Functional Characterization of Bacterial Oligosaccharyltransferases Involved in O-linked Protein Glycosylation

Overview

Journal J Bacteriol

Publisher American Society for Microbiology

Specialty Microbiology

Date 2007 Sep 25

PMID 17890310

Citations 75

Authors

Amirreza Faridmoayer

Messele A Fentabil

Dominic C Mills

John S Klassen

Mario F Feldman

Affiliations

Soon will be listed here.

Abstract

Protein glycosylation is an important posttranslational modification that occurs in all domains of life. Pilins, the structural components of type IV pili, are O glycosylated in Neisseria meningitidis, Neisseria gonorrhoeae, and some strains of Pseudomonas aeruginosa. In this work, we characterized the P. aeruginosa 1244 and N. meningitidis MC58 O glycosylation systems in Escherichia coli. In both cases, sugars are transferred en bloc by an oligosaccharyltransferase (OTase) named PglL in N. meningitidis and PilO in P. aeruginosa. We show that, like PilO, PglL has relaxed glycan specificity. Both OTases are sufficient for glycosylation, but they require translocation of the undecaprenol-pyrophosphate-linked oligosaccharide substrates into the periplasm for activity. Whereas PilO activity is restricted to short oligosaccharides, PglL is able to transfer diverse oligo- and polysaccharides. This functional characterization supports the concept that despite their low sequence similarity, PilO and PglL belong to a new family of "O-OTases" that transfer oligosaccharides from lipid carriers to hydroxylated amino acids in proteins. To date, such activity has not been identified for eukaryotes. To our knowledge, this is the first report describing recombinant O glycoproteins synthesized in E. coli.

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References

Wacker M, Linton D, Hitchen P, Nita-Lazar M, Haslam S, North S . N-linked glycosylation in Campylobacter jejuni and its functional transfer into E. coli. Science. 2002; 298(5599):1790-3. DOI: 10.1126/science.298.5599.1790. View

Lefebre M, Valvano M . Construction and evaluation of plasmid vectors optimized for constitutive and regulated gene expression in Burkholderia cepacia complex isolates. Appl Environ Microbiol. 2002; 68(12):5956-64. PMC: 134411. DOI: 10.1128/AEM.68.12.5956-5964.2002. View

Brennan P . Structure, function, and biogenesis of the cell wall of Mycobacterium tuberculosis. Tuberculosis (Edinb). 2003; 83(1-3):91-7. DOI: 10.1016/s1472-9792(02)00089-6. View

Power P, Roddam L, Rutter K, Fitzpatrick S, Srikhanta Y, Jennings M . Genetic characterization of pilin glycosylation and phase variation in Neisseria meningitidis. Mol Microbiol. 2003; 49(3):833-47. DOI: 10.1046/j.1365-2958.2003.03602.x. View

Banerjee A, Ghosh S . The role of pilin glycan in neisserial pathogenesis. Mol Cell Biochem. 2003; 253(1-2):179-90. DOI: 10.1023/a:1026058311857. View

Kus J, Tullis E, Cvitkovitch D, Burrows L . Significant differences in type IV pilin allele distribution among Pseudomonas aeruginosa isolates from cystic fibrosis (CF) versus non-CF patients. Microbiology (Reading). 2004; 150(Pt 5):1315-1326. DOI: 10.1099/mic.0.26822-0. View

Helenius A, Aebi M . Roles of N-linked glycans in the endoplasmic reticulum. Annu Rev Biochem. 2004; 73:1019-49. DOI: 10.1146/annurev.biochem.73.011303.073752. View

Hegge F, Hitchen P, Aas F, Kristiansen H, Lovold C, Egge-Jacobsen W . Unique modifications with phosphocholine and phosphoethanolamine define alternate antigenic forms of Neisseria gonorrhoeae type IV pili. Proc Natl Acad Sci U S A. 2004; 101(29):10798-803. PMC: 490014. DOI: 10.1073/pnas.0402397101. View

NEUHARD J, Thomassen E . Altered deoxyribonucleotide pools in P2 eductants of Escherichia coli K-12 due to deletion of the dcd gene. J Bacteriol. 1976; 126(2):999-1001. PMC: 233240. DOI: 10.1128/jb.126.2.999-1001.1976. View

10.

Finlay B, Pasloske B, Paranchych W . Expression of the Pseudomonas aeruginosa PAK pilin gene in Escherichia coli. J Bacteriol. 1986; 165(2):625-30. PMC: 214465. DOI: 10.1128/jb.165.2.625-630.1986. View

11.

Virji M, Heckels J, POTTS W, Hart C, Saunders J . Identification of epitopes recognized by monoclonal antibodies SM1 and SM2 which react with all pili of Neisseria gonorrhoeae but which differentiate between two structural classes of pili expressed by Neisseria meningitidis and the distribution of.... J Gen Microbiol. 1989; 135(12):3239-51. DOI: 10.1099/00221287-135-12-3239. View

12.

Liu D, REEVES P . Escherichia coli K12 regains its O antigen. Microbiology (Reading). 1994; 140 ( Pt 1):49-57. DOI: 10.1099/13500872-140-1-49. View

13.

Virji M, Saunders J, SIMS G, Makepeace K, Maskell D, Ferguson D . Pilus-facilitated adherence of Neisseria meningitidis to human epithelial and endothelial cells: modulation of adherence phenotype occurs concurrently with changes in primary amino acid sequence and the glycosylation status of pilin. Mol Microbiol. 1993; 10(5):1013-28. DOI: 10.1111/j.1365-2958.1993.tb00972.x. View

14.

Castric P . pilO, a gene required for glycosylation of Pseudomonas aeruginosa 1244 pilin. Microbiology (Reading). 1995; 141 ( Pt 5):1247-1254. DOI: 10.1099/13500872-141-5-1247. View

15.

Stimson E, Virji M, Makepeace K, Dell A, Morris H, Payne G . Meningococcal pilin: a glycoprotein substituted with digalactosyl 2,4-diacetamido-2,4,6-trideoxyhexose. Mol Microbiol. 1995; 17(6):1201-14. DOI: 10.1111/j.1365-2958.1995.mmi_17061201.x. View

16.

Aebi M, Gassenhuber J, Domdey H, te Heesen S . Cloning and characterization of the ALG3 gene of Saccharomyces cerevisiae. Glycobiology. 1996; 6(4):439-44. DOI: 10.1093/glycob/6.4.439. View

17.

Dykxhoorn D, St Pierre R, Linn T . A set of compatible tac promoter expression vectors. Gene. 1996; 177(1-2):133-6. DOI: 10.1016/0378-1119(96)00289-2. View

18.

Jensen O, Podtelejnikov A, Sagliocco F, Wilm M, Vorm O, Mortensen P . Linking genome and proteome by mass spectrometry: large-scale identification of yeast proteins from two dimensional gels. Proc Natl Acad Sci U S A. 1996; 93(25):14440-5. PMC: 26151. DOI: 10.1073/pnas.93.25.14440. View

19.

Fisher C, Pei G . Modification of a PCR-based site-directed mutagenesis method. Biotechniques. 1997; 23(4):570-1, 574. DOI: 10.2144/97234bm01. View

20.

Marceau M, Forest K, Beretti J, Tainer J, Nassif X . Consequences of the loss of O-linked glycosylation of meningococcal type IV pilin on piliation and pilus-mediated adhesion. Mol Microbiol. 1998; 27(4):705-15. DOI: 10.1046/j.1365-2958.1998.00706.x. View