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N-Glycan structure analysis using lectins and an alpha-mannosidase activity assay
Tomoya O Akama1, Michiko N Fukuda
1Glycobiology Program, Cancer Research Center, The Burnham Institute, La Jolla, CA, USA.
Methods in Enzymology
|November 23, 2006
Summary
Researchers analyzed N-glycan structures in alpha-mannosidase IIx (MX) and alpha-mannosidase II (MII) knockout mice. Methods included mass spectrometry and lectin techniques, revealing insights into glycan processing and enzyme activity.
Area of Science:
- Biochemistry
- Glycobiology
- Molecular Biology
Background:
- Alpha-mannosidase IIx (MX) and alpha-mannosidase II (MII) are homologous enzymes crucial for N-glycan processing.
- Previous studies, particularly involving MII/MX double-knockout mice, have highlighted their critical roles.
Purpose of the Study:
- To analyze the structures of N-glycans synthesized in MII/MX double-knockout mice.
- To characterize the substrate specificity of MX.
- To establish methods for analyzing N-glycans and alpha-mannosidase activity in knockout models.
Main Methods:
- Mass spectrometry analysis
- Two-dimensional high-performance liquid chromatography (HPLC) mapping
- Lectin blot and lectin histochemistry
- Production of soluble MII and MX via mammalian cell transfection
Main Results:
- Detailed analysis of N-glycan structures in mutant mice was achieved.
- Substrate specificity of MX was determined.
- Effective methods for N-glycan analysis and enzyme activity assay were developed.
Conclusions:
- The study provides comprehensive methods for analyzing N-glycans in knockout mice using lectin-based techniques.
- The findings contribute to understanding the roles of MX and MII in N-glycan biosynthesis.
- A protocol for assaying alpha-mannosidase activity using soluble MX is presented.
