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Identification of surface-exposed components of MOMP of Chlamydia trachomatis serovar F
Yan Wang1, Eric A Berg, Xiaogeng Feng
1Section of Infectious Diseases, Department of Medicine, Boston University School of Medicine, Boston, MA 02118, USA.
Abstract:
The identification of surface-exposed components of the major outer membrane protein (MOMP) of Chlamydia is critical for modeling its three-dimensional structure, as well as for understanding the role of MOMP in the pathogenesis of Chlamydia-related diseases. MOMP contains four variable domains (VDs). In this study, VDII and VDIV of Chlamydia trachomatis serovar F were proven to be surface-located by immuno-dot blot assay using monoclonal antibodies (MAbs). Two proteases, trypsin and endoproteinase Glu-C, were applied to digest the intact elementary body of serovar F under native conditions to reveal the surface-located amino acids. The resulting peptides were separated by SDS-PAGE and probed with MAbs against these VDs. N-terminal amino acid sequencing revealed: (1) The Glu-C cleavage sites were located within VDI (at Glu61) and VDIII (at Glu225); (2) the trypsin cleavage sites were found at Lys79 in VDI and at Lys224 in VDIII. The tryptic peptides were then isolated by HPLC and analyzed with a matrix-assisted laser desorption/ionization time-of-flight mass spectrometer and a quadrupole-orthogonal-TOF mass spectrometer coupled with a capillary liquid chromatograph. Masses and fragmentation patterns that correlated to the peptides cleaved from VDI and VDIII regions, and C-terminal peptides Ser333-Arg358 and Ser333-Lys350 were observed. This result demonstrated that these regions are surface-exposed. Data derived from comparison of nonreduced outer membrane complex proteolytic fragments with their reduced fractions revealed that Cys26, 29, 33, 116, 208, and 337 were involved in disulfide bonds, and Cys26 and 337, and 116 and 208 were paired. Based on these data, a new two-dimensional model is proposed.
Insights
This study identifies surface-exposed regions of Chlamydia trachomatis major outer membrane protein (MOMP) using protease digestion and mass spectrometry. These findings are crucial for understanding Chlamydia pathogenesis and developing structural models.
Area of Science:
- Microbiology
- Structural Biology
- Immunology
Background:
- The major outer membrane protein (MOMP) of Chlamydia is a key target for understanding pathogenesis and developing vaccines.
- Identifying surface-exposed regions of MOMP is crucial for structural modeling and understanding Chlamydia-host interactions.
Purpose of the Study:
- To identify and map the surface-exposed variable domains (VDs) of Chlamydia trachomatis serovar F MOMP.
- To elucidate the role of specific amino acid residues and disulfide bonds in MOMP's surface structure.
Main Methods:
- Immuno-dot blot assays using monoclonal antibodies (MAbs) to confirm surface localization of VDII and VDIV.
- Proteolytic digestion of intact elementary bodies with trypsin and endoproteinase Glu-C under native conditions.
- Peptide analysis using SDS-PAGE, N-terminal sequencing, HPLC, and mass spectrometry (MALDI-TOF and LC-Q-TOF).
Main Results:
- VDII and VDIV were confirmed as surface-located.
- Specific cleavage sites for Glu-C (Glu61, Glu225) and trypsin (Lys79, Lys224) were identified within VDI and VDIII.
- Surface-exposed peptides, including C-terminal regions (Ser333-Arg358, Ser333-Lys350), were identified.
- Disulfide bonds involving Cys26, 29, 33, 116, 208, and 337 were characterized, with specific pairings identified (Cys26-Cys337, Cys116-Cys208).
Conclusions:
- The study provides critical data on the surface topology of Chlamydia MOMP.
- A new two-dimensional model of MOMP is proposed based on the experimental findings.
- Understanding MOMP surface exposure is vital for Chlamydia disease mechanism research.
