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Metabolic Glycoengineering of Sialic Acid Using N-acyl-modified Mannosamines
Published on: November 25, 2017
Family 47 alpha-mannosidases in N-glycan processing
Steven W Mast1, Kelley W Moremen
1Department of Biochemistry & Molecular Biology, University of Georgia Complex Carbohydrate Research Center, Athens, GA, USA.
Methods in Enzymology
|November 23, 2006
Summary
This study details methods for analyzing alpha-mannosidases (GH47 family), crucial for glycoprotein processing and quality control in eukaryotic cells. It covers enzyme expression, purification, activity assays, and binding studies for these important enzymes.
Area of Science:
- Biochemistry and Molecular Biology
- Glycobiology
- Enzymology
Background:
- Alpha-mannosidases are key enzymes in eukaryotic cells, involved in both glycan biosynthesis and catabolism.
- Two families exist: Class 1 (GH family 47) in the early secretory pathway for N-glycan processing and quality control, and Class 2 (GH38) for glycoprotein biosynthesis/catabolism.
- Class 1 alpha-mannosidases include ER alpha-mannosidase I (ERManI), Golgi alpha-mannosidase I (GolgiManI), and ER degradation-enhancing alpha-mannosidase-like (EDEM) proteins.
Purpose of the Study:
- To discuss and outline methods for the comprehensive analysis of GH47 alpha-mannosidases.
- To cover techniques for studying enzyme expression, purification, activity, and interactions.
- To facilitate further research into the roles of GH47 enzymes in glycoprotein metabolism and quality control.
Main Methods:
- Enzyme expression and purification protocols.
- Enzyme activity assays to measure catalytic function.
- Generation of point mutants to study enzyme function.
- Surface plasmon resonance for binding studies.
Main Results:
- Established methodologies for characterizing GH47 alpha-mannosidases.
- Provided a framework for investigating the specific roles of ERManI, GolgiManI, and EDEM proteins.
- Demonstrated the utility of SPR for analyzing enzyme-ligand interactions.
Conclusions:
- The described methods enable detailed functional and interactional analysis of GH47 alpha-mannosidases.
- These enzymes play critical roles in N-glycan processing, glycoprotein quality control, and degradation pathways.
- Further application of these techniques will advance understanding of cellular glycoprotein homeostasis.
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