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Evidence that the major outer membrane protein of Chlamydia trachomatis is glycosylated
1Department of Pathobiology, University of Washington, Seattle 98195.
Abstract:
The major outer membrane protein (MOMP) of Chlamydia trachomatis was determined to be a glycoprotein on the basis of susceptibility to glycosidase digestion and the presence of carbohydrate by staining and radiolabeling. The MOMP of the serovar L2 organisms was isolated by electroelution from the protein band excised from the gel after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The incubation of MOMP with N-glycosidase F, an endoglycosidase that cleaves the N-glycan, and periodate resulted in two new molecular weight species. While MOMP treated with N-glycosidase F showed a lower-molecular-weight mobility, the periodate-treated MOMP increased in molecular weight. Both treatments abolished the ability of the MOMP to bind to HeLa cell components. In the immunoblot, the reactivity to the monoclonal antibody specific against the C. trachomatis species was preserved. The endoglycosidase specific to O-linked glycan, endo-alpha-N-acetylgalactosaminidase, had no visible effect on the isolated MOMP. Carbohydrate was detected in the MOMP by p-phenylenediamine staining of the protein band in the gel following SDS-PAGE. Autoradiograms of proteins of chlamydial organisms metabolically labeled with [3H]galactose or [3H]glucosamine and separated by SDS-PAGE revealed the MOMP band. The isolated MOMP was shown to bind specifically to concanavalin A, wheat germ agglutinin, and Dolichos biflorus agglutinin in the lectin binding assay. No binding was observed with Ulex europaeus agglutinin I, soybean agglutinin, or Ricinus communis agglutinin.
Insights
The major outer membrane protein (MOMP) of Chlamydia trachomatis is a glycoprotein. This finding was confirmed through glycosidase digestion and carbohydrate detection methods, impacting its cellular binding properties.
Area of Science:
- Microbiology
- Glycobiology
- Cell Biology
Background:
- The major outer membrane protein (MOMP) is crucial for Chlamydia trachomatis pathogenesis.
- Understanding MOMP's post-translational modifications is key to elucidating its function.
Purpose of the Study:
- To determine if Chlamydia trachomatis MOMP is a glycoprotein.
- To characterize the glycosylation of MOMP and its impact on function.
Main Methods:
- Isolation of MOMP using SDS-PAGE and electroelution.
- Enzymatic digestion with N-glycosidase F and endo-alpha-N-acetylgalactosaminidase.
- Periodate treatment and lectin binding assays.
- Metabolic labeling with radioactive galactose and glucosamine.
- Immunoblotting with species-specific monoclonal antibody.
Main Results:
- MOMP susceptibility to glycosidase digestion and positive carbohydrate staining confirmed its glycoprotein nature.
- N-glycosidase F treatment reduced molecular weight, while periodate treatment increased it.
- Both treatments abolished MOMP binding to HeLa cell components.
- Lectin binding assays showed specific interactions with certain carbohydrate-binding proteins (lectins).
- Monoclonal antibody reactivity was preserved post-treatment.
Conclusions:
- Chlamydia trachomatis MOMP is a glycoprotein, likely N-linked glycosylated.
- Glycosylation of MOMP influences its interaction with host cell components.
- Further investigation into MOMP glycosylation can reveal new therapeutic targets.