Functional implications of corticosteroid-binding globulin N-glycosylation

Area of Science:

• Biochemistry
• Glycobiology
• Proteomics

Background:

• Corticosteroid-binding globulin (CBG) is the primary plasma transporter for glucocorticoids.
• Human and rat CBGs possess six N-glycosylation sites, influencing steroid-binding activity.
• N-glycosylation sites within the reactive center loops (RCLs) are critical for CBG function.

Purpose of the Study:

• To investigate the role of N-glycosylation sites in human and rat CBG steroid-binding.
• To determine the impact of N-glycan presence and composition on CBG's steroid affinity.
• To assess the susceptibility of CBG to proteolytic cleavage by neutrophil elastase and other proteases.

Main Methods:

• Site-directed mutagenesis of conserved N-glycosylation sites (N238 in human, N230 in rat CBG).
• Inhibition of glycosylation using tunicamycin.
• Enzymatic deglycosylation using Endo H and PNGase F.
• Expression of CBG in various cell lines (Lec1, Lec2, CHO-S) with differential glycosylation capabilities.
• Proteolytic assays using neutrophil elastase, chymotrypsin, and LasB.

Main Results:

• Mutations at conserved N-glycosylation sites (N238/N230) significantly disrupt steroid binding.
• Inhibition of glycosylation reduces CBG steroid-binding activity.
• Deglycosylation with Endo H reduces steroid-binding affinity, while PNGase F does not, suggesting deamidation preserves binding.
• CBGs expressed in Lec1 cells exhibit higher steroid-binding affinities compared to those from Lec2 or CHO-S cells.
• Neutrophil elastase cleaves human CBG's RCL more efficiently than chymotrypsin or LasB, reducing steroid-binding capacity.
• Glycosylation at N347 limits RCL cleavage, and other N-glycans offer protection against proteases.

Conclusions:

• The presence and composition of N-glycans, particularly at conserved positions like N238, are critical for maintaining CBG steroid-binding affinity.
• N-glycosylation influences CBG's structural integrity and its susceptibility to proteolytic degradation.
• Specific N-glycans play a protective role against cleavage by proteases like neutrophil elastase.