Mycobacterium tuberculosis 16-kDa Antigen (Hsp16.3) Functions as an Oligomeric Structure in Vitro to Suppress Thermal Aggregation

Abstract

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 ␣-crystallinrelated 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.

NAD synthase NMNAT acts as a chaperone to protect against neurodegeneration

“…Fluorescence analysis was performed with MetaMorph 5.07 (Molecular Devices/Universal Imaging Corp.), Amira 3.0 (TGS, Inc.) and Adobe Photoshop 7.0. The aggregation measurements were performed as described 22 . We searched threedimensional structures on DALI sever 24 (http://www.ebi.ac.uk/dali/); the structure superposition was done with PyMol structure analysis software.…”

“…This assay measures the chaperone's ability to reduce thermally or chemically induced aggregation of a model protein substrate such as citrate synthase or insulin 22 (Fig. 4c).…”

Stabilization of Taq DNA Polymerase at High Temperature by Protein Folding Pathways From a Hyperthermophilic Archaeon, Pyrococcus furiosus

“…The most extensively studied chaperone in P. furiosus is the sHsp, which is an a-crystallin homolog with conserved sequence motifs in common with sHsps and crystallins from all domains of life (Chang et al, 1996;Haley et al, 2000;Kim et al, 1998;Laksanalamai et al, 2001Laksanalamai et al, , 2003van Montfort et al, 2001). Several lines of evidence indicate that sHsps can prevent denatured proteins from aggregating but are unable to refold non-native proteins in a catalytic fashion (Chang et al, 1996;Laksanalamai et al, 2001). Hsp60s on the other hand catalyze ATP-dependent protein folding (Hartl, 1996;Hartl and Hayer-Hartl, 2002).…”

Human T‐ and B‐Cell Reactivity to the 16 kDa α‐Crystallin Protein of Mycobacterium tuberculosis

“…The antigen may stabilize cell structure during long-term survival and permit the bacilli to survive within the low-oxygen environment of the granuloma. The antigen exists in vitro as a trimer of trimers with a molecular weight of 149 000 Ϯ 8000 [7]. It suppresses the thermal aggregation of citrate synthase at 40ЊC, indicating that it can operate as a chaperone in vitro.…”