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Calnexin/Calreticulin and Assays Related to N-Glycoprotein Folding In Vitro

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Lectin Purification and Analysis

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2132))

Abstract

Calnexin (CNX) and calreticulin (CRT) are ER-resident lectin-like molecular chaperones involved in the quality control of secretory or membrane glycoproteins. They can exert molecular chaperone functions via specific binding to the early processing intermediates of Glc1Man9GlcNAc2 oligosaccharides of N-glycoproteins. CNX and CRT have similar N-terminal luminal domains and share the same jelly roll tertiary structure as legume lectins. In addition to the lectin-like interactions, CNX and CRT also suppress the aggregation of non-glycosylated substrates through interaction with hydrophobic peptide parts, suggesting a general chaperone function in glycan-dependent and glycan-independent manners. This chapter describes the isolation and purification of CRT produced in a bacterial expression system. We also introduce in vitro assays to estimate the molecular chaperone functions of CRT via the interaction with monoglucosylated N-glycans using Jack bean α-mannosidase as a target substrate. These assays are valuable in assessing quality control events related to the CNX/CRT chaperone cycle and lectin functions.

This work was supported by a Ministry of Education, Culture, Sports, Science and Technology of Japan Grant-in-Aid for Scientific Research (JP16H06290).

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Authors and Affiliations

  1. Department of Biochemistry, Wakayama Medical University, Wakayama, Japan

    Yoshito Ihara & Midori Ikezaki

  2. Synthetic Cellular Chemistry Laboratory, RIKEN, Saitama, Japan

    Maki Takatani & Yukishige Ito

Authors
  1. Yoshito Ihara
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  2. Midori Ikezaki
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  3. Maki Takatani
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  4. Yukishige Ito
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Correspondence to Yoshito Ihara .

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Editors and Affiliations

  1. Cellular and Molecular Biotechnology Research Institute, National Institute of Advanced Industrial Science and Technology, Tsukuba, Ibaraki, Japan

    Jun Hirabayashi

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Ihara, Y., Ikezaki, M., Takatani, M., Ito, Y. (2020). Calnexin/Calreticulin and Assays Related to N-Glycoprotein Folding In Vitro. In: Hirabayashi, J. (eds) Lectin Purification and Analysis. Methods in Molecular Biology, vol 2132. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0430-4_29

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  • DOI: https://doi.org/10.1007/978-1-0716-0430-4_29

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-0429-8

  • Online ISBN: 978-1-0716-0430-4

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