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J. Biol. Chem., Vol. 283, Issue 38, 26010-26015, September 19, 2008

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The Haemophilus influenzae HMW1 Adhesin Is a Glycoprotein with an Unusual N-Linked Carbohydrate Modification^*

Julia Gross, Susan Grass, Alan E. Davis, Petra Gilmore-Erdmann, R. Reid Townsend^¶, and Joseph W. St. Geme, III¹

From the Departments of Medicine and ^¶Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110 and the Departments of Pediatrics and Molecular Genetics & Microbiology, Duke University Medical Center, Durham, North Carolina 27710

The Haemophilus influenzae HMW1 adhesin mediates adherence to respiratory epithelial cells, a critical early step in the pathogenesis of H. influenzae disease. In recent work, we demonstrated that HMW1 undergoes glycosylation. In addition, we observed that glycosylation of HMW1 is essential for HMW1 tethering to the bacterial surface, a prerequisite for HMW1-mediated adherence to host epithelium. In this study, we examined HMW1 proteolytic fragments by mass spectrometry, achieved 89% amino acid sequence coverage, and identified 31 novel modification sites. All of the modified sites were asparagine residues, in all but one case in the conventional consensus sequence of N-linked glycans, viz. NX(S/T). Liquid chromatography-tandem mass spectrometry analysis using a hybrid linear quadrupole ion trap Fourier transform ion cyclotron mass spectrometer, accurate mass measurements, and deuterium exchange studies established that the modifying glycan structures were mono- or dihexoses rather than the N-acetylated chitobiosyl core that is characteristic of N-glycosylation. This unusual carbohydrate modification suggests that HMW1 glycosylation requires a glycosyltransferase with a novel activity.

Received for publication, March 6, 2008 , and in revised form, May 27, 2008.

^* This work was supported by United States Public Health Service Grants RO1 DC-02873 (to J. W. S.), 2-P41-RR05351 (to the University of Georgia Complex Carbohydrate Research Center), and P41-RR00954 (to the Washington University Mass Spectrometry Resource) and by the William M. Keck Foundation. 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 Figs. 1–5.

¹ To whom correspondence should be addressed: Depts. of Pediatrics and Molecular Genetics & Microbiology, Duke University Medical Center, Children's Health Center, Rm. T901, Durham, NC 27710. Tel.: 919-681-4080; Fax: 919-681-2714; E-mail: j.stgeme{at}duke.edu.

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