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The Haemophilus influenzae HMW1 adhesin is a glycoprotein with an unusual N-linked carbohydrate modification.

ABSTRACT: 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.

SUBMITTER: Gross J

PROVIDER: S-EPMC3258857 | biostudies-literature | 2008 Sep

REPOSITORIES: biostudies-literature

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The Haemophilus influenzae HMW1 adhesin is a glycoprotein with an unusual N-linked carbohydrate modification.

Gross Julia J Grass Susan S Davis Alan E AE Gilmore-Erdmann Petra P Townsend R Reid RR St Geme Joseph W JW

The Journal of biological chemistry 20080711 38

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 ...[more]

PMID: 18621734

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Project description:UnlabelledThe two-partner secretion (TPS) pathway in Gram-negative bacteria consists of a TpsA exoprotein and a cognate TpsB outer membrane pore-forming translocator protein. Previous work has demonstrated that the TpsA protein contains an N-terminal TPS domain that plays an important role in targeting the TpsB protein and is required for secretion. The nontypeable Haemophilus influenzae HMW1 and HMW2 adhesins are homologous proteins that are prototype TpsA proteins and are secreted by the HMW1B and HMW2B TpsB proteins. In the present study, we sought to define the structural determinants of HMW1 interaction with HMW1B during the transport process and while anchored to the bacterial surface. Modeling of HMW1B revealed an N-terminal periplasmic region that contains two polypeptide transport-associated (POTRA) domains and a C-terminal membrane-localized region that forms a pore. Biochemical studies demonstrated that HMW1 engages HMW1B via interaction between the HMW1 TPS domain and the HMW1B periplasmic region, specifically, the predicted POTRA1 and POTRA2 domains. Subsequently, HMW1 is shuttled to the HMW1B pore, facilitated by the N-terminal region, the middle region, and the NPNG motif in the HMW1 TPS domain. Additional analysis revealed that the interaction between HMW1 and HMW1B is highly specific and is dependent upon the POTRA domains and the pore-forming domain of HMW1B. Further studies established that tethering of HMW1 to the surface-exposed region of HMW1B is dependent upon the external loops of HMW1B formed by residues 267 to 283 and residues 324 to 330. These observations may have broad relevance to proteins secreted by the TPS pathway.ImportanceSecretion of HMW1 involves a recognition event between the extended form of the HMW1 propiece and the HMW1B POTRA domains. Our results identify specific interactions between the HMW1 propiece and the periplasmic HMW1B POTRA domains. The results also suggest that the process of HMW1 translocation involves at least two discrete steps, including initial interaction between the HMW1 propiece and the HMW1B POTRA domains and then a separate translocation event. We have also discovered that the HMW1B pore itself appears to influence the translocation process. These observations extend our knowledge of the two-partner secretion system and may be broadly relevant to other proteins secreted by the TPS pathway.

Dataset Information

The Haemophilus influenzae HMW1 adhesin is a glycoprotein with an unusual N-linked carbohydrate modification.

Publications

The Haemophilus influenzae HMW1 adhesin is a glycoprotein with an unusual N-linked carbohydrate modification.

Similar Datasets

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