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Updated: Jul 5, 2026
10:17
Pulse-chase Analysis of N-linked Sugar Chains from Glycoproteins in Mammalian Cells
Published on: April 27, 2010
Inhibition of N-linked glycosylation.
1University of California San Diego, La Jolla, California, USA.
Current Protocols in Immunology
|April 25, 2008
Summary
Researchers used enzyme inhibitors to alter N-linked glycosylation in cultured cells, producing glycoproteins with modified chains. This method helps study oligosaccharide functions on proteins and cells.
Area of Science:
- Biochemistry
- Cell Biology
- Glycobiology
Background:
- N-linked oligosaccharides are crucial post-translational modifications on glycoproteins.
- Understanding the functional roles of these oligosaccharides is essential in cell biology.
Purpose of the Study:
- To describe a method for preventing N-linked glycosylation in cultured cells using enzyme inhibitors.
- To enable the study of functional roles of N-linked oligosaccharides on specific proteins and cells.
Main Methods:
- Determining optimal non-toxic inhibitor concentrations by monitoring protein biosynthesis via [(35)S]methionine incorporation.
- Assessing inhibitor efficacy in hindering oligosaccharide processing using [(3)H]mannose labeling, TCA precipitation, or endo H digestion.
- Utilizing glycoprotein identification methods to measure inhibitor effects on N-linked oligosaccharide chains.
Main Results:
- Successful production of glycoproteins with missing or altered N-linked oligosaccharide chains.
- Established protocols for determining inhibitor concentration and efficacy.
- Provided guidance for analyzing inhibitor impact on specific glycoproteins.
Conclusions:
- Enzyme inhibitors are effective tools for manipulating N-linked glycosylation in cell culture.
- This approach facilitates the investigation of oligosaccharide function in biological systems.
- The described methods support detailed analysis of glycoprotein modification and function.
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