title: Description of a Putative Oligosaccharyl:S-Layer Protein Transferase from the TyrosineO-Glycosylation System ofPaenibacillus alveiCCM 2051T.
abstract: Surface (S)-layer proteins are model systems for studying protein glycosylation in bacteria and simultaneously hold promises for the design of novel, glyco-functionalized modules for nanobiotechnology due to their 2D self-assembly capability. Understanding the mechanism governing S-layer glycan biosynthesis in the Gram-positive bacteriumPaenibacillus alveiCCM 2051Tis necessary for the tailored glyco-functionalization of its S-layer. Here, the putative oligosaccharyl:S-layer protein transferase WsfB from theP. alveiS-layer glycosylation gene locus is characterized. The enzyme is proposed to catalyze the final step of the glycosylation pathway, transferring the elongated S-layer glycan onto distinct tyrosineO-glycosylation sites. Genetic knock-out of WsfB is shown to abolish glycosylation of the S-layer protein SpaA but not that of other glycoproteins present inP. alveiCCM 2051T, confining its role to the S-layer glycosylation pathway. A transmembrane topology model of the 781-amino acid WsfB protein is inferred from activity measurements of green fluorescent protein and phosphatase A fused to defined truncations of WsfB. This model shows an overall number of 13 membrane spanning helices with the Wzy_C domain characteristic ofO-oligosaccharyl:protein transferases (O-OTases) located in a central extra-cytoplasmic loop, which both compares well to the topology of OTases from Gram-negative bacteria. Mutations in the Wzy_C motif resulted in loss of WsfB function evidenced in reconstitution experiments inP. alveiΔWsfB cells. Attempts to use WsfB for transferring heterologous oligosaccharides to its native S-layer target protein inEscherichia coliCWG702 andSalmonella entericaSL3749, which should provide lipid-linked oligosaccharide substrates mimicking to some extent those of the natural host, were not successful, possibly due to the stringent function of WsfB. Concluding, WsfB has all features of a bacterialO-OTase, making it the most probable candidate for the oligosaccharyl:S-layer protein transferase ofP. alvei, and a promising candidate for the firstO-OTase reported in Gram-positives.
authors.affiliation: Department of NanoBiotechnology, NanoGlycobiology Unit, Universität für Bodenkultur Wien, Wien, Austria.
authors.forename: Robin
authors.initials: R
authors.lastname: Ristl
authors.affiliation: Department of NanoBiotechnology, NanoGlycobiology Unit, Universität für Bodenkultur Wien, Wien, Austria.
authors.forename: Bettina
authors.initials: B
authors.lastname: Janesch
authors.affiliation: Department of NanoBiotechnology, NanoGlycobiology Unit, Universität für Bodenkultur Wien, Wien, Austria.
authors.forename: Julia
authors.initials: J
authors.lastname: Anzengruber
authors.affiliation: Department of NanoBiotechnology, NanoGlycobiology Unit, Universität für Bodenkultur Wien, Wien, Austria.
authors.forename: Agnes
authors.initials: A
authors.lastname: Forsthuber
authors.affiliation: Department of NanoBiotechnology, NanoGlycobiology Unit, Universität für Bodenkultur Wien, Wien, Austria.
authors.forename: Johanna
authors.initials: J
authors.lastname: Blaha
authors.affiliation: Department of NanoBiotechnology, NanoGlycobiology Unit, Universität für Bodenkultur Wien, Wien, Austria.
authors.forename: Paul
authors.initials: P
authors.lastname: Messner
authors.affiliation: Department of NanoBiotechnology, NanoGlycobiology Unit, Universität für Bodenkultur Wien, Wien, Austria.
authors.forename: Christina
authors.initials: C
authors.lastname: Schäffer
keywords: Bacterial Glycosylation; Oligosaccharyl Transferase; S-Layer; Trans-Membrane Topology; Tyrosine-O-Glycosylation