Glycobiology Protocols

Volume 347 of the series Methods in Molecular Biology pp 331-342

In Vitro Assays of the Functions of Calnexin and Calreticulin, Lectin Chaperones of the Endoplasmic Reticulum

  • Breanna S. IrelandAffiliated withDepartment of Immunology, University of Toronto
  • , Monika NiggemannAffiliated withDepartment of Biochemistry, University of Toronto
  • , David B. WilliamsAffiliated withDepartment of Immunology, University of TorontoDepartment of Biochemistry, University of Toronto

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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.

Key Words

Aggregation calnexin calreticulin endoplasmic reticulum ERp57 glycoprotein folding lectin molecular chaperone oligosaccharide