Oligosaccharide binding characteristics of the molecular chaperones calnexin and calreticulin

Biochemistry. 1998 Mar 10;37(10):3480-90. doi: 10.1021/bi972465g.


Calnexin and calreticulin are homologous molecular chaperones of the endoplasmic reticulum. Their binding to newly synthesized glycoproteins is mediated, at least in part, by a lectin site that recognizes the early N-linked oligosaccharide processing intermediate, Glc1Man9GlcNAc2. We compared the oligosaccharide binding specificities of calnexin and calreticulin in an effort to determine the basis for reported differences in their association with various glycoproteins. Using mono-, di-, and oligosaccharides to inhibit the binding of Glc1Man9GlcNAc2 to calreticulin and to a truncated, soluble form of calnexin, we show that the entire Glc alpha 1-3Man alpha 1-2Man alpha 1-2Man structure, extending from the alpha 1-3 branch point of the oligosaccharide core, is recognized by both proteins. Furthermore, analysis of the binding of monoglucosylated oligosaccharides containing progressively fewer mannose residues suggests that for both proteins the alpha 1-6 mannose branch point of the oligosaccharide core is also essential for recognition. Consistent with their essentially identical substrate specificities, calnexin and calreticulin exhibited the same relative affinities when competing for binding to the Glc1Man9GlcNAc2 oligosaccharide. Thus, differential glycoprotein binding cannot be attributed to differences in the lectin specificities or binding affinities of calnexin and calreticulin. We also examined the effects of ATP, calcium, and disulfide reduction on the lectin properties of calnexin and calreticulin. Whereas oligosaccharide binding was only slightly enhanced for both proteins in the presence of high concentrations of a number of adenosine nucleotides, removal of bound calcium abrogated oligosaccharide binding, an effect that was largely reversible upon readdition of calcium. Disulfide reduction had no effect on oligosaccharide binding by calnexin, but binding by calreticulin was inhibited by 70%. Finally, deletion mutagenesis of calnexin and calreticulin identified a central proline-rich region characterized by two tandem repeat motifs as a segment capable of binding oligosaccharide. This segment bears no sequence homology to the carbohydrate recognition domains of other lectins.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Adenosine Triphosphate / pharmacology
  • Amino Acid Sequence
  • Animals
  • Binding Sites
  • Calcium-Binding Proteins / chemistry
  • Calcium-Binding Proteins / genetics
  • Calcium-Binding Proteins / metabolism*
  • Calnexin
  • Calreticulin
  • Carbohydrate Sequence
  • Cell Line
  • Cricetinae
  • Dithiothreitol / pharmacology
  • Egtazic Acid / pharmacology
  • Endoplasmic Reticulum / metabolism
  • In Vitro Techniques
  • Mice
  • Molecular Chaperones / chemistry
  • Molecular Chaperones / genetics
  • Molecular Chaperones / metabolism*
  • Molecular Sequence Data
  • Oligosaccharides / chemistry
  • Oligosaccharides / metabolism*
  • Peptide Mapping
  • Protein Binding
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Ribonucleoproteins / chemistry
  • Ribonucleoproteins / genetics
  • Ribonucleoproteins / metabolism*
  • Sequence Deletion


  • Calcium-Binding Proteins
  • Calreticulin
  • Molecular Chaperones
  • Oligosaccharides
  • Recombinant Fusion Proteins
  • Ribonucleoproteins
  • Calnexin
  • Egtazic Acid
  • Adenosine Triphosphate
  • Dithiothreitol