Asn347 Glycosylation of Corticosteroid-binding Globulin Fine-tunes the Host Immune Response by Modulating Proteolysis by Pseudomonas aeruginosa and Neutrophil Elastase

Zeynep Sumer-Bayraktar,Oliver C Grant,Vignesh Venkatakrishnan,Robert J Woods,Nicolle H Packer,Morten Thaysen-Andersen

doi:10.1074/jbc.m116.735258

Zeynep Sumer-Bayraktar, Oliver C Grant + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m116.735258

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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.

Highlights

This study offers new molecular insight into host- and pathogen-based manipulation of the human immune system
During inflammation, Corticosteroid-binding globulin (CBG) acts as a negative acute-phase protein [16], where the level of intact functionally active CBG in circulation is reduced due to the increased rate of proteolytic cleavage of this glucocorticoid carrier with a concomitant cortisol release [17] and an IL-6-driven reduction in CBG synthesis [18, 19]
The reduced cortisol affinity of cleaved CBG increases the local concentration of free cortisol, which is beneficial toward resolving inflammation in affected tissues by the anti-inflammatory effects of cortisol [17, 29]

Summary

Introduction

This study offers new molecular insight into host- and pathogen-based manipulation of the human immune system. 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 Asn347 glycosylation as a function of digestion time.

Results

Conclusion