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. 2008 Nov 28:8:208.
doi: 10.1186/1471-2180-8-208.

Functional characterization of a small heat shock protein from Mycobacterium leprae

Nirmala Lini  1 , Elengikal Abdul Azeez Rehna, Sugathan Shiburaj, Jayapal Jeya Maheshwari, Nallakandy Panagadan Shankernarayan, Kuppamuthu Dharmalingam
Affiliations

Functional characterization of a small heat shock protein from Mycobacterium leprae

Nirmala Lini et al. BMC Microbiol. .

Abstract

Background: Small heat shock proteins are ubiquitous family of stress proteins, having a role in virulence and survival of the pathogen. M. leprae, the causative agent of leprosy is an uncultivable organism in defined media, hence the biology and function of proteins were examined by cloning M. leprae genes in heterologous hosts. The study on sHsp18 was carried out as the knowledge about the functions of this major immunodominant antigen of M. leprae is scanty.

Results: The gene encoding Mycobacterium leprae small heat shock protein (sHsp18) was amplified from biopsy material of leprosy patients, and cloned and expressed in E. coli. The localization and in vitro characterization of the protein are detailed in this report. Data show that major portion of the protein is localized in the outer membrane of E. coli. The purified sHsp18 functions as an efficient chaperone as shown by their ability to prevent thermal inactivation of restriction enzymes SmaI and NdeI. Physical interaction of the chaperone with target protein is also demonstrated. Size exclusion chromatography of purified protein shows that the protein can form multimeric complexes under in vitro conditions as is demonstrated for several small heat shock proteins.

Conclusion: The small heat shock protein sHsp18 of M. leprae is a chaperone and shows several properties associated with other small heat shock proteins. Membrane association and in vitro chaperone function of sHsp18 shows that the protein may play a role in the virulence and survival of M. leprae in infected host.

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Figures

Figure 1
Figure 1
Comparison of the localization and solubilisation in urea of sHsp18 protein (A) and Mce* protein (B). To check whether the sHsp18 protein forms inclusion bodies on over expression, the protein profile of the soluble and insoluble protein fractions were checked on a 12% SDS-PAGE. Lanes contain the soluble fraction (lane 1), the insoluble fraction (lane 2) of the crude sonication extract, and the supernatants after incubation of the insoluble fraction with suspension buffer containing 2 M urea (lane 3), 4 M urea (lane 4), 6 M urea (lane 5), and 8 M urea (lane 6). C&D. 2D gel analysis to show the presence of sHsp18 in the outer membrane fractions of E. coli. 45 μg protein was loaded on a 7 cm IPG strip with a pI range covering 4–7. After the second dimension, the gel was stained with colloidal coomassie. sHsp18 and OmpA (marker for outer membrane fraction) have been shown. 'C' represents outer membrane fraction and 'D' represents inner membrane fraction.
Figure 2
Figure 2
(A). Self aggregation of sHsp18. sHsp18 protein was purified by denaturing method and dialyzed against 50 mM PBS containing 200 mM NaCl for 12 hrs. Dialyzed protein was precipitated by acetone precipitation. The protein was dissolved in 1.5 M urea and applied to the column equilibrated against PBS and eluted the fractions in PBS. The bands corresponding to oligomeric forms were indicated by dotted arrows and position of molecular weight markers are indicated with solid arrows. (B). Non-denaturing Gel analysis of MagneHis Purified sHsp18 protein. Confirmation of the oligomeric form was done by native gel electrophoresis. Lanes represent High Molecular weight marker (lane 1), 10 μg of Non-denatured BSA showing oligomers (lane 2), 5 and 10 μg of purified protein under non-denatured condition (lanes 3–4), 5 and 10 μg of purified protein under denatured condition (lanes 5–6).
Figure 3
Figure 3
(A). Comparison of in vitro chaperone activity of α-crystallin and purified sHsp18. Enzymes (2 U) were heat inactivated (SmaI at 37°C for 90 min and NdeI at 45°C for 90 min) in the presence or absence of molecular chaperones (0.2 μg) and assayed for the cleavage of 1 μg of plasmid DNA. Lanes represent, λHind III marker (lane 1), digested plasmid (lane 2), uncut plasmid (lane 3), plasmid digested with- heat inactivated SmaI (lane 4), heat inactivated NdeI (lane 5), heat inactivated SmaI in the presence of sHsp18 (lane 6), heat inactivated SmaI in the presence of α-crystallin (lane 7), heat inactivated NdeI in the presence of sHsp18 (lane 8) and heat inactivated NdeI in the presence of α-crystallin (lane 9). (B). sHsp18 can act as a molecular chaperone in wide range of physiological temperatures. 0.2 μg sHsp18 or BSA was incubated with 2 U of SmaI at different temperatures for 90 min, and the cleavage of plasmid DNA was assayed at 25°C for 3 hrs. Lanes represent, λHind III marker (lane 1), uncut plasmid (lane 2), plasmid digested with SmaI as control (lane 3), plasmid incubated with heat inactivated SmaI without and with sHsp18 at 30°C (lanes 4–5); at 35°C (lanes 6–7), at 40°C (lanes 8–9), at 45°C (lanes 11–12), plasmid incubated with SmaI with BSA at 35°C, 40°C and 45°C (lanes 12–14). (C). Preheating of molecular chaperones does not affect chaperone activity. Lanes represent, λHind III marker (lane 1), undigested plasmid (lane 2), plasmid digested with- SmaI (lane 3), SmaI with preheated sHsp18 at 100°C for 5 min (lane 4) and with preheated α-crystallin at 100°C for 5 min (lane 5).
Figure 4
Figure 4
(A). sHsp18 interacts with SmaI. Heat inactivated SmaI was added to, sHsp18 bound MagneHis particles as per methods. sHsp18 was able to interact and refold the heat inactivated SmaI and restore its biological activity. But in the control, heat inactivated SmaI without sHsp18 did not show any activity. Lanes represent, λ Hind III marker (lane 1), undigested plasmid (lane 2), plasmid DNA digested with heat inactivated SmaI added to sHsp18 bound MagneHis particles (lane 3), plasmid DNA treated with heat inactivated SmaI added to MagneHis particles (lane 4). (B) SmaI do not bind directly to MagneHis particles. Native SmaI was incubated with MagneHis particles for 5 min and the supernatant and pellet fractions were taken for restriction assay. Lanes represent, λ Hind III marker (lane 1). Plasmid DNA was digested with SmaI added MagneHis pellet fraction (lane 2), plasmid DNA digested with supernatant fraction (lane 3) and control digest (lane 4).
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