Asn347-Glycosylation of Corticosteroid Binding Globulin Fine-tunes the Host Immune Response by Modulating Proteolysis by <em>P. aeruginosa</em> and Neutrophil Elastase — ASN Events
  1. Schedule
  2. Symposium One: Students of the APS (SoAPS)
  3. Asn347-Glycosylation of Corticosteroid Binding Globulin Fine-tunes the Host Immune Response by Modulating Proteolysis by P. aeruginosa and Neutrophil Elastase

Asn347-Glycosylation of Corticosteroid Binding Globulin Fine-tunes the Host Immune Response by Modulating Proteolysis by P. aeruginosa and Neutrophil Elastase (#003)

Zeynep Sumer Bayraktar 1 , Oliver Grant 2 , Vignesh Venkatakrishnan 1 , Robert J. Woods 2 , Nicolle H. Packer 1 , Morten Thaysen-Andersen 2
  1. Chemistry and Biomolecular Sciences, Macquarie University, Sydney, NSW, Australia
  2. Complex Carbohydrate Research Center, The University of Georgia, Athens, Georgia, USA

Corticosteroid binding globulin (CBG) delivers anti-inflammatory cortisol to inflamed tissues upon elastase-based proteolysis of an exposed reactive center loop (RCL). However, the molecular basis of the RCL proteolysis by co-existing host and bacterial elastases and mechanisms regulating these events in inflamed and infected tissues remain unknown. We document that the RCL-localized Asn347-glycosylation is ideally positioned to regulate the RCL cleavage by human neutrophil elastase (NE) and P. aeruginosa elastase (PAE) and that it does so 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 N-glycosylation status as a function of NE/PAE incubation time. The site-specific (Val344-Thr345) and rapid (seconds-minutes) NE-based RCL proteolysis was significantly antagonized by several volume-enhancing Asn347-glycan features i.e. site occupancy, triantennary β1,4/6-GlcNAc branching, β-galactosylation and α1,6-fucosylation and strongly augmented by Asn347 α2,3-sialylation (all P < 0.05). In contrast, the inefficient (minutes-hours) PAE-based RCL cleavage occurring equally well at Thr345-Leu346 and Asn347-Leu348 was completely antagonized by the Asn347-glycan, but received beneficial electrostatic interactions from sialoglycans of other CBG glycosylation sites. In addition, virulent P. aeruginosa growth was augmented by the free cortisol, which may explain the weak PAE efficiency in cleaving glycosylated RCL during host infection. Finally, molecular dynamics simulations of relevant CBG Asn347-glycoforms, carried out using a homology model of uncleaved CBG, largely confirmed the experimental observations and suggested additional interesting relationships between Asn347-glycosylation and RCL cleavage. In conclusion, we are the first to show that site-specific CBG N-glycosylation regulates the bioavailability of cortisol in inflamed environments by fine-tuning the RCL proteolysis by endogenous and exogenous elastases via different mechanisms. This study offers new molecular insight into host- and pathogen-based manipulation of the human immune system.