Mycobacterium tuberculosis heat 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 mM ATP 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 tuberculosis can 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][6][7]. It was shown that MTB HSP 16.3, initially described as the immunodominant 14-or 16-kDa antigen [8][9][10][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 asso...
Mycobacterium tuberculosis heat 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 mM ATP 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 tuberculosis can 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][6][7]. It was shown that MTB HSP 16.3, initially described as the immunodominant 14-or 16-kDa antigen [8][9][10][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 asso...
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