Role of disulfide bonds in the oligomeric structure and protease resistance of recombinant and native Treponema pallidum surface antigen 4D

Abstract

Recombinant Treponema pallidum surface antigen 4D isolated from Escherichia coli formed a proteaseresistant ordered ring structure composed of 19,000-dalton subunits. On gradient sodium dodecyl sulfatepolyacrylamide gel electrophoresis, the higher oligomers of recombinant 4D migrated with molecular masses that were nearly multiples of the 190,000-dalton basic ordered ring. Reduction at room temperature with 2-mercaptoethanol converted the 190,000-dalton ordered ring and the higher oligomers to a 160,000-dalton form and the dissociated monomer. A 190,000-dalton form of 4D was identified in sodium dodecyl sulfate-solubilized T. pallidum after reduction at room temperature. Disulfide bonds stabilized both native and recombinant 4D oligomers against dissociation by heating in detergent without a reducing agent. Electron microscopy of recombinant 4D revealed that the characteristic ordered ring structure was maintained after reduction. Reduction of 4D under conditions that preserved the ordered ring structure did not affect the resistance of the molecule to digestion with proteinase K. The properties of 4D suggest that it may fulfill an important structural role in the T. pallidum outer membrane.Detailed study of the surface proteins of Treponema pallidum recently has been made possible by the expression of treponemal antigens in Escherichia coli (21,26,31,32). We have recently described a recombinant T. pallidum protein, designated 4D, which is a protease-resistant, 190,000-dalton ordered ring structure composed of 19,000-dalton monomers (6, 7). Several lines of evidence suggest that the native 4D antigen plays a role in the pathogenesis of syphilis. 4D and immunologically related molecules have been detected only in T. pallidum and the other pathogenic treponemes (7, 23; unpublished data). Rabbit antisera to the purified recombinant molecule immobilize T. pallidum cells in vitro (7), and 4D antiserum has been used to demonstrate the surface location of the native 4D antigen by immunoelectron microscopy (22). Rabbits immunized intravenously with the recombinant protein develop significant partial protection against subsequent intradermal challenge (L. Borenstein, J. Radolf, T. Fehniger, J. Miller, and M. Lovett, submitted for publication).Previously we demonstrated that molecules identical to both the 19,000-dalton monomer and the 90,000-dalton proteinase K limit-digestion product of recombinant 4D could be identified in T. pallidum immunoblots reacted with recombinant 4D antiserum (7). The native form of 4D, however, was not identified. In this report we demonstrate that disulfide bonds mediate the higher oligomeric structure of both native and recombinant 4D. After reduction with 2-metcaptoethanol, the 190,000-dalton form of native 4D could be identified. We MATERIALS AND METHODS T. pallidum Nichols was maintained by intratesticular passage in New Zealand White rabbits without the use of cortisone acetate. T. pallidum for immunoblots was obtained from rabbits given daily intramuscular injections of cortisone ac...

Evidence for divalent cation (Ca2+)-stabilized oligomeric proteins and covalently bound protein-peptidoglycan complexes in the outer membrane of Rhizobium leguminosarum

“…A second characteristic of some of the outer membrane proteins of R. leguminosarum is the formation of SDSand heat-resistant oligomers, which require divalent cations for their stability. Outer membrane porins of other gramnegative bacteria also form SDS-resistant oligomers, mostly trimers (16,17), which are probably mostly stabilized by electrostatic interactions, whereas stabilization by disulfide bridges has been described for Treponema pallidum (20). However, in contrast to the oligomers described here, they are denatured in SDS at 100°C and usually even at lower temperatures (13).…”

Reactivity of Antibodies from Syphilis Patients to a Protein Array Representing theTreponema pallidumProteome

“…If the development of the humoral immune response between secondary and early latent syphilis in humans coincides with protective immunity, then the 11 proteins that exhibited reactivity only during early latency are of great interest. Four of the 11 proteins, including TP0163 (Mn 2ϩ /Mg 2ϩ ABC transport, periplasmic binding protein TroA), TP0216 (heat shock protein 70), TP0292 (conserved hypothetical protein), and TP1038 (bacterioferrin), have been previously identified as antigens (3,17,20). Five of the seven remaining novel antigens were also identified as antigens in our previous analysis using sera collected from rabbits (13).…”

Selective release of the Treponema pallidum outer membrane and associated polypeptides with Triton X-114

“…Immunoblots were prepared that contained samples of T. pallidum extracted with TX-114 in the presence or absence of 50 mM DTT and fractionated by the phase partitioning procedure. Figure 8 shows the results when the immunoblots were probed with affinity-purified anti-4D antiserum (23) mixed with an anti-47-kDa monoclonal antibody (15). The 4D antigen, detected as a 19-kDa monomer and 34-kDa dimer, remained with the insoluble cytoplasmic cylinders (Fig.…”