Functional implications of corticosteroid-binding globulin N-glycosylation.

Marc Simard,Geoffrey L Hammond,Caroline Underhill

doi:10.1530/jme-17-0234

Abstract

Corticosteroid-binding globulin (CBG) is a plasma carrier of glucocorticoids. Human and rat CBGs have six N-glycosylation sites. Glycosylation of human CBG influences its steroid-binding activity, and there are N-glycosylation sites in the reactive center loops (RCLs) of human and rat CBGs. Proteolysis of the RCL of human CBG causes a structural change that disrupts steroid binding. We now show that mutations of conserved N-glycosylation sites at N238 in human CBG and N230 in rat CBG disrupt steroid binding. Inhibiting glycosylation by tunicamycin also markedly reduced human and rat CBG steroid-binding activities. Deglycosylation of fully glycosylated human CBG or human CBG with only one N-glycan at N238 with Endo H-reduced steroid-binding affinity, while PNGase F-mediated deglycosylation does not, indicating that steroid binding is preserved by deamidation of N238 when its N-glycan is removed. When expressed in N-acetylglucosaminyltransferase-I-deficient Lec1 cells, human and rat CBGs, and a human CBG mutant with only one glycosylation site at N238, have higher (2–4 fold) steroid-binding affinities than when produced by sialylation-deficient Lec2 cells or glycosylation-competent CHO-S cells. Thus, the presence and composition of an N-glycan in this conserved position both appear to influence the steroid binding of CBG. We also demonstrate that neutrophil elastase cleaves the RCL of human CBG and reduces its steroid-binding capacity more efficiently than does chymotrypsin or the Pseudomonas aeruginosa protease LasB. Moreover, while glycosylation of N347 in the RCL limits these activities, N-glycans at other sites also appear to protect CBG from neutrophil elastase or chymotrypsin.

Highlights

Corticosteroid-binding globulin (CBG) is a plasma glycoprotein produced by the liver
We substituted N238 in human CBG with Asp, but the mutated protein was not secreted into the culture medium by CHO cells and accumulated within the cells, presumably as a misfolded and partially degraded protein
We base this on the observation that the CBG N238D that accumulates in the CHO cell pellets runs close to the 37 kDa size marker, whereas we would have expected it to run in excess of 42 kDa because its five other glycosylation sites are intact (Fig. 2B)

Summary

Introduction

Corticosteroid-binding globulin (CBG) is a plasma glycoprotein produced by the liver. It binds glucocorticoids and progesterone preferentially and determines the amounts of these steroids that are non-protein bound and accessible to target cells (Lewis et al 2005, Lin et al 2010, Perogamvros et al 2012, Bolton et al 2014, Lei et al 2015). CBG is decorated by as many as six N-glycans that account for about 30% of its overall mass (Sumer-Bayraktar et al 2011), and at least four of the consensus sites for N-glycosylation are in conserved positions.

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Results

Conclusion