title: Asn347 Glycosylation of Corticosteroid-binding Globulin Fine-tunes the Host Immune Response by Modulating Proteolysis by Pseudomonas aeruginosa and Neutrophil Elastase.
abstract: Corticosteroid-binding globulin (CBG) delivers anti-inflammatory cortisol to inflamed tissues upon elastase-based proteolysis of the exposed reactive center loop (RCL). However, the molecular mechanisms that regulate the RCL proteolysis by co-existing host and bacterial elastases in inflamed/infected tissues remain unknown. We document that RCL-localized Asn(347) glycosylation fine-tunes the RCL cleavage rate by human neutrophil elastase (NE) and Pseudomonas aeruginosa elastase (PAE) by different mechanisms. NE- and PAE-generated fragments of native and exoglycosidase-treated blood-derived CBG of healthy individuals were monitored by gel electrophoresis and LC-MS/MS to determine the cleavage site(s) and Asn(347) glycosylation as a function of digestion time. The site-specific (Val(344)-Thr(345)) and rapid (seconds to minutes) NE-based RCL proteolysis was significantly antagonized by several volume-enhancing Asn(347) glycan features (i.e. occupancy, triantennary GlcNAc branching, and α1,6-fucosylation) and augmented by Asn(347) NeuAc-type sialylation (all p < 0.05). In contrast, the inefficient (minutes to hours) PAE-based RCL cleavage, which occurred equally well at Thr(345)-Leu(346) and Asn(347)-Leu(348), was abolished by the presence of Asn(347) glycosylation but was enhanced by sialoglycans on neighboring CBG N-sites. Molecular dynamics simulations of various Asn(347) glycoforms of uncleaved CBG indicated that multiple Asn(347) glycan features are modulating the RCL digestion efficiencies by NE/PAE. Finally, high concentrations of cortisol showed weak bacteriostatic effects toward virulent P. aeruginosa, which may explain the low RCL potency of the abundantly secreted PAE during host infection. In conclusion, site-specific CBG N-glycosylation regulates the bioavailability of cortisol in inflamed environments by fine-tuning the RCL proteolysis by endogenous and exogenous elastases. This study offers new molecular insight into host- and pathogen-based manipulation of the human immune system.
authors.affiliation: From the Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney, New South Wales 2109, Australia and.
authors.forename: Zeynep
authors.initials: Z
authors.lastname: Sumer-Bayraktar
authors.affiliation: the Complex Carbohydrate Research Center, University of Georgia, Athens, Georgia 30602.
authors.forename: Oliver C
authors.initials: OC
authors.lastname: Grant
authors.affiliation: From the Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney, New South Wales 2109, Australia and.
authors.forename: Vignesh
authors.initials: V
authors.lastname: Venkatakrishnan
authors.affiliation: the Complex Carbohydrate Research Center, University of Georgia, Athens, Georgia 30602.
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Woods
authors.affiliation: From the Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney, New South Wales 2109, Australia and.
authors.forename: Nicolle H
authors.initials: NH
authors.lastname: Packer
authors.affiliation: From the Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney, New South Wales 2109, Australia and morten.andersen@mq.edu.au.
authors.forename: Morten
authors.initials: M
authors.lastname: Thaysen-Andersen
mesh_terms: D001216:Asparagine / Q000276:immunology; D001426:Bacterial Proteins / Q000276:immunology*; D006031:Glycosylation; D054884:Host-Pathogen Interactions / Q000276:immunology*; D006801:Humans; D006854:Hydrocortisone / Q000276:immunology*; D019272:Leukocyte Elastase / Q000276:immunology*; D059748:Proteolysis; D011550:Pseudomonas aeruginosa / Q000502:physiology*; D014156:Transcortin / Q000276:immunology*
chemical_list: D001426:Bacterial Proteins; C534935:SERPINA6 protein, human; D001216:Asparagine; D014156:Transcortin; D019272:Leukocyte Elastase; D006854:Hydrocortisone
keywords: P. aeruginosa elastase; Pseudomonas aeruginosa (P. aeruginosa); corticosteroid-binding globulin; cortisol; glycoprotein; glycosylation; molecular dynamics; neutrophil elastase; proteolysis; reactive center loop