Acylation of the 47-kilodalton major membrane immunogen of Treponema pallidum determines its hydrophobicity

Abstract

The 47-kilodalton (kDa) major integral membrane immunogen of Treponema pallidum was recently found to be a proteolipid. Similar two-dimensional electrophoretic mobilities and common hydrophobic properties displayed by the native (T. pallidum) and recombinant (Escherichia coli) 47-kDa antigens suggested that the recombinant antigen also possesses covalently bound lipid. Both intact E. coli and E. coli minicells acylated the 47-kDa antigen; immunoprecipitation with a monoclonal antibody specific for the 47-kDa immunogen supported the contention that the acylated product of E. coli corresponds to the cloned T. pallidum antigen. Triton X-114 phase partitioning was used to compare the relative hydrophobicities of 47-kDa molecules synthesized by in vitro and in vivo protein translation systems. The products synthesized by T. pallidum, intact E. coli, or E. coli minicells were hydrophobic, while the protein synthesized in an E. coli cell-free translation system was hydrophilic. Processing experiments with E. coli suggested that the primary gene translation product of the protein is not synthesized in a precursor form, unlike other bacterial proteolipids. These results indicate that the hydrophobicity of the 47-kDa integral membrane protein is conferred substantially by the covalently attached lipid(s). The biochemical similarities between the native and recombinant 47-kDa proteolipids will provide a foundation for future investigations into the structure and immunogenicity of this integral membrane protein of T. pallidum.

Analysis of the N-terminal region of the 47-kilodalton integral membrane lipoprotein of Treponema pallidum

“…In fact, earlier DNA sequence analysis suggested that the molecule was markedly hydrophilic (absence of membrane-spanning domains) (15). The subsequent finding of acylation (3,4) clarified the previously observed hydrophobic character of the molecule (5,26). However, in the DNA sequence reported by Hsu et al (15), no sequences characteristic of signal peptides or of a bacterial lipoproteinspecific signal peptidase II processing site were readily identifiable in the DNA or corresponding amino acid sequences.…”

“…Figure 6 shows that the electrophoretic mobility of the nonacylated form of the protein is similar to that of the native 47-kDa antigen. Previous studies had shown that the 47-kDa antigen migrates as a doublet on SDS-PAGE because of occasional translational readthrough of the first of two stop codons in the mRNA (4). The larger of these two forms of the protein is visible only when the gel is overloaded (4,5).…”

“…MAb 11E3 (immunoglobulin G 2b) is specific for the T. pallidum 47-kDa lipoprotein (18,24). Antiserum to the carboxy-terminal region corresponding to the region between the two stop codons (peptide 2) was generated from synthetic peptides and has been previously described (4). Horseradish peroxidase conjugates of goat anti-mouse, rabbit anti-goat, goat anti-rabbit, and goat anti-human immunoglobulins were purchased from Jackson Immunoresearch Laboratories, Inc. (West Grove, Pa.).…”

“…However, in the DNA sequence reported by Hsu et al (15), no sequences characteristic of signal peptides or of a bacterial lipoproteinspecific signal peptidase II processing site were readily identifiable in the DNA or corresponding amino acid sequences. Studies using the processing inhibitors carbonyl cyanide m-chlorophenylhydrazone and ethanol did not reveal a precursor form of this protein (4). Interestingly, attempts to visualize a precursor form of the T. pallidum 35.5-kDa lipoprotein (16).…”

Molecular cloning, expression, and DNA sequence analysis of the gene that encodes the 16-kilodalton outer membrane lipoprotein of Serpulina hyodysenteriae

“…It is proposed that this outer membrane lipoprotein of S. hyodysentenae be designated SmpA (Serpulina membrane protein A) and that the gene that encodes it be designated, smpA. T. pallidum also possesses membrane-located lipoproteins (5,6,31), although these lipoproteins do not appear to be present on the surface of the spirochete (8). On the other hand, the variable lipoproteins (Vmp) of Borrelia hernsii are surface orientated and confer the property of antigenic variation on the organism, thus enabling the organism to evade the immune mechanisms of the host (2,30).…”