stored at)70 °C
Immunodetection and quantitation of p26
Protein samples electrophoresed in 12.5% SDS
polyacryl-amide gels were either stained with Coomassie blue or
transferred to nitrocellulose Blots were rinsed briefly with
Tris/NaCl/Pi(0.01MTris/HC1, 0.14MNaC1, pH 7.4) and
stained with 0.2% Ponceau-S in water to confirm protein
transfer For immunodetection, membranes were blocked
45 min in 5% milk powder dissolved in Tris/NaCl/Pi/
Tween (Tris/NaCl/Pi with 0.1% Tween 20), followed by
incubation for 30 min at room temperature with either
anti-p26 Ig [57] or anti-(His6tag) Ig (Santa Cruz
Biotech-nology, Inc., Santa Cruz, CA, USA) diluted in HST buffer
(0.01M Tris/HC1, 1M NaC1, 0.5% Tween 20, pH 7.4)
Blots were washed twice in HST buffer, then in Tris/NaCl/
Pi/Tween, prior to incubation for 30 min with horseradish
peroxidase (HRP)-conjugated goat anti-(rabbit IgG) Ig
(Jackson Immunochemicals, Inc.) diluted in HST buffer
Membranes were washed twice in HST buffer, twice in
Tris/NaCl/P/Tween and once in Tris/NaCl/P, with each
wash for 5 min Immunoconjugates were detected by the enhanced chemiluminescence (ECL) procedure (Amersham Pharmacia Biotech) following manufacturer’s instructions The p26 bands were scanned with a BioRad Model GS-670 Imaging Densitometer and analyzed in MOLECU-LAR ANALYST Values so obtained were compared with those comprising the linear portion of a standard curve established for quantitation of p26 The standard curve was prepared by electrophoresing different amounts of cell free extract from Artemia cysts containing p26 in 12.5% SDS polyacrylamide gels, blotting to nitrocellulose and probing with antip26 antibody before scanning Each densitometer value (arbitrary units) was plotted against the amount of cell free extract protein in the gel lane from which the density measurement was made
Centrifugation of p26 in sucrose gradients Sucrose gradients were formed in 0.1M Tris/glycine (pH 7.4) by layering 5 mL of 10% sucrose on 5 mL of 50% sucrose and centrifuging at 200 000 g for 3 h at 15°C Four-hundred microliters of cell free extract from bacteria grown at 30°C was loaded per gradient and centrifuged at
200 000 g for 21 h at 4°C in a Beckman SW41 Ti rotor Additionally, 400 lL of p26 purified from Artemia cysts [57], and molecular mass markers of 29 kDa (carbonic anhydrase), 66 kDa (bovine serum albumin), 150 kDa (alcohol dehydrogenase), 200 kDa (a-amylase), 443 kDa (apoferritin), and 669 kDa (thyroglobulin) (Sigma) were centrifuged on gradients Tube bottoms were punctured with a 25-gauge needle, 1 mL samples were collected, and
75 lL from each fraction was mixed with 25 lL of 4· SDS polyacrylamide gel treatment buffer Twenty microliters of each sample was then electrophoresed in 12.5% SDS polyacrylamide gels, blotted to nitrocellulose and probed with antibody to p26 Each molecular mass marker, located
by reading the A280of gradient fractions, tended to occur
in several samples, thus each marker was centrifuged separately The position of the peak tube for each marker
is indicated in the figures
Thermotolerance ofE coli BL21(DE3) expressing p26 Two milliliters of Luria–Bertani broth containing
50 lgmL)1 ampicillin and 1 mM IPTG was inoculated with a single colony of E coli BL21(DE3) transformed with either full-length or truncated p26 cDNA in pET21(+), and incubated at 30°C for 8–9 h Immediately before heat shock, 0.5 mL of culture was diluted 1 : 10 in fresh medium supplemented with 25 lgmL)1 ampicillin Cultures were incubated at 54°C in a water bath, 100 lL samples were removed after 0, 15, 30, 45 and 60 min of heat shock, diluted in cold LB broth and maintained on ice prior to plating in duplicate on LB agar Colonies were counted after 20–24 h at 37°C and all p26 constructs were tested a minimum of three times for thermotolerance induction To verify the presence of p26, 500 lL of each IPTG induced culture was removed prior to heating, cells were collected by centrifugation for 20 s at top speed in a microcentrifuge, resuspended in 50 lL of treatment buffer, placed in a boiling water bath for 3 min, and frozen at )20 °C before electrophoresis in SDS/polyacrylamide gels, blotting to nitrocellulose and immunodetection
Trang 4R E S U L T S
Cloning of full-length and truncated p26 cDNAs
Six cDNA products were generated by PCR using
selected primers and p26-3-6-3 as template (Table 1)
The cDNAs include: p26-full, the full-length p26 cDNA;
p26-ND36, lacks N-terminal residues 1–36; p26-ND60,
lacks N-terminal residues 1–60; p26-CD40, lacks
C-termi-nal residues 153–192; p26-CD10, lacks C-termiC-termi-nal residues
183–192; p26-alpha, lacks residues 1–60 and 153–192,
thereby corresponding to the a-crystallin domain That
the cDNA fragments in pET21(+) were of the proper size
was confirmed by restriction digestion with BamHI and
XhoI Additionally, primers p26-1Bam-s and
p26-192Xho-as (Table 1) amplified only the cDNA in p26-full, and
amplification of constructs with the primers employed for
production of their respective inserts yielded PCR
prod-ucts of the expected length That is, the fragments were
the same size as those released from pET21(+) by
restriction digestion and to those obtained during the
initial PCR As final verification of identity, and to see if
errors were introduced during PCR amplification, each
p26 cDNA cloned in pET21(+) was sequenced and its
deduced amino-acid sequence determined (not shown)
With one exception, deduced amino-acid sequences of
cDNA products were identical, exclusive of engineered
deletions, to full-length p26 Construct p26-ND60 had a
modified nucleotide at position 407 (numbered as in
full-length p26-3-6-3) that caused a Val136Ala substitution
Each p26 cDNA had cytosine at position 324, whereas
adenine was reported for p26-3-6-3, and cytosine at
position 354 was replaced by thymine Neither of these
changes modified the deduced amino-acid sequence of
p26 The p26 cDNAs cloned in pET21(+) and utilized in
subsequent experiments are represented schematically in
Fig 1
Synthesis of full-length and truncated p26
inE coli BL21(DE3)
Cell free extracts prepared from E coli transformed with
p26 cDNAs in pET21(+) and induced at 30°C with IPTG
were electrophoresed in SDS/polyacrylamide gels and either
stained with Coomassie blue (Fig 2A) or blotted to
nitrocellulose and probed with antibody to p26 (Fig 2B)
Only p26-ND36 yielded an additional band visible in stained
gels (Fig 2A, lane 2) In agreement with this observation,
immunostaining of blots with antibody to p26 gave a strong
reaction with p26-ND36, while bands of lesser intensity were
obtained for p26-full, p26-CD40 and p26-CD10 (Fig 2B)
Extracts from bacteria transformed with p26-ND60 and
p26-alpha in pET21(+) usually failed to produce visible
bands when Western blots were stained with anti-p26 Ig
(Fig 2B), although very weak bands appeared occasionally
(not shown) The relative amounts of p26 in lysates of
transformed E coli were determined by incubating Western
blots with anti-p26 Ig, taking care to ensure that density
measurements were within the linear range of film exposure
(Fig 2C) The ratio of full: ND36: CD40:
p26-CD10 was 2 : 16 : 1 : 2.5 Similar quantities of each p26
variant were produced by transformed bacteria incubated at
30 and 37°C, but much of the p26 at the higher temperature
pelleted upon centrifugation at 12 000 g for 15 min (Fig 3)
In contrast, full-length and truncated p26 polypeptides synthesized at 30°C were almost completely soluble, thus bacteria used for subsequent analysis were grown at this temperature Occasionally, expressed proteins appeared as doublets (Fig 3B, lane 2, 3D, lane 4) but the reason is unknown
Centrifugation of bacterially expressed p26
in sucrose gradients The sedimentation patterns of p26 proteins encoded by pET21(+) constructs, and detectable on Western blots with anti-p26 Ig, varied upon centrifugation of cell free extracts
in sucrose gradients (Fig 4) For example, p26-CD40 (Fig 4C) existed mainly as monomers, whereas p26-CD10 (Fig 4D) and p26-ND36 (Fig 4B) were in protein com-plexes larger than monomers but smaller on average than
Fig 1 Schematic representation of p26 cDNAs cloned in pET21(+) Results obtained by cloning full-length and truncated derivatives of p26 in the prokaryotic expression vector, pET21(+), are summarized The p26 cDNAs were cloned into the BamHI and XhoI sites of pET21(+), and the constructs were used to transform E coli BL21(DE3) MCS, multiple cloning site; Amp, ampicillin resistance; ori, origin of replication; lac1, lac operator repressor gene; f1 origin, filamentous phage origin of replication Additional description of clones is available in Table 1.
Trang 5those seen in E coli transformed with pET21(+) containing
full-length p26 cDNA (Fig 4A) p26 purified from Artemia
cysts ocurred as oligomers (not shown) and in cell extracts
from cysts (Fig 4E) the p26 complexes were slightly larger
in size than these produced by recombinant p26 As revealed
by sedimentation patterns therefore truncated p26 variants
detectable by antip26 antibody exhibited limited ability to
oligomerize and/or to interact with other proteins, whereas
full-length p26 in cell free extracts from E coli and Artemia
formed much larger protein complexes, perhaps due to oligomer assembly as demonstrated for purified p26
Thermotolerance ofE coli expressing full-length and truncated p26 cloned in pET21(+)
Bacteria transformed with p26 containing constructs dem-onstrated greater thermotolerance than cells that had incorporated only pET21(+) (Fig 5) Maximum tolerance occurred in bacteria expressing p26-full, but this was only marginally better than protection conferred by p26-ND36 and p26-ND60, which in turn was greater than the resistance afforded by p26-CD40, p26-CD10 and p26-alpha The insert (Fig 5) indicates either that p26 occurred in cell free extracts from only four transformed cultures, although all exhibited enhanced heat tolerance, or that p26 variants produced by p26-ND60 and p26-alpha were present but recognized poorly by antip26 antibody To determine if recognition of p26-ND60 and p26-alpha encoded polypeptides by antibody
to p26 was problematic, corresponding cDNA fragments were inserted into pRSETC and used to transform E coli IPTG induced bacteria containing full-length p26, p26-ND60 and p26-alpha in pRSETC produced polypeptides of appropriate size that reacted with antibody to the (His)6
epitope tag, while only the product of p26-full reacted with anti-p26 Ig (Fig 6) Thus, antibody to p26 reacted poorly with polypeptides encoded by p26-ND60 and p26-alpha, demonstrating that enhanced thermotolerance conferred by these two constructs correlated with synthesis of p26 polypeptides Additionally, when tested by centrifugation
on sucrose gradients, the size of full-length p26-His6 (Fig 7A) was close to those produced by recombinant full-length p26 lacking His6, while results with the truncated p26 polypeptides (Fig 7B,C) were similar to those for p26-CD40, yielding mostly monomers As with the other truncated derivatives of p26, the induction of thermotoler-ance by p26-ND60 and p26-alpha did not depend on oligomer formation
Fig 2 Expression of pET21(+) containing p26 cDNA in transformed
bacteria Cell free protein extracts were prepared from E coli
trans-formed with pET21(+) containing full-length and truncated p26
cDNAs and grown in the presence of IPTG at 30 °C Samples were
electrophoresed in 12.5% SDS polyacrylamide gels and either stained
with Coomassie blue (A) or transferred to nitrocellulose and probed
with antibody to p26 using the ECL procedure (B) Each lane received
15 lL of cell free extract in A and 10 lL in B M, molecular mass
markers of 97, 66, 43, 31, 22 and 14 kDa; 1, p26-full; 2, p26-ND36; 3,
p26-ND60; 4, p26-CD40; 5, p26-CD10; 6, p26-alpha; 7, pET21(+).
Arrowhead, p26-ND36 Panel C, Western blots containing lysates of
transformed E coli BL21(DE3) grown at 30 °C and induced with
IPTG were probed with antibody to p26 Films were scanned and
absorbance of the p26 band in each lane, in arbitrary units,
deter-mined The amounts of sample applied to the gel were: p26-full, 5 lL;
p26-ND36, 1 lL; p26-CD40, 10 lL; p26-CD10, 5 lL The lanes in
which p26 is not visible each received 10 lL of lysate.
Fig 3 Solubility of p26 synthesized in transformed bacteria E coli BL21(DE3) transformed with p26 cDNA in pET21(+) and induced with IPTG were grown at either 30 °C (A,B) or 37 °C (C,D) for 5 h, following which soluble (A,C) and insoluble (B,D) fractions were prepared Twenty microliters of each sample was electrophoresed in 12.5% polyacrylamide gels, blotted to nitrocellulose and probed with antibody to p26 by the ECL procedure Lane 1, 5 lg of cell free extract protein from Artemia cysts; 2, p26-full; 3, p26-ND36; 4, p26-CD40; 5, p26-CD10.
Trang 6D I S C U S S I O N
Restriction digestion, PCR amplification and sequencing
confirmed the identity of p26 cDNA fragments cloned in
pET21(+) and pRSETC The deduced amino-acid
sequence for each construct was identical to the
corre-sponding region encoded by 3-6-3 [58], except for
p26-ND60, which had a Val136Ala substitution Alanine and
valine are similar amino acids, indicating this modification is
unlikely to affect p26 Two other nucleotides were altered in
all constructs, but neither rendered an amino-acid
conver-sion Because each construct was amplified from the same
p26-3-6-3 preparation, these changes probably represent
errors in the original sequence
SDS/PAGE of extracts from IPTG induced bacteria gave
a visible protein band of the expected size for p26-ND36, but not for other cDNA fragments cloned in pET21(+), upon
Fig 4 Centrifugation of p26 in sucrose gradients Four hundred ll of
extract from E coli BL21(DE3) transformed with p26 cDNA cloned
in pET21(+) and 400 lL of cell free extract from Artemia cysts were
centrifuged in sucrose gradients Samples from gradient fractions were
electrophoresed in 12.5% SDS polyacrylamide gels, transferred to
nitrocellulose, and reacted with antibody to p26 using the ECL
pro-cedure The top of each gradient is to the left of the figure, and
numbers across the top indicate successive samples taken from
gradi-ents (A) p26-full; (B) p26-ND36; (C) p26-CD40; (D) p26-CD10; (E)
p26 in cell free extract from Artemia The positions from left to right of
molecular mass markers representing 29, 66, 150, 200, 443 and
669 kDa are indicated by arrows.
Fig 5 Thermotolerance of transformed bacteria Transformed E coli BL21(DE3), grown as described in Materials and methods, were incubated at 54 °C for the times indicated, plated in duplicate on LB agar and incubated at 37 °C for 20 h Colonies were counted and the log 10 values of colony forming units (cfu) per mL were plotted against the length of heat shock in min The results shown are the average of three independent experiments Bacteria containing only pET21(+) did not survive 60 min of heat shock and the curve was terminated at
45 min Insert, 10 lL of cell lysate from each heat shocked culture was electrophoresed in 12.5% SDS polyacrylamide gels, blotted to nitro-cellulose and probed with anti-p26 Ig, a procedure repeated for each heat shock experiment 1, full; 2, ND36; 3, ND60; 4, p26-CD40; 5, p26-CD10; p26-alpha.
Fig 6 Expression of p26 cDNA cloned in pRSETC Cell free protein extracts were prepared from E coli transformed with either pRSETC (A,C) or pET21(+) (B,D) containing p26 cDNA Duplicate samples were electrophoresed in 12.5% SDS polyacrylamide gels, transferred
to nitrocellulose and probed with antibody to either p26 (A,B) or the (His) 6 epitope tag encoded by pRSETC (C,D) Each lane received 7.5 lL of extract 1, p26-full; 2, p26-ND60; 3, p26-alpha; 4, vector only.
Trang 7Coomassie blue staining Probing of Western blots with p26
specific antibodies demonstrated the products of four
constructs in extracts of IPTG induced bacteria, but
polypeptides corresponding to p26-ND60 and p26-alpha
were either absent or recognized poorly by anti-p26 Ig
Western blots of extracts from E coli transformed with
p26-full, p26-ND36 and p26-alpha cloned in pRSETC and
probed with antip26 antibody gave results identical to those
just described However, antibody to His6 revealed the
epitope tag, and thus p26-ND36 and p26-alpha, in duplicate
samples from pRSETC transformed E coli Interestingly,
mammalian Cos1 cells transiently transfected with
p26-ND60 and p26-alpha were immunofluorescently labeled
with antip26 antibody, even though the p26 variants were
not detectable on Western blots of extracts from these cells
(data not shown) The combined results support the
conclusion that bacteria transformed with pET21(+)
containing p26-ND60 and p26-alpha produced p26, but it
was poorly recognized by anti-p26 Ig after electrophoresis
and transfer to nitrocellulose Equally important, full-length
and truncated derivatives of p26 from transformed E coli
were almost completely soluble at 30°C, but less so at
37°C, indicating that bacteria grown at the lower
tempe-rature are more likely to give an accurate portrayal of p26 function than are cells incubated at 37°C
Small heat shock/a-crystallin proteins generally exist as large oligomers when purified [4,8,39,40,57], but there are exceptions [19,20,22] In this study, p26, either purified from Artemiaembryos (not shown) or in cell free extracts, was shown by sucrose gradient centrifugation to exist in protein complexes as large as 670 kDa Liang et al [57] demon-strated that purified p26 assembled into oligomers of
670 kDa, and full-length p26 in cell free extracts from transfected Cos1 cells is also in a complex of similar mass (unpublished data) Full-length p26 synthesized in E coli yielded protein complexes somewhat smaller on average than those in extracts from Artemia, and by way of comparison, small heat shock/a-crystallin proteins pro-duced in transformed bacteria usually reside as oligomers similar in size to those in cells from which the expressed cDNA was obtained [17,24,37,61] The reluctance of full-length p26 to form complexes as large as those in Artemia may reflect improper post-translational processing of the protein in E coli On the other hand, trivial explanations for the slightly reduced mass are either that Triton X-100 used during protein preparation affects quaternary structure or that oligomerization of p26 and/or its interaction with other proteins is concentration dependent The former possibility was not investigated systematically, but preliminary data suggest detergent does not affect the ability of p26 to form large complexes in bacterial extracts Published results vary
in terms of how the concentration of small heat shock/ a-crystallin proteins influences oligomer assembly For example, a-crystallin tends to oligomerize readily, even at low concentrations [40,42], while Hsp20 oligomerization is concentration dependent [19] Expression of full-length p26 cDNA in pRSETC was more than for pET21(+), and the average size of p26 complexes resolved in sucrose gradients increased, perhaps as a consequence of greater oligomer-ization due to higher p26 concentration
The absence in cell free extracts of high molecular mass complexes when p26 lacks either part or all of the N-terminus favours a role for this region in oligomer assembly Reinforcing this proposal, tetramers are the maximum size attained by Hspl2.2 and 12.3 from Caenor-habditis elegans [22], and like p26-ND36, the N-terminal domains of these proteins are short High molecular mass oligomers are not detected after N-terminal deletion of
C elegansHsp16.2, although dimers and possibly tetramers are present [18] Additionally, Hspl2.6 from C elegans, with 16 fewer N-terminal residues than Hspl6.2, is mono-meric [20] Eliminating the 56 N-terminal residues from aA-crystallin, but not the first 19, reduced oligomer mass, as did removal of 87 N-terminal residues from Hsp27 [25] and
33 residues from Hsp25 [33], although the latter modifica-tion was small In contrast, loss of 42 residues from the N-terminus of a rice small heat shock/a-crystallin protein increased oligomer size [23] Deleting the last 16 C-terminal residues of C elegans Hspl6.2 had limited effect on quaternary structure [18], as is true for dispensing with 10 C-terminal residues from aA-crystallin [25] and 18 C-terminal residues from Hsp25 [15] In contrast, oligomer formation by p26 lacking C-terminal residues was compro-mised, signifying this region is important for oligomeriza-tion Although caution is required because the function of p26, a eukaryotic protein, was examined in E coli, the
Fig 7 Centrifugation of p26 in sucrose gradients Four hundred
microliters of extract from E coli BL21(DE3) transformed with p26
cDNA cloned in pET21(+) was centrifuged in sucrose gradients.
Samples from gradient factions were electrophoresed in 12.5% SDS
polyacrylamide gels, transferred to nitrocellulose, and reacted with
antibody to the His 6 epitope tag The top of each gradient is to the left
of the figure, and numbers across the top indicate successive samples
from gradients A, p26-full, B, p26-ND60; C, p26-alpha The positions
from left to right of molecular mass markers representing 29, 66, 150,
200, 443 and 669 kDa are indicated by arrows.
Trang 8results corroborate the idea that N-terminal domains of
small heat shock/a-crystallin proteins aid construction of
oligomers from smaller building blocks arising by
interac-tions between residues within a-crystallin domains
[7,8,21,62] Contrary to other reports, the C-terminal
extension is also implicated in oligomerization, but the
exact nature of its role is uncertain
Small heat shock/a-crystallin proteins confer
thermotol-erance on prokaryotic and eukaryotic organisms
[23,27–33] The construct used previously to examine
induction of thermotolerance by full-length Artemia p26
encoded N-terminal, nonp26 residues, missing from the
construct employed herein [49], but the outcome was
similar in each case Moreover, loss of N-terminal residues
did not drastically change the ability of p26 to confer
thermotolerance on E coli, suggesting this domain and the
assembly of large oligomers are not required for protection
In agreement, bacteria transformed with Hsp25, and
Hsp25 lacking 33 N-terminal amino-acid residues, are
equally heat tolerant [33] Removal of C-terminal
exten-sions from C elegans Hspl6.2, murine Hsp25 and human
aA-crystallin reduced, but did not extinguish small heat
shock/a-crystallin protein chaperone activity in vitro
[15,16,18], as is true when hydrophobic residues are placed
in the region [17] Loss of the C-terminal extension lowered
protein solubility, consistent with the notion that this
region is a solubilizing agent [14–17] Eliminating the
C-terminal extension had little effect on p26 solubility
when bacteria were grown at 30°C and 37 °C, although
testing at higher temperatures may be informative
Additionally, the C-terminal extension of p26 is required
for full induction of thermotolerance in E coli and thus
may be necessary for chaperoning in vitro
Oligomerization in the context of thermotolerance, as
described in this study, is not as thoroughly investigated as
the association between quaternary structure and
chaper-oning in vitro, where an increase in oligomer mass
gene-rally enhances protection of client proteins [18–20,22,63]
However, oligomer mass is not the only determinant of
small heat shock/a-crystallin protein function As a case in
point, oligomers lacking chaperone activity arise from
chimeric a-crystallins [63] Also, insertion of a peptide
[41,64] and change of a single residue [10,11,13,65] lead to
enlarged oligomers with curtailed chaperone action in vitro
In other work, dissociation of oligomers was a prerequisite
for chaperoning in vitro [66], and disassembly of active units
from an oligomeric (storage) state of a-crystallin was
proposed, upon structural analysis of aA-crystallin by
site-directed spin labelling, as a model for chaperone function
[67] Such results downplay oligomerization as a prerequisite
for protection against stress In this vein, monomeric
a-crystallin at low chaperone to target ratios protects lens
sorbitol dehydrogenase enzyme activity upon heating [46],
while the N-terminal portion of a small heat shock/
a-crystallin protein confers thermotolerance on cells [29]
and prevents aggregation of stressed proteins in vitro [12]
These polypeptides are unlikely to oligomerize, either in vivo
or in vitro Clearly, our data strengthen the notion that small
heat shock/a-crystallin proteins function in vivo when not in
large oligomers Whether this signals nonspecific effects on
proteins, an interplay with membranes as reported recently
[68], or mechanistic differences between thermotolerance
and molecular chaperoning in vitro awaits purification of
truncated p26 derivatives and testing in a defined system, experiments now in progress
A C K N O W L E D G E M E N T S
The work was supported by a Natural Sciences and Engineering Research Council of Canada Research Grant and a Nova Scotia Health Research Foundation New Opportunity Grant to T H M.
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