(Received for publication, August 25, 1995; and in revised form, December 20, 1995)
From the
Tuberculosis continues to be a major disease threatening
millions of lives worldwide. Several antigens of Mycobacterium
tuberculosis, identified by monoclonal antibodies, have been
cloned and are being exploited in the development of improved vaccines
and diagnostic reagents. We have expressed and purified the 16-kDa
antigen, an immunodominant antigen with serodiagnostic value, which has
been previously cloned and shown to share low sequence homology with
the -crystallin-related small heat shock protein family.
Sedimentation equilibrium analytical ultracentrifugation and dynamic
light scattering demonstrate the formation of a specific oligomer, 149
± 8 kDa, consisting of approximately nine monomers. In 4 M urea, a smaller oligomer of 47 ± 6 kDa (or trimer) is
produced. Analysis by electron cryomicroscopy reveals a triangular
shaped oligomeric structure arising from the presence of three
subparticles or globules. Taken together, the data suggest an antigen
complex structure of a trimer of trimers. This antigen, independent of
ATP addition, effectively suppresses the thermal aggregation of citrate
synthase at 40 °C, indicating that it can function as a molecular
chaperone in vitro. A complex between the antigen and
heat-denatured citrate synthase can be detected and isolated using high
performance liquid chromatography. We propose to rename the 16-kDa
antigen Hsp16.3 to be consistent with other members of the small heat
shock protein family.
There has been a recent increase in the number of reported cases
of tuberculosis (TB), ()an old scourge and still the leading
cause of death among infectious diseases. In addition, multidrug
resistant strains of Mycobacterium tuberculosis, the bacterium
that causes TB, have been isolated (for reviews, see Bloom and
Murray(1992) and Nowak(1995)). Approximately one-third of the
world's population harbors M. tuberculosis, and the
World Heath Organization (WHO) predicts that, by the year 2005, TB will
kill 4 million people per year (Nowak, 1995). Mycobacteria are daunting
organisms to study, and the molecular pathogenesis of TB is still
poorly understood (Bloom and Murry, 1992).
In the process of
screening for strain-specific antibodies, as well as developing subunit
vaccines and efficient diagnostic tests, many murine monoclonal
antibodies (mAbs) have been generated against various mycobacterial
antigens (Coates et al., 1981; Engers et al., 1986;
Ivanyi et al., 1988; Jackett et al., 1988). Using
these mAbs and recombinant DNA techniques, genes of more than a dozen
mycobacterial antigens have been cloned (Young et al., 1990;
Verbon et al., 1992). Interestingly, several of these major
antigens have sequence homologies to the conserved heat shock proteins
(Young et al., 1990; Verbon et al., 1992; Kong et
al., 1993). Two of these antigens, the 18 kDa antigen from Mycobacterium leprae and the 16-kDa antigen from M.
tuberculosis, show sequence homology with the
-crystallin-related small heat shock proteins (Nerland et
al., 1988; Verbon et al., 1992). The 16-kDa antigen of M. tuberculosis has previously been referred to as 14K antigen
(Engers et al., 1986; Jackett et al., 1988; Young et al., 1990) and major membrane protein (Lee, et
al., 1992) in the literature. This protein is one of the prominent
antigens of M. tuberculosis defined by mAb TB68 (Coates et
al., 1981), as well as F23-49, and F24-2 (Engers et al.,
1986). It carries epitopes restricted to tubercle bacilli (i.e. M.
tuberculosis, Mycobacterium africanum, and Mycobacterium bovis) on the basis of B-cell recognition
(Coates et al., 1981) and is effective for diagnostic uses
(Kingston et al., 1987; Jackett et al., 1988; Ivanyi et al., 1988; Verstijnen et al., 1989). The cellular
location is unknown, although it is proposed to be on the outside of
the cell wall (Schoningh et al., 1990; Verstijnen et
al., 1989). This antigen, which is capable of generating
cell-mediated immune responses, also contains T-cell epitopes which are
cross-reactive with M. leprae antigens (Kingston et
al., 1987). It is the cell-mediated immune system which plays the
dominant role in defense against mycobacteria.
We have previously reported that another immunodominant antigen of M. tuberculosis, the 38-kDa antigen, is a phosphate-binding protein which serves as an initial receptor for active transport (Chang et al., 1994). Here we describe the subcloning, overexpression, and purification of the 16-kDa antigen. Verbon et al.(1992) previously cloned the gene and further noted low level sequence homology with small heat shock proteins (smHsp). We also present direct evidence that the purified recombinant 16-kDa antigen forms a specific oligomer and can function as a molecular chaperone in vitro, consistent with its homology to smHsps.
Figure 1: Purification profile of the recombinant 16-kDa Antigen of M. tuberculosis. Proteins were separated by SDS-polyacrylamide gel electrophoresis (8-25% acrylamide gradient) and stained with Coomassie Brilliant Blue (PhastGel system, Pharmacia). Lanes 1 and 5, molecular weight markers; 2, supernatant after sonication and centrifugation; 3 and 4, purified proteins after column chromatography with DEAE and PEI(NH), respectively.
The 16-kDa antigen was overexpressed (Fig. 1, lane 2) and purified using two anion exchange columns, DEAE cellulose and PEI(NH) silicon. The 16-kDa antigen was eluted at 0.15 M NaCl from the DEAE column and 0.75 M sodium acetate from the PEI(NH) column (lanes 3 and 4) and had an approximate isoelectric point of 5.1. The protein was approximately 95% homogenous after these two columns (Fig. 1, lane 4). The average yield was about 15 mg of 16-kDa antigen from 1 liter of LB culture.
The 10 residues at the N terminus of the purified recombinant 16-kDa antigen were determined to be: Ala-Thr-Thr-Leu-Pro-Val-Gln-Arg-His-Pro, confirming the sequence from the gene except that the N-terminal Met residue is missing. Lee et al.(1992) also observed no Met after protein sequencing. We observed similar Met cleavage with another M. tuberculosis antigen (the 38-kDa antigen expressed in the same system) (Chang et al., 1994).
The molecular mass obtained from sedimentation equilibrium was a single species of 151 ± 7 kDa. Dynamic light scattering measurements produced a consistent result of 147 ± 8 kDa. These results indicate the existence of an oligomer of nine subunits. However, the apparent molecular mass estimated from gel filtration is 210 ± 10 kDa. Gel filtration depends on the shape of the protein, and interactions between the protein of interest or standard proteins and the column matrix. In contrast, sedimentation equilibrium provides a more accurate method for the determination of the native molecular weight of a protein, independent of hydrodynamic properties (Laue and Rhodes, 1990).
Electron cryomicroscopic
examination of the 16-kDa antigen indicated a triangular shaped
particle (Fig. 2A). Further image processing revealed
that this shape is due to the presence of three globules or lobes (Fig. 2B). Fig. 2B shows 7 of the class
averages and their corresponding S images (see
``Experimental Procedures''). The class averages were
computed from 2,900 particle images from two micrographs of comparable
defocus (4 µm). These images have been subjected to two rounds
of alignment and classification. The S images support the
statistical reliability of each of these class averages. Since the
protein molecules are presumably oriented randomly on the electron
microscopic grid, the class averages represent different projections of
the molecule. Fig. 2B shows members of the final class
averages with the most obvious trimeric appearance and the least
intra-class variance. The edge dimension of the class average with the
most apparent 3-fold symmetry is
100 Å. In the absence of an
atomic resolution structure, we cannot yet delineate its quaternary
configuration. However, given the evidence that the protein tends to
form a 9-subunit complex, these class average images suggest that the
16-kDa oligomer is a trimer of trimers. This oligomeric structure is
supported by the finding that the molecular weight of the antigen in 4 M urea or 1 M guanidine HCl was reduced to
approximately one-third of the original size (47 ± 6 kDa) as
determined by dynamic light scattering. This suggests that the three
globules (each a complex of three 16.1-kDa subunits) can be
disassembled, and that the total number of subunits in the entire
complex is an integral multiple of three. In view of all these
observations, we propose that the 16-kDa antigen forms a specific
nine-subunit complex with a calculated total mass of 144.9 kDa, based
on a monomer mass of 16,100 Da determined by mass spectrometry (Lee et al., 1992).
Figure 2: Electron cryomicroscopic images of the Hsp16.3. Panel A, 100 keV electron image of ice-embedded 16-kDa antigen taken at a defocus value of 4 µm. The contrast of the image is sufficiently high to visualize an individual oligomer. Since the protein molecules can be oriented differently, its projection images appear differently. A recurrent feature is the trimeric shape as highlighted by arrowheads. Panel B, 7 class averages and the corresponding S images computed from 2900 particle images after iterative steps of alignment, multivariate statistical analysis, and automatic classification. Each class is an average of from 30 to 40 individual particle images. The white contrast denotes the protein density on the top row and area of invariance among members of each class in the bottom row.
Our results, as shown in Fig. 3, Fig. 4, and Fig. 5, clearly indicate that the 16-kDa antigen oligomer effectively inhibits the thermal aggregation of CS at 39.5 °C. No suppression of CS aggregation is observed by adding either ribonuclease A or lysozyme under the same assay conditions (Fig. 4). The same holds true for the addition of bovine serum albumin (data not shown). Addition of ATP up to 10 mM with magnesium (up to 1 mM) did not enhance the chaperone activity of the 16-kDa antigen, consistent with other members of the smHsp family.
Figure 3:
Thermal aggregation of CS is prevented by
addition of Hsp16.3. One µM concentration of CS dimer was
incubated in a spectrophotometer cell thermostatted at 39.5 °C in
50 mM HEPES, pH 7.0, with various concentrations of Hsp16.3.
Aggregation of CS was monitored by measuring apparent light scattering (A) every 30 s. Curves show CS alone
(-), or with 0.55 µM, 1.10 µM,
2.21 µM, 4.41 µM, or 8.83 µM Hsp16.3. Shown is representative data from one experiment. Hsp16.3
alone does not detectably aggregate in this
assay.
Figure 4:
Suppression of CS thermal aggregation by
Hsp16.3 is specific. All curves represent the mean of duplicate or
triplicate data. Experimental conditions are identical to those shown
in Fig. 3. The rate of CS aggregation in the presence of 8.8
µM Hsp16.3 (7.6 10
OD/min) is
70 times slower than the rate of CS alone (0.052 OD/min). On the y axis, the rate of CS aggregation by itself is assigned a value of
one. (
-
) various concentrations of Hsp16.3 added
to 1 µM CS at 39.5 °C. (
-
)
bovine pancreatic RNase A as a control for Hsp16.3.
(
-
) chicken egg white lysozyme as a control
for Hsp16.3 The x axis gives the concentration of Hsp16.3 or
RNase A or lysozyme in the assay (CS is always 1 µM). All
proteins were dialyzed against 50 mM HEPES, pH 7.0, before
use. Neither lysozyme nor RNase A aggregate in this
assay.
Figure 5: Time course of Hsp16.3 addition to preheated CS. CS (1 µM) was added to the thermostatted cell, then Hsp16.3 added at a concentration of 2.21 µM after various periods of time, as indicated by arrows. (-) CS and Hsp16.3 added at the same time, Hsp16.3 added 2.5 min after CS, Hsp16.3 added 5 min after CS, 10 min after, and 15 min delay after CS addition.
The 16-kDa antigen also inhibits CS aggregation when added at various times during the incubation of CS alone (Fig. 5). Aggregation is inhibited regardless of how long it was allowed to proceed before antigen addition. Since thermal inactivation of CS is essentially complete after only 10 min (Fig. 6), this suggests that the 16-kDa antigen interacts with a partially unfolded intermediate of CS rather than the native state. Hsp90 was also found to interact with unfolding intermediates of CS at 43 °C (Jakob et al., 1995).
Figure 6:
Hsp16.3 does not protect CS against
thermal inactivation nor assist in reactivation. One µM of
CS was incubated at 39.5 °C in 50 mM HEPES, pH 7.0 with 1
mM dithiothreitol in the presence () or absence (
)
of 8.83 µM Hsp16.3. Samples were shifted to 25 °C
after 20 min. The enzymatic activity of CS was determined at the
indicated times.
Figure 7: Complex formation between heat-denatured CS and Hsp16.3. Proteins were mixed in a siliconized microcentrifuge tube and heated at 40 °C for the time indicated. Mixture was spun then loaded onto a TSKgel column and eluted with 0.2 M NaCl, 50 mM HEPES, pH 7.0. Detection was by absorbance at 280 nm. The average retention times of peaks one, two, and three were 10.1, 13.1, and 16.0 min, respectively (Panel A). The other two peaks with greater retention times are contaminants of the CS solution, as they are present when CS is chromatographed alone and do not change after heating. HPLC peaks were isolated and concentrated using Centricon 30, then subjected to SDS-PAGE analysis (Panel B) demonstrated that peak 3 (lane 4) was CS, peak 2 (lane 3) was Hsp16.3, and peak 1 (lane 2) was a complex of the two proteins. Numbers on the left are the molecular mass, in kilodaltons, of the protein standards (lane 1). Arrowheads on the right indicate the position of CS (43.5-kDA monomer) and Hsp16.3 (16.1-kDa monomer).
The small heat shock protein family is the most abundant and diverse group of Hsps, members of which have been discovered in organisms from bacteria to plants to humans. The chaperone activity of smHsps is ATP-independent in contrast to the Hsp60 and Hsp70 families. While the average monomeric molecular mass of smHsps is 15-30 kDa, they usually form 200-800 kDa homo- or hetero-oligomeric complexes (for reviews, see de Jong et al.(1993), Jakob and Buchner (1994), and Arrigo and Landry(1994), the size of which may be controlled by phosphorylation (Kato et al., 1994).
Our
results strongly suggest that the recombinant 16-kDa antigen of M.
tuberculosis forms a specific multisubunit complex and can
function as a molecular chaperone in vitro in an
ATP-independent manner. We will therefore refer to the 16-kDa antigen
as Hsp16.3, so named in the Swiss Protein Databank by Verbon et al. (accession no. A42651), in order to be consistent with other
members of the small heat shock protein family. Recently it has been
observed that -crystallin and a few related smHsps (murine Hsp25,
human Hsp27, pea Hsp18.1, and Hsp17.7) can also function as molecular
chaperones (Horwitz, 1992; Jakob et al., 1993; Merck et
al., 1993; Lee at al., 1995). The family of smHsps are
much less conserved than the other families of Hsps (Hsp90, Hsp70, and
Hsp60) (de Jong et al., 1993; Jakob and Buchner, 1994). The
studies presented here provide evidence that the family of small heat
shock proteins share common functionality as molecular chaperones.
Additionally, preheating Hsp16.3 at 40 °C enhances suppression of
CS aggregation (data not shown). The mechanism of this interesting
phenomenon is currently being investigated.
Interestingly, Hsp16.3 does not protect citrate synthase from thermal inactivation at 40 °C, although aggregation is completely suppressed (Fig. 3). Hsp18.1 and Hsp17.7 from pea similarly prevented CS aggregation but did not protect CS activity at 45 °C (Lee et al., 1995). We also observed that adding Hsp16.3 into preheated CS halted aggregation (Fig. 5). This strongly suggests, along with the activity data (Fig. 6), that Hsp16.3 binds to partially unfolded intermediates (which are inactive enzymatically) but not native proteins. While this small heat shock protein is effective in suppressing aggregation, other proteins may be needed for the refolding to the native state in vivo (see Jakob and Buchner (1994)).
In light of the molecular
weight analysis indicating a mass of approximately 145 kDa for the
Hsp16.3 complex, which could be dissociated into trimers in the
presence of denaturants, combined with the result of the analysis by
electron cryomicroscopy, we conclude that the complex is a trimer of
trimers. We are not aware of any other smHsp whose complex structure
has been as well defined as that of the Hsp16.3 (Fig. 2B). Indeed, although controversial, several
different models of the quaternary structure of -crystallin have
been proposed (for review, see de Jong et al.(1993)).
Interestingly, electron microscopic images of negatively stained
Hsp17.7 from pea, which is composed of 12 subunits, revealed both round
and triangular structures (Lee et al., 1995). If the
triangular structure is proven to be similar to the one established for
the Hsp16.3, then it is possible that the Hsp17.7 is a trimer of
tetramers.
The expression of a homolog of Hsp16.3 in Mycobacterium habana, the 18-kDa antigen, is significantly increased when subjected to heat shock (Lamb et al., 1990). The stress inducibility of Hsp16.3 in M. tuberculosis is not well established and needs to be further investigated (Young and Garbe, 1991).
The size of the Hsp16.3 oligomer under our analytical conditions did not change within the concentration range of 0.4 to 15 mg/ml (data not shown). A similar observation was made with recombinant murine Hsp25 by Behlke et al.(1991). Previous studies suggested that smHsps in mammalian and chicken cells exist in a variety of dynamic states, depending upon the physiological status of the cells (Collier and Schlesinger, 1986; Arrigo et al., 1988). Whether this is the case for Hsp16.3 in M. tuberculosis cells is still unknown.
Lee et al.(1992) found that Hsp16.3 is a major membrane protein, but not likely to be an intrinsic membrane protein judging from its behavior in detergent. In other words, this protein is probably peripherally associated with the membrane. Monoclonal antibody detection did not reveal any secreted Hsp16.3 in culture supernatant (Abou-Zeid, 1988; Verbon et al., 1990). Two smHsps, produced in response to heterologous protein expression in E. coli, were found to be tightly associated with inclusion bodies (Allen et al., 1992). In higher organisms, smHsps were found in the cytoplasm, nuclear and in various organelles (for a review, see Arrigo and Landry(1994)). In vivo location and crystallographic structure analysis of Hsp16.3 are aims for future studies.
Our results demonstrate that the 16-kDa antigen (Hsp16.3) exists as a specific oligomer, a trimer of trimers, and can function as a molecular chaperone in vitro. Hsp16.3 is easily expressed and provides a good model system for further studies of the function of small heat shock proteins and molecular chaperones in general.