Sputum protein N-glycosylation in cystic fibrosis: a sweet response to bacterial colonisation

Thesis by publication.Bibliography: leaves 253-268.Introduction -- Cystic fibrosis and bacterial colonisation define the sputum N-glycosylation phenotype -- Investigating the link between clinical CF-typed Pseudomonas aeruginosa isolates and PAO1 laboratory strain with pauci-mannosidic N-glycans -- Proteome and N-glycoproteome of Cystic Fibrosis sputum links the pauci-mannosidic N-glycans with abundant neutrophil proteins -- Conclusions and future directions.Cystic fibrosis (CF) is a prevalent autosomal recessive disease characterised by chronicinfection and inflammation in the lungs. The damaged lungs secrete excess mucus that isexpectorated as sputum. The lung mucus is the primary site of microbial infection, withPseudomonas aeruginosa as one of the major infecting pathogens in CF patients. Alteration inglycosylation of mucins (heavily O-glycosylated proteins) has been shown to play a role inbacterial binding to the mucus in the lungs of CF patients. However, there has been littlereported on the N-glycosylation of the protein complement of CF sputum.We have characterized the total secreted protein N-glycome of sputum derived from five CFpatients, two non-CF patients infected with pathogens (iNCF) and two pathogen free non-CF(NCF) patients. The glycosylation difference between CF and iNCF, compared to betweeniNCF and NCF, sputum separates the role played by the CF disease itself in regulating Nglycanexpression as distinct from the effects of bacterial colonisation. A significant increasein unusual and truncated pauci-mannosidic N-glycans and a decrease in complex and hybridN-glycans on the sputum proteins were observed in CF and iNCF sputum compared to NCFsputum, suggesting a common effect of infection.To address the link between the variation in CF sputum N-glycans and bacterial colonisation,four P. aeruginosa clinical strains (PASS1, PASS2, PASS3 and PASS4) were isolated fromsputum of CF patients. We showed that secreted bacterial exoglycosidases were not thecause of the trimming of the N-glycans to pauci-mannosidic structures and that the bacterialproteins do not carry pauci-mannose N-glycans. The presence of the pauci-mannose Nglycanson the sputum proteins were thus not directly of bacterial origin.Proteomic analysis revealed an up-regulation of proteins involved in the immune response(specifically neutrophil proteins) in bacterial colonised (CF and iNCF) compared with noninfected(NCF) sputum. State-of-art site-specific N-glycoproteomic analysis of CF, iNCF andNCF sputum proteins showed that the abundant proteins of neutrophil origin, such asmyeloperoxidase and azurocidin, carried these truncated pauci-mannosidic N-glycans.Whilst abundant knowledge regarding mucin O-glycosylation is available in the literature,this study provides new structural N-glycan and site-specific N-glycoproteome informationto aid in understanding the complex cellular and molecular environment of the CF affectedrespiratory tract.Mode of access: World wide web1 online resource (xi, 269 leaves diagrams, graphs, table