Two protein lysine methyltransferases methylate outer membrane protein B from Rickettsia

Abstract

Rickettsia prowazekii, the etiologic agent of epidemic typhus, is a potential biological threat agent. Its outer membrane protein B (OmpB) is an immunodominant antigen and plays roles as protective envelope and as adhesins. The observation of the correlation between methylation of lysine residues in rickettsial OmpB and bacterial virulence has suggested the importance of an enzymatic system for the methylation of OmpB. However, no rickettsial lysine methyltransferase has been characterized. Bioinformatic analysis of genomic DNA sequences of Rickettsia identified putative lysine methyltransferases. The genes of the potential methyltransferases were synthesized, cloned, and expressed in Escherichia coli, and expressed proteins were purified by nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography. The methyltransferase activities of the purified proteins were analyzed by methyl incorporation of radioactively labeled S-adenosylmethionine into recombinant fragments of OmpB. Two putative recombinant methyltransferases (rRP789 and rRP027-028) methylated recombinant OmpB fragments. The specific activity of rRP789 is 10- to 30-fold higher than that of rRP027-028. Western blot analysis using specific antibodies against trimethyl lysine showed that both rRP789 and rRP027-028 catalyzed trimethylation of recombinant OmpB fragments. Liquid chromatography-tandem mass spectrometry (LC/MS-MS) analysis showed that rRP789 catalyzed mono-, di-, and trimethylation of lysine, while rRP027-028 catalyzed exclusively trimethylation. To our knowledge, rRP789 and rRP027-028 are the first biochemically characterized lysine methyltransferases of outer membrane proteins from Gram-negative bacteria. The production and characterization of rickettsial lysine methyltransferases provide new tools to investigate the mechanism of methylation of OmpB, effects of methylation on the structure and function of OmpB, and development of methylated OmpB-based diagnostic assays and vaccine candidates.

Fig 1

Molecular models of the SAM binding domains of R. prowazekii Madrid E RP789 and R. prowazekii RP22 RP027-028. Molecular models of the putative SAM binding domains RP789(Y48–L213) and RP027-028(K3–L185) with bound SAM were generated using the amino acid sequences of RP789 and RP027-028 and the Web server of 3DLigandSite. The protein is in ribbons, and SAM is in ball and stick models. Figures were generated using Chimera.

Fig 2

Time courses of methylation of recombinant rOmpB(AN) and rOmpB(K) by rRP789 (A) or rRP027-028 (B). (A) rRP789 catalyzed methylation of 2 μM rOmpB(AN) (●) or 1 μM rOmpB(K) (■) in the presence of 0.16 mM [³H]SAM (68 mCi/mmol), 0.2 mM DTT, and 8.3 mM sodium phosphate (pH 8.0). (B) rRP027-028 catalyzed methylation of rOmpB(AN) (—●—) and rOmpB(K) (—■—) under the same conditions as for panel A. The concentrations of rRP789 and rRP027-028 were equal to the concentration of the rOmpB fragment. Controls containing AN only (--●--), K only (--■--), and methyltransferase enzyme only () are also shown.

Fig 3

Western blot analysis of rRP789 and rRP027-028 catalyzed methylation of rOmpB(AN) and rOmpB(K). Western blot analysis was carried out for rOmpB(AN) and rOmpB(K) before and after methylation by rRP789 and rRP027-028. The upper panel is an SDS-polyacrylamide gel, and the lower panel is the Western blot. Reaction mixtures included 0.15 μM rRP789 or rRP027-028, 7 μg of rOmpB(AN) or rOmpB(K), and 0.64 mM SAM in 8.3 mM sodium phosphate (pH 8.0) and water in a total volume of 50 μl. The samples “AN only” (lane 1) and “K only” (lane 4) did not contain any enzyme, and the samples “rRP789 only” and “rRP027-028” (lanes 7 and 8, respectively) did not contain a protein substrate.