The Haemophilus influenzae HMW1 adhesin is a glycoprotein with an unusual N-linked carbohydrate modification.
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.
Gross
Julia
J
Department of Medicine, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Grass
Susan
S
Davis
Alan E
AE
Gilmore-Erdmann
Petra
P
Townsend
R Reid
RR
St Geme
Joseph W
JW
D018829:Adhesins, Bacterial / Q000378:metabolism*; D000595:Amino Acid Sequence; D001422:Bacterial Adhesion; D002241:Carbohydrates / Q000737:chemistry*; D004847:Epithelial Cells / Q000166:cytology; D006023:Glycoproteins / Q000737:chemistry*; D006031:Glycosylation; D006193:Haemophilus influenzae / Q000378:metabolism*; D013058:Mass Spectrometry; D008956:Models, Chemical; D008969:Molecular Sequence Data; D011134:Polysaccharides / Q000737:chemistry; D017434:Protein Structure, Tertiary; D017550:Spectroscopy, Fourier Transform Infrared; D014357:Trypsin / Q000737:chemistry
D018829:Adhesins, Bacterial; D002241:Carbohydrates; D006023:Glycoproteins; C117431:HMW1 protein, bacteria; D011134:Polysaccharides; D014357:Trypsin