title: Functional identification of a galactosyltransferase critical to Bacteroides fragilis Capsular Polysaccharide A biosynthesis.
abstract: Capsular Polysaccharide A (CPSA), a polymer of a four-sugar repeating unit that coats the surface of the mammalian symbiont Bacteroides fragilis, has therapeutic potential in animal models of Multiple Sclerosis and other autoinflammatory diseases. Genetic studies have demonstrated that CPSA biosynthesis is dependent primarily on a single gene cluster within the B. fragilis genome. However, the precise functions of the individual glycosyltransferases encoded by this cluster have not been identified. In this report each of these glycosyltransferases (WcfQ, WcfP, and WcfN) have been expressed and tested for their function in vitro. Using a reverse phase high performance liquid chromatography (HPLC) assay, WcfQ and WcfP were found to transfer galactose from uridine diphosphate (UDP)-linked galactose (Gal) to N-acetyl-4-amino-6-deoxy-galactosamine (AADGal) linked to a fluorescent mimic of bactoprenyl diphosphate, the native isoprenoid anchor for bacterial polysaccharide biosynthesis. The incorporation of galactose to form a bactoprenyl-linked disaccharide was confirmed by radiolabel incorporation and mass spectrometry (MS) of purified product. Using varying concentrations of UDP-Gal and enzyme, WcfQ was found to be the most effective protein at transferring galactose, and is the most likely candidate for in vivo incorporation of the sugar. WcfQ also cooperated in the presence of three preceding biosynthetic enzymes to form an isoprenoid-linked disaccharide in a single-pot reaction. This work represents a critical step in understanding the biosynthetic pathway responsible for the formation of CPSA, an unusual and potentially therapeutic biopolymer.
authors.affiliation: Department of Chemistry, University of North Carolina at Charlotte, 9201 University City Blvd., Charlotte, NC 28223, United States; Center for Biomedical Engineering and Sciences, University of North Carolina at Charlotte, 9201 University City Blvd., Charlotte, NC 28223, United States. Electronic address: Jerry.Troutman@uncc.edu.
authors.forename: Jerry M
authors.initials: JM
authors.lastname: Troutman
authors.affiliation: Department of Biology, University of North Carolina at Charlotte, 9201 University City Blvd., Charlotte, NC 28223, United States.
authors.forename: Sunita
authors.initials: S
authors.lastname: Sharma
authors.affiliation: Department of Chemistry, University of North Carolina at Charlotte, 9201 University City Blvd., Charlotte, NC 28223, United States.
authors.forename: Katelyn M
authors.initials: KM
authors.lastname: Erickson
authors.affiliation: Department of Chemistry, University of North Carolina at Charlotte, 9201 University City Blvd., Charlotte, NC 28223, United States.
authors.forename: Christina D
authors.initials: CD
authors.lastname: Martinez
mesh_terms: D016667:Bacterial Capsules / Q000737:chemistry; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D001441:Bacteroides fragilis / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005690:Galactose / Q000031:analogs & derivatives / Q000378:metabolism; D015870:Gene Expression; D016680:Genome, Bacterial; D016695:Glycosyltransferases / Q000235:genetics* / Q000378:metabolism; D005810:Multigene Family; D011135:Polysaccharides, Bacterial / Q000096:biosynthesis* / Q000737:chemistry; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013729:Terpenes / Q000378:metabolism; D014530:Uridine Diphosphate / Q000031:analogs & derivatives / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D011135:Polysaccharides, Bacterial; D011994:Recombinant Proteins; D013729:Terpenes; D014530:Uridine Diphosphate; D016695:Glycosyltransferases; D005690:Galactose
keywords: Capsule; Exopolysaccharide; Glycosyltransferase; Isoprenoid; Polysaccharide