Functional Characterization of Proteins from a Putative Chondroitin Sulfate A Degrading Operon and the Crystal Structure of an Exo-N-Acetylgalactosamine-4-sulfatase from Tannerella forsythia
Master of Science (MSc)
Biological and Chemical Sciences
Faculty of Science
Dr. Michael Suits
Dr. Geoff Horsman
Dr. Matthew Smith
Advanced periodontitis has been shown to have strong associations with the residence of a bacterial triad of Porphyromonas gingivalis, Tannerella forsythia, and Treponema denticola also known as the red complex. These bacteria were previously thought to be asaccharolytic, making them better suited to survive in deep in periodontal pockets where access to carbohydrates from food is limited. Information gathered from genomic analysis suggested that upregulation of an operon (BFO2285-BFO2295, BFO3043) could confer the capacity for T. forsythia to degrade chondroitin-4-sulfate, the principle glycosaminoglycan of alveolar bone tissue. Bioinformatics analysis suggested the operon was similar to an operon found in Bacteroidetes thetaiotaomicron which is an organism closely related to T. forsythia. Using these regions of synteny, a model of the putative chondroitin sulfate A degrading pathway and predicted protein localization of each protein was constructed based on B. thetaiotaomicron. BFO2285, BFO2290, and BFO2291 were recombinantly expressed and purified from Escherichia coli. BFO2291 and BFO2290 were both crystallized, and the crystal structure of BFO2290 was determined to a resolution of 2.85 Å showing a conserved α/β fold typical of bacterial carbohydrate arylsulfatases. Michaelis-Menten kinetic characterization of BFO2290 determined that it was an exolytic-O4 sulfatase, with a reported Km of 0.75 mg/ml ± 0.60 mg/ml, Kcat of 3.74 min-1 ± 0.88 min-1, and Vmax of 7.48 μM/min ± 1.76 μM/min. BFO2285 was believed to have been successfully purified, however wasn’t crystallized and lacked detectable catalytic activity. Analysis conducted using fluorophore assisted carbohydrate electrophoresis (FACE) suggested that both BFO2291 and activated BFO2290 were involved in the putative chondroitin sulfate A degrading pathway. BFO2291 was shown to be active as an endolytic chondroitin sulfate A lyase, which generated a variety of fragment sizes of chondroitin sulfate A, while activated BFO2290 was shown to be an exolytic N-acetylgalactosamine-4-sulfatase. However, FACE analysis and attempted kinetic assays conducted using BFO2285 suggested the protein was catalytically inactive, and the experiments conducted to determine its role in the putative chondroitin sulfate A degrading pathway remain inconclusive. The characterizations proteins of this putative chondroitin sulfate A degrading mechanism from T. forsythia suggest that T. forsythia has the capacity to degrade chondroitin sulfate A, which could be linked to alveolar bone loss in patients with severe periodontitis.
Nguyen, Peter, "Functional Characterization of Proteins from a Putative Chondroitin Sulfate A Degrading Operon and the Crystal Structure of an Exo-N-Acetylgalactosamine-4-sulfatase from Tannerella forsythia" (2020). Theses and Dissertations (Comprehensive). 2332.
Available for download on Friday, September 29, 2023