Sequence analysis of the 47-kilodalton major integral membrane immunogen of Treponema pallidum
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P L Hsu1, N R Chamberlain, K Orth
1Department of Pathology, University of Texas Health Science Center, Houston 77030.
Abstract:
The complete primary amino acid sequence for the 47-kilodalton (kDa) major integral membrane immunogen of Treponema pallidum subsp. pallidum was obtained by using a combined strategy of DNA sequencing (of the cloned gene in Escherichia coli) and N-terminal amino acid sequencing of the native (T. pallidum subsp. pallidum-derived) antigen. An open reading frame believed to encode the 47-kDa antigen comprised 367 amino acid codons, which gave rise to a calculated molecular weight for the corresponding antigen of 40,701. Of the 367 amino acids, 113 (31%) were sequenced by N-terminal amino acid sequencing of trypsin and hydroxylamine cleavage fragments of the native molecule isolated from T. pallidum subsp. pallidum; amino acid sequence data had a 100% correlation with that of the amino acid sequence predicted from DNA sequencing of the cloned gene in E. coli. Although no consensus sequences for the initiation of transcription or translation were readily identifiable immediately 5' to the putative methionine start codon, a 63-base-pair PstI fragment located 159 nucleotides upstream was required for expression of the 47-kDa antigen in E. coli. The 47-kDa antigen sequence did not reveal a typical leader sequence. The overall G+C content for the DNA corresponding to the structural gene was 53%. Hydrophilicity analysis identified at least one major hydrophilic domain of the protein near the N terminus of the molecule which potentially represents an immunodominant epitope. No repetitive primary sequence epitopes were found. The combined data provide the molecular basis for further structural and functional studies regarding the role of the antigen in the immunopathogenesis of treponemal disease.
Insights
Researchers determined the amino acid sequence of a key Treponema pallidum antigen using DNA and protein sequencing. This 47-kilodalton antigen
Area of Science:
- Microbiology and Immunology
- Molecular Biology
- Genetics
Background:
- Treponema pallidum subsp. pallidum is the causative agent of syphilis.
- Integral membrane proteins of T. pallidum are crucial for understanding its pathogenesis and developing diagnostics.
- The 47-kilodalton (kDa) antigen is a major immunogen in T. pallidum infections.
Purpose of the Study:
- To elucidate the complete primary amino acid sequence of the 47-kDa major integral membrane immunogen of Treponema pallidum subsp. pallidum.
- To correlate DNA sequence data with N-terminal amino acid sequencing of the native antigen.
- To identify potential immunodominant epitopes and understand the molecular basis of antigen function.
Main Methods:
- Combined strategy of DNA sequencing of the cloned gene in Escherichia coli.
- N-terminal amino acid sequencing of the native antigen isolated from T. pallidum subsp. pallidum.
- Analysis of antigen expression in E. coli, including identification of regulatory DNA fragments.
- Hydrophilicity analysis to predict potential immunodominant epitopes.
Main Results:
- The complete primary amino acid sequence of the 47-kDa antigen was determined, comprising 367 codons with a calculated molecular weight of 40,701 Da.
- Amino acid sequence data from native fragments showed 100% correlation with the sequence predicted from the cloned gene.
- A 63-base-pair PstI fragment upstream of the gene was found to be essential for antigen expression in E. coli.
- Hydrophilicity analysis revealed a potential immunodominant epitope near the N-terminus; no repetitive epitopes were identified.
Conclusions:
- The study provides the complete molecular sequence of a major T. pallidum immunogen.
- The findings establish a molecular basis for future studies on the antigen's structure, function, and role in syphilis pathogenesis.
- This detailed sequence information is vital for developing targeted diagnostics and potential vaccines against syphilis.