Glycoforms modify the dynamic stability and functional activity of an enzyme

Area of Science:

• Biochemistry
• Glycobiology
• Protein Science

Background:

• Glycoproteins exist as diverse glycoforms, with varying oligosaccharides at glycosylation sites.
• Bovine pancreatic ribonuclease B (RNase B) is a natural mixture of five glycoforms differing in oligomannose sugar structures.

Purpose of the Study:

• To isolate and analyze individual RNase B glycoforms to determine how specific sugars influence protein structure, dynamics, stability, and function.
• To investigate the impact of glycosylation on protein properties.

Main Methods:

• Isolation of pure RNase B glycoforms using exoglycosidase digestion.
• Analysis of glycoforms by capillary electrophoresis.
• Measurement of amide proton exchange rates to assess protein dynamics.
• Proteolytic resistance assays (Pronase).
• Enzymatic activity assays and molecular modeling.

Main Results:

• Electrophoretically pure single glycoforms of RNase B were successfully prepared.
• Glycosylation decreased overall molecular flexibility without altering 3D structure.
• Individual glycoforms exhibited variations in dynamic stability.
• All glycoforms showed increased resistance to Pronase compared to unglycosylated RNase A.
• A 4-fold variation in functional activity was observed among different glycoforms, potentially due to steric factors.

Conclusions:

• Specific oligosaccharide structures on RNase B glycoforms modulate protein dynamics, stability, and enzymatic function.
• Glycosylation plays a critical role in determining the functional properties of glycoproteins.
• Steric effects of sugars are important in modulating protein-ligand interactions.