Identification of surface-exposed components of MOMP of Chlamydia trachomatis serovar F.
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.
Wang
Yan
Y
Section of Infectious Diseases, Department of Medicine, Boston University School of Medicine, Boston, MA 02118, USA.
Berg
Eric A
EA
Feng
Xiaogeng
X
Shen
Li
L
Smith
Temple
T
Costello
Catherine E
CE
Zhang
You-xun
YX
D000595:Amino Acid Sequence; D015153:Blotting, Western; D002473:Cell Wall / Q000737:chemistry* / Q000378:metabolism; D002692:Chlamydia trachomatis / Q000737:chemistry* / Q000378:metabolism; D019295:Computational Biology; D003545:Cysteine / Q000378:metabolism; D015151:Immunoblotting; D008958:Models, Molecular; D008969:Molecular Sequence Data; D018272:Porins / Q000737:chemistry* / Q000378:metabolism; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D020539:Sequence Analysis, Protein; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D018272:Porins; C071677:omp1 protein, Chlamydia trachomatis; D003545:Cysteine