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J. Biol. Chem., Vol. 281, Issue 38, 27712-27723, September 22, 2006

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Neisseria gonorrhoeae Type IV Pili Undergo Multisite, Hierarchical Modifications with Phosphoethanolamine and Phosphocholine Requiring an Enzyme Structurally Related to Lipopolysaccharide Phosphoethanolamine Transferases^*

Finn Erik Aas, Wolfgang Egge-Jacobsen^¶1, Hanne C. Winther-Larsen, Cecilia Løvold, Paul G. Hitchen^||, Anne Dell^||2, and Michael Koomey³

From the Centre for Molecular Biology and Neuroscience, Department of Molecular Biosciences, University of Oslo, 0316 Oslo, Norway, ^¶Biotechnology Centre of Oslo, University of Oslo, 0317 Oslo, Norway, and ^||Division of Molecular Biosciences, Faculty of Natural Sciences, Imperial College, London SW7 2AZ, United Kingdom

The zwitterionic phospho-forms phosphoethanolamine and phosphocholine are recognized as influential and important substituents of pathogen cell surfaces. PilE, the major pilin subunit protein of the type IV pilus (Tfp) colonization factor of Neisseria gonorrhoeae undergoes unique, post-translational modifications with these moieties. These phospho-form modifications have been shown to be O-linked alternately to a specific, conserved serine residue of PilE. However, the enzymes and precursors involved in their addition are unknown, and the full spectrum of PilE post-translational modifications has yet to be defined. Here, an intact protein-based mass spectrometric approach was integrated with bioinformatics and reverse genetics to address these matters. Specifically we show that a protein limited in its distribution to pathogenic Neisseria species and structurally related to enzymes implicated in phosphoethanolamine modification of lipopolysaccharide is necessary for PilE covalent modification with phosphoethanolamine and phosphocholine. These findings strongly suggest that protein phospho-form modification is mechanistically similar to processes underlying analogous modifications of prokaryotic saccharolipid glycans. We also show that PilE undergoes multisite and hierarchical phospho-form modifications and that the stoichiometries of site occupancy can be influenced by PilE primary structure and the abundance of the pilin-like protein PilV. Together, these findings have important implications for the structure and antigenicity of PilE.

Received for publication, May 5, 2006 , and in revised form, July 5, 2006.

^* This work was supported by both a Storforsk ("big research") grant from the Research Council of Norway and the Consortium for Advanced Microbial Sciences and Technologies national technology platform funded through the Functional Genomics program of the Research Council of Norway. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. S1 and Table 1.

² A Biotechnology and Biological Sciences Research Council Professorial Fellow.

¹ Present address and to whom correspondence may be addressed: Centre for Molecular Biology and Neuroscience and Dept. of Molecular Biosciences, University of Oslo, 0316 Oslo, Norway. Tel.: 47-22857295; Fax: 47-22856041; E-mail: w.m.egge-jacobsen{at}biotek.uio.no. ³ To whom correspondence may be addressed: Dept. of Molecular Biosciences, University of Oslo, 0316 Oslo, Norway. Tel.: 47-22854091; Fax: 47-22856041; E-mail: johnk{at}imbv.uio.no.

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