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Characterization and subcellular localization of human neutral class II alpha-mannosidase [corrected]
Elina Kuokkanen1, Wesley Smith, Marika Mäkinen
1Institute of Biotechnology, University of Helsinki, FIN-00014, Finland.
Glycobiology
|August 8, 2007
Summary
Neutral alpha-mannosidase, a cytosolic enzyme, plays a role in breaking down oligosaccharides. Studies show it
Area of Science:
- Biochemistry
- Cell Biology
- Enzymology
Background:
- Neutral alpha-mannosidase (a glycosyl hydrolase family 38 enzyme) is implicated in hydrolyzing cytosolic free oligosaccharides.
- Its precise subcellular localization and function remain under investigation, with prior studies suggesting both cytosolic and ER association.
Purpose of the Study:
- To determine the subcellular localization of neutral alpha-mannosidase using immunofluorescence microscopy.
- To characterize the human recombinant enzyme's activity with natural substrates to clarify its biological function.
Main Methods:
- Immunofluorescence microscopy was employed to visualize neutral alpha-mannosidase distribution.
- Fluorescent recovery after photobleaching (FRAP) was used to assess enzyme diffusion in the cytosol.
- Characterization of the purified recombinant enzyme involved activity assays with various substrates and cations.
Main Results:
- Immunofluorescence revealed neutral alpha-mannosidase is granularly distributed in the cytosol, not in the ER, lysosomes, or autophagosomes.
- FRAP indicated a slower-than-expected two-phased diffusion, suggesting cytosolic complex formation.
- The recombinant enzyme, a tetramer with neutral pH optimum, hydrolyzed Man(9)GlcNAc to Man(5)GlcNAc, with Fe(2+) being a potent activator.
Conclusions:
- Subcellular localization and enzyme characterization support neutral alpha-mannosidase's role in hydrolyzing soluble cytosolic oligomannosides.
- The enzyme's cytosolic complexation may influence its activity or localization.
- Specific cation activation profiles highlight unique properties of the human neutral alpha-mannosidase.

