N-Glycan structure analysis using lectins and an alpha-mannosidase activity assay.
Alpha-mannosidase IIx (MX) and alpha-mannosidase II (MII) are homologous enzymes whose critical roles in N-glycan processing were established in large part by analysis of the MII/MX double-knockout mouse. To analyze the structures of N-glycans synthesized in the mutant mice, we employed lectin blot and lectin histochemistry in addition to mass spectrometry analysis and two-dimensional high-performance liquid chromatography (HPLC) mapping. We also produced soluble MII and MX by transfecting mammalian cells with expression vectors and determined substrate specificity of MX. This chapter describes methods using lectins to analyze N-glycans in knockout mice and provides a protocol to assay alpha-mannosidase activity using soluble MX.
Akama
Tomoya O
TO
Glycobiology Program, Cancer Research Center, The Burnham Institute, La Jolla, CA, USA.
Fukuda
Michiko N
MN
D000818:Animals; D000831:Animals, Newborn; D019556:COS Cells; D002522:Chlorocebus aethiops; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D004789:Enzyme Activation; D006651:Histocytochemistry / Q000379:methods*; D016591:Histocytological Preparation Techniques / Q000379:methods*; D037102:Lectins / Q000737:chemistry*; D013058:Mass Spectrometry; D051379:Mice; D018345:Mice, Knockout; D011134:Polysaccharides / Q000032:analysis / Q000737:chemistry*; D013379:Substrate Specificity; D043323:alpha-Mannosidase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification
D037102:Lectins; D011134:Polysaccharides; D043323:alpha-Mannosidase