Abstract
Virulence of the gastric pathogen Helicobacter pylori (Hp) is directly linked to the pathogen's ability to glycosylate proteins; for example, Hp flagellin proteins are heavily glycosylated with the unusual nine-carbon sugar pseudaminic acid, and this modification is absolutely essential for Hp to synthesize functional flagella and colonize the host's stomach. Although Hp's glycans are linked to pathogenesis, Hp's glycome remains poorly understood; only the two flagellin glycoproteins have been firmly characterized in Hp. Evidence from our laboratory suggests that Hp synthesizes a large number of as-yet unidentified glycoproteins. Here we set out to discover Hp's glycoproteins by coupling glycan metabolic labeling with mass spectrometry analysis. An assessment of the subcellular distribution of azide-labeled proteins by Western blot analysis indicated that glycoproteins are present throughout Hp and may therefore serve diverse functions. To identify these species, Hp's azide-labeled glycoproteins were tagged via Staudinger ligation, enriched by tandem affinity chromatography, and analyzed by multidimensional protein identification technology. Direct comparison of enriched azide-labeled glycoproteins with a mock-enriched control by both SDS-PAGE and mass spectrometry-based analyses confirmed the selective enrichment of azide-labeled glycoproteins. We identified 125 candidate glycoproteins with diverse biological functions, including those linked with pathogenesis. Mass spectrometry analyses of enriched azide-labeled glycoproteins before and after cleavage of O-linked glycans revealed the presence of Staudinger ligation-glycan adducts in samples only after beta-elimination, confirming the synthesis of O-linked glycoproteins in Hp. Finally, the secreted colonization factors urease alpha and urease beta were biochemically validated as glycosylated proteins via Western blot analysis as well as by mass spectrometry analysis of cleaved glycan products. These data set the stage for the development of glycosylation-based therapeutic strategies, such as new vaccines based on natively glycosylated Hp proteins, to eradicate Hp infection. Broadly, this report validates metabolic labeling as an effective and efficient approach for the identification of bacterial glycoproteins.
Highlights
Helicobacter pylori (Hp)1 infection poses a significant health risk to humans worldwide
1 The abbreviations used are: Hp, Helicobacter pylori; MOE, metabolic oligosaccharide engineering; Ac4GlcNAz, peracetylated Nazidoacetylglucosamine; Ac4GlcNAc, peracetylated N-acetylglucosamine; GlcNAc, N-acetylglucosamine; mudPIT, multidimensional protein identification technology; PBS, phosphate buffered saline; ureA, urease alpha subunit; ureB, urease beta subunit; carbohydrate-active enzymes (CAZy), carbohydrate active enzymes; Phos-FLAG, phosphine conjugated to a FLAG peptide (DYKDDDDK); Phos-FLAG-His6, phosphine conjugated to a FLAG-His6 peptide (DYKDDDDKHHHHHH); TCEP, tris(2-carboxyethyl)phosphine; HBFA, heptafluorobutyric acid; FBS, fetal bovine serum; HPLC-Chip/Q-TOFMS, High Performance Liquid Chromatographic-Chip Quadrupole Time-of-Flight Mass Spectrometer; nanoESI, nano-electrospray ionization; HexNAz, N-azidoacetylhexosamine; HexNAc, N-acetylhexosamine; Collision-induced dissociation (CID), collision-induced dissociation
The two flagellin glycoproteins have been firmly characterized in Hp [7] to date
Summary
Helicobacter pylori; MOE, metabolic oligosaccharide engineering; Ac4GlcNAz, peracetylated Nazidoacetylglucosamine; Ac4GlcNAc, peracetylated N-acetylglucosamine; GlcNAc, N-acetylglucosamine; mudPIT, multidimensional protein identification technology; PBS, phosphate buffered saline; ureA, urease alpha subunit; ureB, urease beta subunit; CAZy, carbohydrate active enzymes; Phos-FLAG, phosphine conjugated to a FLAG peptide (DYKDDDDK); Phos-FLAG-His, phosphine conjugated to a FLAG-His peptide (DYKDDDDKHHHHHH); TCEP, tris(2-carboxyethyl)phosphine; HBFA, heptafluorobutyric acid; FBS, fetal bovine serum; HPLC-Chip/Q-TOFMS, High Performance Liquid Chromatographic-Chip Quadrupole Time-of-Flight Mass Spectrometer; nanoESI, nano-electrospray ionization; HexNAz, N-azidoacetylhexosamine; HexNAc, N-acetylhexosamine; CID, collision-induced dissociation. Insights into Hp’s pathogenesis have revealed that Hp’s glycan pseudaminic acid is a bona fide target of therapeutic intervention This is one of a number of examples linking protein glycosylation to virulence in medically significant bacterial pathogens [13, 14]. The unusual structures of bacterial glycans, which often contain amino- and deoxy-carbohydrates exclusively found in bacteria [12, 23,24,25], hampers their identification using existing tools Though methods such as the use of glycan-binding reagents [20, 24, 26, 27] and periodic acid/hydrazide glycan labeling [15] have successfully detected glycoproteins in a range of bacteria, they present limitations. This work validates metabolic oligosaccharide engineering as a complementary method to discover bacterial glycoproteins
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