Distinct contributions of the lectin and arm domains of calnexin to its molecular chaperone function

J Biol Chem. 2009 Feb 6;284(6):3433-44. doi: 10.1074/jbc.M804866200. Epub 2008 Dec 12.

Abstract

Calnexin is a Ca2+-binding transmembrane chaperone of the endoplasmic reticulum that recognizes Glc1Man5-9GlcNAc2 oligosaccharides on folding glycoproteins as well as non-native elements of the polypeptide backbone. This latter mode of recognition enables calnexin to suppress the aggregation of both glycosylated and nonglycosylated substrates. The luminal portion of calnexin (S-Cnx) consists of two domains, a globular lectin domain and an extended arm domain. To understand the function of these domains during the interaction of calnexin with non-native protein conformers, we tested deletion mutants of S-Cnx for their abilities to suppress the aggregation of nonglycosylated firefly luciferase. The arm domain alone exhibited no capacity to suppress aggregation. However, stepwise truncation of the arm domain in S-Cnx resulted in a progressive reduction in aggregation suppression potency to the point where the globular domain alone exhibited 25% potency. To characterize the polypeptide-binding site, we used hydrophobic peptides that were competitors of the ability of S-Cnx to suppress luciferase aggregation. Direct binding experiments revealed a single site of peptide binding in the globular domain (Kd = 0.9 microm) at a location distinct from the lectin site. Progressive truncation of the arm domain in S-Cnx had no effect on the binding of small peptides but reduced the binding affinity of S-Cnx for large, non-native protein substrates. Because protein substrates exhibited no binding to the isolated arm domain, our findings support a model in which calnexin suppresses aggregation through a polypeptide-binding site in its globular domain, with the arm domain enhancing aggregation suppression by sterically constraining large substrates.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Calnexin / chemistry*
  • Calnexin / genetics
  • Calnexin / metabolism
  • Dogs
  • Lectins / chemistry*
  • Lectins / genetics
  • Lectins / metabolism
  • Molecular Chaperones / chemistry*
  • Molecular Chaperones / genetics
  • Molecular Chaperones / metabolism
  • Mutation
  • Oligosaccharides / chemistry
  • Oligosaccharides / genetics
  • Oligosaccharides / metabolism
  • Protein Binding / physiology
  • Protein Structure, Tertiary / physiology

Substances

  • Lectins
  • Molecular Chaperones
  • Oligosaccharides
  • Calnexin