In vitro assays of the functions of calnexin and calreticulin, lectin chaperones of the endoplasmic reticulum.
Calnexin and calreticulin are molecular chaperones of the endoplasmic reticulum (ER) whose folding-promoting functions are directed predominantly toward aspargine-linked glycoproteins. This is a consequence of calnexin and calreticulin being lectins with specificity for the early oligosaccharide (OS)-processing intermediate, Glc1Man9GlcNAc2. In addition, they interact with non-native conformers of glycoprotein polypeptide chains to prevent aggregation and recruit the thiol oxidoreductase ERp57 to catalyze glycoprotein disulfide formation/isomerization. In vitro assays of these functions have contributed greatly to our understanding of how calnexin and calreticulin promote glycoprotein folding. This chapter describes the isolation of Glc1Man9GlcNAc2 OS, as well as the assay used to measure OS binding. Furthermore, details are provided of assays that detect ERp57 binding by calnexin and calreticulin, as well as the abilities of these chaperones to suppress the aggregation of non-native protein substrates.
Ireland
Breanna S
BS
Department of Immunology, University Toronto, Toronto, Ontario, Canada.
Niggemann
Monika
M
Williams
David B
DB
D037281:Calnexin / Q000032:analysis / Q000378:metabolism*; D037282:Calreticulin / Q000032:analysis / Q000378:metabolism*; D002240:Carbohydrate Sequence; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D006031:Glycosylation; D037102:Lectins / Q000378:metabolism; D008351:Mannans / Q000378:metabolism; D008967:Molecular Biology / Q000379:methods*; D018832:Molecular Chaperones / Q000378:metabolism*; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000302:isolation & purification / Q000378:metabolism; D019704:Protein Disulfide-Isomerases / Q000378:metabolism
D037282:Calreticulin; D037102:Lectins; D008351:Mannans; D018832:Molecular Chaperones; D009844:Oligosaccharides; C475461:mannosyl(9)-N-acetylglucosamine2; D037281:Calnexin; D019704:Protein Disulfide-Isomerases; C085868:PDIA3 protein, human