MTB HSP 16.3 displays several characteristics of small heat shock proteins sHsps: its expression is increased in response to stress, it protects against protein aggregation in vitro, and
Trang 1Functional similarities between the small heat shock proteins
Melissa M Valdez1, John I Clark1,2, Gabrielle J S Wu3and Paul J Muchowski4
1
Departments of Biological Structure, and2Ophthalmology, University of Washington, Seattle, WA, USA;3Seattle Genetics, Bothell,
WA, USA;4Department of Pharmacology, University of Washington, Seattle, WA, USA
Mycobacterium tuberculosisheat shock protein 16.3 (MTB
HSP 16.3) accumulates as the dominant protein in the latent
stationary phase of tuberculosis infection MTB HSP 16.3
displays several characteristics of small heat shock proteins
(sHsps): its expression is increased in response to stress, it
protects against protein aggregation in vitro, and it contains
the core Ôa-crystallinÕ domain found in all sHsps In this study
we characterized the chaperone activity of recombinant
MTB HSP 16.3 in several different assays and compared the
results to those obtained with recombinant human
aB-crystallin, a well characterized member of the sHsp
family Recombinant MTB HSP 16.3 was expressed in
Escherichia coli and purified to apparent homogeneity
Similar to aB-crystallin, MTB HSP16.3 suppressed citrate
synthase aggregation and in the presence of 3.5 mMATP the chaperone activity was enhanced by twofold ATP stabilized MTB HSP 16.3 against proteolysis by chymotrypsin, and no effect was observed with ATPcS, a nonhydrolyzable analog
of ATP Increased expression of MTB HSP 16.3 resulted in protection against thermal killing in E coli at 48°C While the sequence similarity between human aB-crystallin and MTB HSP 16.3 is only 18%, these results suggest that the functional similarities between these proteins containing the core Ôa-crystallinÕ domain are much closer
Keywords: ATP; human aB-crystallin; molecular chaperone; Mycobacterium tuberculosis HSP 16.3; small heat shock proteins
One-third of the world’s population is infected with latent
inactive tuberculosis and active tuberculosis is the leading
cause of death due to an infectious disease [1] Each year,
new infections occur in 54 million people; 6.8 million people
develop clinical disease, and 2.4 million cases result in death
[2] There is still limited knowledge of the molecular
pathogenesis of the latent stage of this organism [3]
Individuals who have been infected with Mycobacterium
tuberculosiscan harbor stable dormant bacilli for decades
before developing an active infection later in life [4] Recent
reports indicate an important role for M tuberculosis
(MTB) heat shock protein (HSP) 16.3 in the survival of
MTB during prolonged periods of infection [5–7] It was
shown that MTB HSP 16.3, initially described as the
immunodominant 14- or 16-kDa antigen [8–11], was a
major component in tuberculosis infection in humans and
played an important role in enhancing protein stability and
survival [5] Eighty-five percent of patients with active
tuberculosis showed a positive reaction to this antigen,
suggesting that this protein expressed in vivo had a key role
in MTB infection [11,12] The 14K antigen was later
renamed MTB HSP 16.3 [13] MTB HSP 16.3 accumulates
to become the dominant protein in the latent stationary phase of M tuberculosis infection [7] Over-expression of HSP 16.3 in log phase growth of M tuberculosis slowed the growth rate and protected against stationary phase autolysis
in vitro [7] MTB HSP 16.3 has been characterized as a membrane associated protein [12] having sequence homo-logy to other proteins in the small heat shock protein (sHsp) family [11,14] All sHsps share sequence similarity in a conserved 80–100 amino-acid Ôa-crystallinÕ domain region found in the C-terminus which is thought to be important for chaperone functions [14–16] MTB HSP 16.3 has been shown to contain an oligomeric, active structure which may form a trimer of trimers and possesses in vitro molecular chaperone activity [13]
Up-regulation of large and small sHsps is thought to be a universal response to stress In vitro, human aB-crystallin and other sHsps function as molecular chaperones by suppressing unfolding and aggregation of polypeptides in response to stress [17,18] MTB HSP 16.3 modulates its chaperone activity by exposing hydrophobic surfaces and demonstrates conformational flexibility allowing maximum interactions with denaturing proteins [19] A recent paper reports that the only universally conserved leucine residue among all the members of the sHsp family plays an important role in molecular chaperone activity of MTB HSP 16.3 and oligomeric structure formation [20] It has been reported that the chaperone activity of MTB HSP 16.3
is independent of the effects of ATP [13,19] In contrast, molecular chaperones of the large heat shock protein families suppress protein unfolding and aggregation during stress and participate in the refolding of denatured proteins
in vitro, in an ATP-dependent manner [21,22] While the molecular chaperone effects of the sHsp do not require ATP, the activity of human-aB crystallin was enhanced with
Correspondence to J I Clark, Department of Biological Structure,
Box 357420, University of Washington, Seattle, WA 98195-7420,
USA Fax: +1 206 543 1524, Tel.: 1 206 685 0950,
E-mail: clarkji@u.washington.edu
Abbreviations: MTB HSP 16.3, Mycobacterium Tuberculosis heat
shock protein 16.3; sHsps, small heat shock proteins; IPTG, isopropyl
thio-b- D -galactoside; CFUs, colony forming units; CS, citrate
synthase.
(Received 12 July 2001, revised 17 January 2002, accepted 25 January
2002)
Trang 2ATP [23] Recent reports confirmed the effect of ATP on
sHsps using bovine a-crystallin to stabilize partially
dena-tured proteins during reactivation in an ATP-dependent
manner [24] and indicating the involvement of ATP in
substrate release [25] In this study, the effect of ATP on
recombinant MTB HSP 16.3 was compared with the effect
of ATP on recombinant human aB-crystallin
We report the expression and purification of recombinant
MTB HSP 16.3 from E coli and studies of its function as a
molecular chaperone MTB HSP 16.3 was compared with
aB-crystallin in vivo and in vitro biochemically and in
functional assays Although only 18% sequence identity is
shared between the two sHsps, MTB HSP 16.3 functioned as
effectively as aB-crystallin as a molecular chaperone in vitro
The molecular chaperone activity of recombinant MTB
HSP 16.3 was enhanced in the presence of ATP which is
consistent with previous findings of the effect of ATP on
recombinant human aB-crystallin [23] The expression of
MTB HSP 16.3 in E coli exposed to high temperatures
resulted in a very impressive level of survival Our results
suggest the chaperone activity of MTB HSP 16.3 may play an
important role in the survival and stability of M tuberculosis
M A T E R I A L S A N D M E T H O D S
Expression and purification of MTB HSP 16.3
HSP 16.3 was subcloned into the pET-20b(+) expression
vector which was provided by H McHaourab (Department
of Molecular Physiology and Biophysics, Vanderbilt
University School of Medicine, Nashville, TN, USA) The
pET-20b(+)-HSP 16.3 expression plasmid was used to
transform E coli BL21 (DE3) competent cells (Novagen,
Inc., Milwaukee, WI, USA) The expression of HSP 16.3
was based on a method described previously [26] One litre
of Luria–Bertani broth containing 10 g NaCl, 5 g yeast
extract, and 10 g tryptone (DIFCO Laboratories), pH 7.0
with 50 lgÆmL)1carbenicillian was inoculated with 10 mL
of an overnight culture containing the
pET-20b(+)-HSP16.3 vector The flasks were incubated for a total of
3 h at 37°C until D600 ¼ 0.8–1.0 The cells were then
induced with 0.4 mMisopropyl thio-b-D-galactoside (IPTG)
for another 4 h Cells were then harvested by sedimentation
and frozen at)20 °C until further use Cell pellets of the
pET-20b(+)-HSP 16.3 were then lysed with 10 mL lysis
buffer (20 mMTris/HCl pH 7.0) and transferred to a small
beaker which was placed in ice Forty microliters of 50 mM
phenylmethanesulfonyl fluoride and 400 lL 10 mgÆmL)1
lysozyme were added with constant stirring for 10 min;
20 mg deoxycholic acid were then added with an additional
10 min of stirring The mixture was removed from the ice
bath and 200 lL of 1 mgÆmL)1DNAse was added with
stirring for 30 min The sample was then placed in a 50-mL
tube and centrifuged at 18 000 g for 1 h The supernatant
from this sample was transferred to a new beaker with
constant stirring at room temperature with the addition of
400 lL 5% polyethylenimine and 800 lL 200 mM
dithio-threitol for 10 min The sample was then centrifuged at
35 000 g for 2 h at 4°C The supernatant was decanted and
the insoluble pellet was discarded The supernatant was then
ready for purification using the Pharmacia FPLC system
The supernatant containing the soluble protein was
filtered through a 0.22 lm filter and was loaded onto a High
Trap Q Anion Exchange Column (Pharmacia), pre-equil-ibrated with Buffer A (20 mM Bis/Tris, pH 6.5) The proteins were eluted using a linear gradient of 0–1.0M NaCl The protein fractions were analyzed using SDS/PAGE (Invitrogen) Proteins were analyzed on a 4–12% polyacrylamide electrophoretic gel in the presence of 0.1% SDS and Mes buffer and were stained with Coomasie blue R-350 (Amersham Pharmacia) Fractions containing the 16.3-kDa protein were then pooled and concentrated using a 10 000 molecular mass cut-off concentrator (Amicon) Concentrated protein (5 mL) was loaded onto
a Phenyl Superose Hydrophobic Interaction Column, preequilibrated with a buffer containing 50 mM sodium phosphate, and 1.0M ammonium sulfate, pH 7.0 The protein was then eluted using 50 mM sodium phosphate,
pH 7.0 The protein fractions were analyzed by SDS/PAGE and all fractions containing the protein were pooled and concentrated to 500 lL The concentrated sample was applied to a Superdex 200 HR 10/30 size exclusion column (Pharmacia) and purified by gel filtration Fractions containing the protein were collected, concentrated, and then quantified for total protein concentration using the Bradford method (Bio-Rad)
To confirm protein purification of HSP 16.3, a Western immunoblot analysis was performed using monoclonal antibody IT-4 (a-16 kDa) provided by D Sherman (Department of Pathobiology, University of Washington, Seattle, WA, USA) Detection of protein was performed using NEN Western Blot Chemiluminescence Reagents (NEN Life Science Products Inc.) The sequence of the purified recombinant HSP 16.3 was confirmed by
MS Automatic Edman sequencing was used with an applied Biosystems model 470 A automatic protein sequencer
In vivo cell viability experiment with HSP 16.3 at 48 °C
A XbaI–XhoI fragment containing the entire coding sequence of HSP 16.3 was excised from pET20b(+)-HSP 16.3 and ligated into pET16b (Pharmacia) digested with the same restriction enzymes Restriction mapping analysis was performed on pET16b-HSP 16.3 to ensure that the HSP 16.3 gene was inserted in the proper orientation, and the coding sequence of HSP 16.3 was verified by DNA sequence analysis The pET16b-HSP 16.3 vector was then transformed into BL21 (DE3) competent cells
For the thermal killing experiment, an equal number of cells were grown containing either pET16b-HSP 16.3 vector, pET16b (empty vector control), or pET16b-aB (positive control) Equal numbers of cells from overnight cultures were inoculated into 50 mL of L-broth medium containing 100 lgÆlL)1carbenicillin and grown at 37°C until they reached an D600¼ 0.8 Protein expression was then induced with 1.0 mM IPTG After a 2 h induction, samples were shifted to a shaking water bath at 48°C Samples were removed at 3-h time points postinduction and scored for cell viability by plating on Luria–Bertani broth plates containing carbenicillin Cell viability was determined by counting the number of colony forming units (CFUs) on each plate after heat shock at 48°C relative to the starting number of CFUs formed in each culture prior to heat shock Total protein lysates from cells that expressed HSP 16.3 and control cultures were
Trang 3analyzed by SDS/PAGE as described above The
experi-ment was repeated four times using duplicates of each cell
culture
Chaperone assays
The thermal unfolding and aggregation of citrate synthase
(CS; Roche Molecular Biochemicals) at 45°C was
deter-mined by measuring the absorption from light scattering at
320 nm in a Beckman Spectrophotometer over a period of
30 min Native CS was diluted to a 15 lM working
solution containing 20 mM Tris/HCl (pH 7.4), and
100 mM NaCl To test the molecular chaperone effect,
different molar ratios of HSP 16.3 were diluted into a
reaction buffer containing 100 mM Tris/HCl (pH 7.4),
100 mM NaCl in the presence or absence of ATP (final
volume 400 lL) For testing the effects of ATP and the
nonhydrolyzable ATP analog ATPcS, the reaction buffer
was equilibrated with 3.5 mM ATP (or ATPcS), 3.5 mM
MgCl2 and 10 mM KCl before addition of HSP 16.3 or
CS The protection from thermal aggregation of CS at
45°C with HSP 16.3 was also compared with the same
molar ratios of aB-crystallin (predicted from monomeric
molecular weights)
Chymotrypsin digestion of HSP 16.3
Chymotrypsin digestion with HSP 16.3 was based on the
methods used for GroEL and aB-crystallin [27,28] In
summary, for each reaction 70 lg MTB HSP 16.3 were
diluted into a final volume of 100 lL buffer containing
100 mM Tris/HCl, pH 7.4, 3.5 mM MgCl2, 10 mM KCl,
and 0.01% Tween-20 For reactions with ATP or ATPcS,
a final concentration of 3.5 mM ATP or ATPcS was
added to the reaction mixture To each sample at time
point 0, 0.17, 0.51 or 1.36 lg chymotrypsin was added
from a stock solution of 0.17 mgÆmL)1 Samples were
maintained at 37°C for the duration of the experiment
Immediately after chymotrypsin addition, 13.5 lL of the
reaction mixture was removed and quenched with 1.5 lL
100 mM phenylmethanesulfonyl fluoride, and placed on
ice At 5-min time points, 13.5-lL aliquots were removed
and treated identically to the zero time point sample
Samples were analyzed by SDS/PAGE analysis as
described above
R E S U L T S
Expression and purification of recombinant MTB
HSP 16.3 inE coli
Figure 1 is the SDS/PAGE and Western immunoblot
analyses of the expression and purification of MTB
HSP 16.3 Induction of protein expression with IPTG
resulted in the appearance of a protein band at
approximately 16.3 kDa (Fig 1A, lanes 2 and 3) The
expressed MTB HSP 16.3 was purified by a combination
of anion exchange, hydrophobic interaction and size
exclusion chromatography (lanes 4–6) Western
immuno-blot analysis of recombinant MTB HSP 16.3 was
performed using a monoclonal antibody [IT-4
(a-16 kDa)] raised against native MTB HSP 16.3
(Fig 1B) IT-4 (a-16 kDa) recognized the recombinant
MTB HSP 16.3 from E coli and did not react with recombinant aB-crystallin (Fig 1B) The purification yield obtained from 1 L of Luria–Bertani broth culture was between 10 and 30 mg of MTB HSP 16.3
Fig 1 Expression and Purification of MTB HSP 16.3 (A) The expression and purification of MTB HSP 16.3 was analyzed by SDS/ PAGE using 4–12% Bis/Tris polyacrylamide gels in the presence of Mes buffer Lanes 1 and 7, molecular mass markers; lane 2, expression protein in E coli cells not induced by IPTG; lane 3, protein expression
in E coli after induction with IPTG; lane 4, following purification on the High trap Q anion exchange column; lane 5, MTB HSP 16.3 enriched after purification using a Phenyl Superose Hydrophobic Interaction Column; lane 6, following Tris/HCl buffer exchange of MTB HSP 16.3 on a Superdex 200 Size Exclusion column (to remove salt) A protein assay performed after desalting the sample showed that the yields varied between 10 and 30 mgÆL)1of MTB HSP 16.3 cell culture (B) SDS/PAGE (left) and Western immunoblot (right) on a 4–12% polyacrylamide gel of recombinant MTB HSP 16.3 and recombinant human aB-crystallin Lane 1, molecular mass markers; lane 2, MTB HSP 16.3; lane 3, aB-crystallin In the Western immu-noblot, the IT-4 antibody to MTB HSP 16.3 detected recombinant MTB HSP 16.3 in lane 2 only No reactivity with anti-(MTB HSP 16.3) Ig was observed in lane 3, which contained human aB-crystallin (right side).
Trang 4Amino-acid sequence determination of recombinant
MTB HSP 16.3
The sequence of the purified recombinant MTB HSP 16.3
was confirmed by MS The 12 residues at the N-terminus of
MTB HSP 16.3 were determined to be:
Ala-Thr-Thr-Leu-Pro-Val-Gly-Arg-His-Pro-Arg-Ser The N-terminal Met
residue was not identified by protein sequencing, as
observed previously [12,13]
Protection of cell viability at 48 °C inE coli by MTB
HSP 16.3 and aB-crystallin
To characterize the protective effect of MTB HSP 16.3
over-expression in vivo, a thermal killing assay in E coli was
used (Fig 2A) The number of CFUs were counted in
cultures of E coli with and without MTB HSP 16.3 and
aB-crystallin shifted to 48°C The proportion of viable cells that survived heat shock at 48°C was plotted at three time points (t ¼ 0, 3 and 6 h after 48 °C heat shock) Cells that expressed MTB HSP 16.3 or aB-crystallin were resistant to thermal killing at 48°C over the 6-h time course of the experiment (Fig 2A) After 3 hs, the viability of cells that contained the empty control vector decreased by four orders
of magnitude, while the viability of cells that over-expressed MTB HSP 16.3 or aB-crystallin decreased only slightly Within 6 h of heat shock at 48°C, no viable cells were observed in cells containing the aB-crystallin vector that were not induced for protein expression The viability by
6 h, decreased dramatically in cells containing the pET 16b control vector as measured by the CFUs In contrast, the viability of cells that over-expressed MTB HSP 16.3 decreased by two orders of magnitude after the 6-h heat shock The protective effect of the induced aB-crystallin was stronger than MTB HSP 16.3 SDS/PAGE analysis showed strong expression of MTB HSP 16.3 and aB-crystallin at time points 0, 3, and 6 h following heat shock at 48°C (Fig 2B,C)
In vitro chaperone activity of MTB HSP 16.3 and aB-crystallin
In Fig 3, we observed the effects of different concentrations
of MTB HSP 16.3 in the presence and absence of ATP on the aggregation of CS In the absence of added MTB HSP 16.3, aggregation of CS increased after a short delay to reach a maximum after approximately 25 min at 45°C
Fig 2 Cell viability of MTB HSP 16.3 The pET 16b-MTB HSP 16.3 vector, the control pET 16b-aB vector and the pET 16b vector con-taining no inserted gene were expressed at 37 °C and induced with IPTG when the cell cultures reached D 600 ¼ 0.8 After induction for
2 h and heat shock to 48 °C, the cells were incubated for a further 6 h Samples were taken at concurrent and sequential time points beginning
at the time of heat shock, plated and CFU counted The proportions of viable cells expressing the pET 16b-MTB HSP 16.3 vector and the two control vectors were plotted for 0, 3 and 6 h following heat shock At
48 °C, the proportion of surviving cells expressing pET 16b vector only
or aB-crystallin uninduced cells was negligible and viability of cell cultures decreased more than fourfold (A) By 6 h post heat shock, the cultures that over-expressed MTB HSP 16.3 and the aB-crystallin in-duced cells remained viable Protein expression was analyzed by SDS/ PAGE on a 4–12% polyacrylamide gel in the presence of 0.1% SDS and Mes buffer (B,C) Lanes 1 and 6 are molecular mass markers Lanes 2–5 show protein expression in cells containing the pET 16b vector alone at selected times from 0 to 6 h Lane 2 is the protein expression in cells containing the pET 16b vector alone not induced with IPTG Lanes 3–5 are the pET 16b vector at the zero, 3 and 6 h time points after 2 h of induction and post heat shock at 48 °C Lane 7
of (B) is protein expression in cells containing the pet 16b-MTB HSP 16.3 vector not induced with IPTG Lanes 8–10 are protein expression for the pET 16b-MTB HSP 16.3 vector at time point zero, 3 and 6 h after 2 h of induction and post heat shock (C) SDS/PAGE of cells containing aB-crystallin induced and not induced with IPTG Lane 2–5 show aB-crystallin uninduced at the 0, 3 and 6 h time points Lane 7 contains cell homogenates of aB-crystallin not induced Lanes 8–10 contain aB-crystallin induced with IPTG at the 0, 3 and 6 h time points The cells expressing high levels of MTB HSP 16.3 or aB-crystallin (lanes 8–10) survived in culture at 48 °C (A).
Trang 5(Fig 3A) With increasing ratios of MTB HSP 16.3 to CS, a concentration-dependent suppression of aggregation was observed over the 30-min period (Fig 3A) Complete protection against aggregation was observed at a ratio of
15 : 1 MTB HSP 16.3:CS (monomer : monomer) The addition of 3.5 mM ATP enhanced the effect of MTB-HSP 16.3 on CS aggregation by approximately twofold (Fig 3B) ATPcS, a nonhydrolyzable analog of ATP, did not enhance the effect of MTB HSP 16.3 on CS aggregation (Fig 3C) The chaperone activity of MTB HSP 16.3 was next compared to aB-crystallin at identical molar ratios (Fig 4) In general, aB-crystallin was more effective as a molecular chaperone than MTB HSP 16.3 under the conditions of these experiments (Fig 4) Complete suppres-sion of CS aggregation by MTB HSP16.3 required a molar ratio of 15 : 1, while aB-crystallin required a molar ratio of
5 : 1 for complete suppression of aggregation
Chymotrypsin proteolysis of MTB HSP 16.3
in the absence and presence of ATP MTB HSP 16.3 was digested with chymotrypsin in the absence and presence of ATP at 42°C (Fig 5A–C) Proteolysis of MTB HSP 16.3 increased with chymotrypsin concentration as expected (data not shown) Each individ-ual lane is a sample of MTB HSP 16.3 plus chymotrypsin
Fig 4 Comparison of molecular chaperone activity between recombin-ant MTB HSP 16.3 and recombinrecombin-ant human aB-crystallin The molecular chaperone activity of MTB HSP 16.3 on CS aggregation was compared to the effect of human aB-crystallin on CS aggregation The aggregation of CS was measured in the presence of different concentrations of MTB HSP 16.3 or human aB-crystallin after a 30-min period The bar graphs measure the aggregation of CS in arbitrary units vs the ratios of MTB HSP 16.3/CS and human aB-crystallin/CS With increased ratios of the molecular chaperone protein to CS, there was increased protection against CS aggregation Recombinant human aB-crystallin demonstrated better protection against CS aggregation than MTB HSP 16.3 At the 15 : 1 molar ratio
of MTB HSP 16.3/CS, protection against CS aggregation was almost complete Similar protection was observed at a ratio of 5 : 1 for human aB-crystallin/CS.
Fig 3 Molecular Chaperone Activity of MTB HSP 16.3 To test the
molecular chaperone activity of MTB HSP 16.3, a series of
aggrega-tion assays was performed using CS with and without ATP over a
30-min period The aggregation of CS was measured with the addition
of different concentrations of MTB HSP 16.3 and in the presence or
absence of ATP and ATP analogs (A) Aggregation of CS is plotted in
arbitrary units against time in the presence of increasing ratios of MTB
HSP 16.3 to CS With the increase of MTB HSP 16.3, there was an
increase of protection against thermal aggregation of CS [d, CS alone;
j, HSP 16.3/CS (5 : 1); ,, HSP 16.3/CS (10 : 1); , HSP 16.3/CS
(12 : 1); s, HSP 16.3/CS (15 : 1)] (B) Aggregation of CS plotted in
arbitrary units against time in the absence and presence of ATP at two
different molar concentrations of MTB HSP 16.3 In the presence of
ATP, the molecular chaperone effect of MTB HSP 16.3 was enhanced
for aggregation of CS and maximum suppression of aggregation was
observed at a molar ratio of 10 : 1 HSP 16.3 : CS [d, CS alone;
,, HSP 16.3 : CS (5 : 1); j, HSP 16.3 : CS (5 : 1) + ATP; s,
HSP 16.3 : CS (10 : 1); , HSP 16.3 : CS (10 : 1) + ATP] (C)
Control for the effect of ATP on the molecular chaperone activity of
MTB HSP 16.3 When 3.5 m M MgCl 2 and 1 m M KCl were added to a
solution containing MTB HSP 16.3 and CS no effect on chaperone
activity was observed The results using ATPcS and MgCl 2 with KCl
suggest the importance of hydrolysis of ATP for chaperone activity of
MTB HSP 16.3 [d, CS alone; ,, HSP 16.3 : CS (10 : 1) + 1 m M
KCl and 3.5 m M MgCl 2 ; s, HSP 16.3 : CS (10 : 1); , HSP 16.3 : CS
(10 : 1) + 3.5 m M ATPcS; j, HSP 16.3 : CS (10 : 1) + 3.5 m M
ATP].
Trang 6taken at 5-min time intervals over a 30-min period Nearly
all intact MTB HSP 16.3 was degraded after 15 min in the
absence of ATP (Fig 5A) In the presence of 3.5 mMATP,
MTB HSP 16.3 was stabilized against proteolysis, and
intact MTB HSP 16.3 could be detected even after 30 min
of proteolysis (Fig 5B) The digestion pattern of MTB
HSP 16.3 was similar in the absence and presence of ATP,
where two major proteolytic fragments at Mr 8 and
13 kDa were observed The specificity of the effect of ATP
on stabilization of MTB HSP 16.3 against proteolysis was
confirmed with nonhydrolyzable ATP analog ATPcS,
which had no stabilizing effect against proteolysis (Fig 5C)
D I S C U S S I O N
Our results demonstrated similar functions for recombinant
MTB HSP 16.3 and human aB-crystallin as molecular
chaperones, although the sequence identity between MTB
HSP 16.3 and aB-crystallin is only 18% (Fig 6) MTB
HSP 16.3 is a sHsp that contains the conserved core
Ôa-crystallinÕ domain shared by members of the sHsp family
[14–16] M tuberculosis HSP 16.3 was expressed and
puri-fied from E coli for the comparative characterization on the
molecular chaperone activity in vivo and in vitro with human
aB-crystallin, the well characterized archetype of the sHsp
family of molecular chaperones [14,16,29]
In vivo,the protective effect of MTB HSP 16.3 expression
on the survival of E coli in a thermal killing assay at 48°C
was impressive (Fig 2A) At 48°C, there was
approxi-mately two orders of magnitude difference between
survi-ving cells expressing MTB HSP 16.3 and controls without
MTB HSP 16.3 expression The results for MTB HSP 16.3
are consistent with previous reports with other sHsps
[23,30,31] The protective effect of aB crystallin on cell
survival was stronger than MTB HSP 16.3 In previous
in vivostudies, over-expression of HSP 16.3 at the end of
log-phase growth in M tuberculosis resulted in an enhanced
resistance to autolysis [5] Our results showing a protective
effect of MTB HSP 16.3 against thermal killing in E coli
are consistent with previous studies on the importance of MTB HSP 16.3 expression in M tuberculosis [5–7] Although the role of MTB HSP 16.3 is not completely understood, these experiments suggest that MTB HSP 16.3 may provide protection against cell death in M tuberculosis
In an in vitro aggregation assay using CS as a target protein, MTB HSP 16.3 was effective as a chaperone, although less effective than aB-crystallin in suppressing CS aggregation It is possible that additional cofactors found only in M tuberculosis cytosol could increase the chaperone activity of MTB HSP 16.3 It is also likely that the efficiency
of MTB HSP 16.3 as a chaperone may be improved using target proteins that are native to M tuberculosis
The effects of ATP on the chaperone activity of MTB HSP 16.3 were similar to aB-crystallin, a sHsp whose
Fig 6 Sequence alignment of recombinant MTB HSP 16.3 and recombinant human aB-crystallin Amino-acid sequence alignment between recombinant MTB HSP 16.3 and human aB-crystallin was aligned using the MULTALIN MULTIPLE SEQUENCE ALIGNMENT program (PBIL, France) with the help of S Yarfitz (University of Washington Health Sciences Library, Seattle, WA, USA) Shading indicates chemically identical and similar amino-acids residues ( BOXSHADE
program from the European Molecular Biology Network) Residues highlighted black indicate amino-acid residues that are chemically identical and residues highlighted gray indicate amino-acid residues that are chemically similar Between MTB HSP 16.3 and human aB-crystallin there was an 18% sequence identity and an overall 30% shared sequence similarity between the two proteins The conserved core a-crystallin domain observed in proteins of the sHsp spans resi-dues of E67–I161 in the aB-crystallin sequence.
Fig 5 The chymotrypsin proteolysis of MTB HSP 16.3 SDS/PAGE used 4–12% Bis/Tris polyacrylamide gels in the presence of Mes buffer (A–C) Arrows indicate the MTB HSP 16.3 band Lane 1 of each gel contains the molecular mass markers Each individual lane is a sample of MTB HSP 16.3 plus 0.51 lg chymotrypsin taken at 5-min intervals over a 30-min period MTB HSP 16.3 was readily degraded by chymotrypsin and nearly all intact protein was degraded by 15 min (A) In the presence of 3.5 m M ATP, MTB HSP 16.3 was stabilized against proteolysis by chymotrypsin and intact MTB HSP 16.3 remained after 30 min of digestion (B) In the presence of the non hydrolyzable analogue of ATP, ATPcS, there was no stabilization of MTB HSP 16.3 against chymotrypsin proteolysis (C), consistent with the effect of ATPcS on the molecular chaperone function of MTB HSP 16.3 reported in Fig 3.
Trang 7chaperone function was enhanced by ATP [23] In separate
reports, ATP increased the refolding of xylose reductase by
total a-crystallin [34], increased the binding of a-crystallin to
lens membranes, and inhibited the chaperone activity of a
plant sHsp [35,36] Consistent with previous studies using
aB-crystallin, the present results demonstrated that ATP
enhanced the chaperone effect of MTB HSP 16.3 by
twofold in the CS aggregation assay However, the specific
role of ATP in the chaperone function of sHsps has been
controversial [18,23,28,29] Previous reports indicated that
the chaperone activity of MTB HSP 16.3 may be ATP
independent [13,19] while structural studies demonstrated
an interaction between ATP and total bovine a-crystallin
using equilibrium binding studies, intrinsic tryptophan
fluorescence and31P NMR [23,25,34,35,37] aB-Crystallin
has also been reported to display an autokinase activity
[38–40] Recent reports suggest that ATP may participate in
the release of target peptides from aA-crystallin [24,25]
Here, MTB HSP 16.3 chaperone activity was measured in a
Tris/HCl buffer system, while previous studies of MTB
HSP 16.3 were performed in either a Hepes/HCl or sodium
phosphate buffer systems [13,19] The conditions used in
this study were the same as those used successfully to
demonstrate the ATP effect on human aB-crystallin [23]
In separate experiments the chymotrypsin proteolytic
digestion pattern of MTB HSP 16.3 in the presence and
absence of ATP was evaluated Similar to aB-crystallin [28]
and Hsp27 [32], chymotrypsin cleavage sites in MTB
HSP 16.3 appeared to be shielded in the presence of ATP
The similarity of the chymotrypsin digestion pattern for
MTB HSP 16.3 to previous studies with aB-crystallin and
Hsp27 may indicate similar domain structures and assembly
properties that are stabilized in the presence of ATP As
with aB-crystallin, ATPcS (a nonhydrolyzable ATP analog)
did not enhance the chaperone function of MTB HSP 16.3,
and did not protect against its proteolysis by chymotrypsin
Although there is only 18% sequence identity, the core
Ôa-crystallinÕ domain in MTB HSP16.3 may have functional
significance similar to that of aB-crystallin [28]
MTB HSP 16.3 of M tuberculosis may be ideally suited
for studies of the structure and function of the core
Ôa-crystallinÕ domain of sHsps because the quaternary
structure is more monodisperse than aB-crystallin and
other sHsps, that are known to have highly variable
quaternary structures [41] The crystal structure of HSP
16.5 from Methanococcus janaschii demonstrates a
mono-mer containing a core domain that consists largely of
b sheets [42] The molecules of HSP 16.5 form dimers that
assemble into a spherical complex of octahedral symmetry,
while MTB HSP 16.3 is reported to consist of a trimer of
trimers [13] Spin labeling of MTB HSP 16.3 in solution is
consistent with a core domain consisting of a twofold
symmetric interface between subunits that involved two
b strands in the core a-crystallin domain interacting in an
antiparallel fashion [43] While previous mutagenesis studies
demonstrated the functional importance of the core
Ôa-crystallinÕ domain in sHsps [28,29,33], the structural basis
for the function of the conserved core Ôa-crystallinÕ domain
remains to be defined
New strategies are needed to understand the precise
mechanism that allows the tubercle bacilli of M tuberculosis
to survive long-term dormancy This study supports the
hypothesis that the conserved core Ôa-crystallinÕ domain in
MTB HSP16.3 may be important for long-term dormancy
in M tuberculosis Future in vivo studies of specific inter-actions between MTB HSP 16.3 and other latent stage proteins will lead to a better understanding of the molecular chaperone activity of MTB HSP 16.3 Further structure– function analyses including the determination of an atomic resolution model of MTB HSP 16.3 are needed Crystallo-graphic studies of HSP 16.3 could be used to determine sites for interactions with other proteins and/or ATP Structure– function studies on MTB HSP 16.3 may have important implications for therapeutic drug discovery for the eradica-tion of bacilli in the latent stage of human M tuberculosis infection
A C K N O W L E D G E M E N T S
We thank H Mchaourab for the kind gift of the HSP 16.3 clone,
D Sherman for the kind gift of the monoclonal antibody IT-4 (a-16 kDa), and S Yarfitz for technical assistance with the Multalin Sequence alignment program We also thank J Clark and C Ganders for technical assistance This work was supported by National Eye Institute Grant EY0452 (to J I C.).
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