Protein tyrosine O-glycosylation – A rather unexplored prokaryotic glycosylation system

Department of NanoBiotechnology, ViennaInstitute of BioTechnology, Universität für Bodenkultur Wien, A-1190 Vienna,Austria.
Glycobiology (Impact Factor: 3.15). 03/2010; 20(6):787-98. DOI: 10.1093/glycob/cwq035
Source: PubMed


Glycosylation is a frequent and heterogeneous posttranslational protein modification occurring in all domains of life. While protein N-glycosylation at asparagine and Oglycosylation at serine, threonine or hydroxyproline residues have been studied in great detail, only few data are available on O-glycosidic attachment of glycans to the amino acid tyrosine. In this study, we describe the identification and characterization of a bacterial protein tyrosine O-glycosylation system. In the Gram-positive, mesophilic bacterium Paenibacillus alvei CCM 2051T, a polysaccharide consisting of [→3)-β-D-Galp-(1[α-DGlcp-(1→6)] →4)-β-D-ManpNAc-(1→] repeating units is O-glycosidically linked via an adaptor with the structure -[GroA-2→OPO2→4-β-D-ManpNAc-(1→4)] →3)-α-LRhap-(1→3)-α-L-Rhap-(1→3)-α-L-Rhap-(1→3)-β-DGalp-(1→ to specific tyrosine residues of the S-layer protein SpaA. A ~24.3-kb S-layer glycosylation (slg) gene cluster encodes the information necessary for the biosynthesis of this glycan chain within 18 open reading frames (ORF). The corresponding translation products are involved in the biosynthesis of nucleotide-activated monosaccharides, assembly and export as well as in the transfer of the completed polysaccharide chain to the S-layer target protein. All ORFs of the cluster, except those encoding the nucleotide sugar biosynthesis enzymes and the ATP binding cassette (ABC) transporter integral transmembrane proteins, were disrupted by the insertion of the mobile group II intron Ll. LtrB, and S-layer glycoproteins produced in mutant backgrounds were analyzed by mass spectrometry. There is evidence that the glycan chain is synthesized in a process comparable to the ABC-transporter-dependent pathway of the lipopolysaccharide O-polysaccharide biosynthesis.Furthermore, with the protein WsfB, we have identified an O-oligosaccharyl:protein transferase required for the formation of the covalent β-D-Gal→Tyr linkage between the glycan chain and the S-layer protein. © The Author 2010. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: [email protected]
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    • "The gene pamA (Gene ID: 18058116) from the pam gene cluster was disrupted in the genome of P. larvae DSM25430 via a recently described strategy (Zarschler et al. 2009). Vector pTT_wsfA243 (Zarschler et al. 2010) was used for constructing a targetron vector for targeted group II intron insertion at position 1080 from the start codon of pamA of the pam gene cluster. Retargeting of the LI.LtrB targetron of vector pTT_wsfA243 prior to transformation into P. larvae was performed following the manufacturer's protocol and essentially as already described for disruption of the P. larvae S-layer gene splA (Poppinga et al. 2012). "
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    ABSTRACT: Paenibacillus larvae is the etiological agent of American Foulbrood (AFB) a world-wide distributed devastating disease of the honey bee brood. Previous comparative genome analysis and more recently, the elucidation of the bacterial genome, provided evidence that this bacterium harbors putative functional nonribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs) and therefore, might produce nonribosomal peptides (NRPs) and polyketides (PKs). Such biosynthesis products have been shown to display a wide-range of biological activities such as antibacterial, antifungal or cytotoxic activity. Herein we present an in silico analysis of the first NRPS/PKS hybrid of P. larvae and we show the involvement of this cluster in the production of a compound named paenilamicin (Pam). For the characterization of its in vitro and in vivo bioactivity, a knock-out mutant strain lacking the production of Pam was constructed and subsequently compared to wild-type species. This led to the identification of Pam by mass spectrometry. Purified Pam-fractions showed not only antibacterial but also antifungal and cytotoxic activities. The latter suggested a direct effect of Pam on honey bee larval death which could, however, not be corroborated in laboratory infection assays. Bee larvae infected with the non-producing Pam strain showed no decrease in larval mortality, but a delay in the onset of larval death. We propose that Pam, although not essential for larval mortality, is a virulence factor of P. larvae influencing the time course of disease. These findings are not only of significance in elucidating and understanding host–pathogen interactions but also within the context of the quest for new compounds with antibiotic activity for drug development.
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    • "In the case of P. alvei CCM 2051 T , the corresponding enzyme is assumed to be the predicted membrane protein WsfB, the only protein from the slg gene locus, to which (in addition to WsaA, which is encoded in a very short upstream coding sequence) no distinct function could be attributed in the glycan biosynthesis so far. The architectture of the slg gene locus of P. alvei is similar to that of G. stearothermophilus, even though it is not a polycistronic cluster as is the case for G. stearothermophilus [17]. Sequence based annotation of the slg gene locus-enzymes identified WsfB as the putative O-OTase in this organism. "
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    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 bacterium Paenibacillus alvei CCM 2051(T) is necessary for the tailored glyco-functionalization of its S-layer. Here, the putative oligosaccharyl:S-layer protein transferase WsfB from the P. alvei S-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 tyrosine O-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 in P. alvei CCM 2051(T), 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 of O-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 in P. alvei ΔWsfB cells. Attempts to use WsfB for transferring heterologous oligosaccharides to its native S-layer target protein in Escherichia coli CWG702 and Salmonella enterica SL3749, 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 bacterial O-OTase, making it the most probable candidate for the oligosaccharyl:S-layer protein transferase of P. alvei, and a promising candidate for the first O-OTase reported in Gram-positives.
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    • "Glycosides of (2S,4R)-4-hydroxyproline (Hyp), tyrosine (Tyr) and hydroxylysine (Hyl) (Figure 2), however, do not undergo this elimination since they are not β-hydroxyacids. Glycosides of Tyr have long been known but are not so common (Zarschler et al. 2010). Glycosides of (2S,5R)-hydroxylysine are found predominantly in collagens and apparently only in mammals (Spiro et al. 1971; Butler 2008). "
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