Kapoor, Mili and Honnappa, Srinivas and Eaazhisai, Kandiah and Emiliano, Gemma and Lars, Ellgaard and Oscarson, Stefan and Ari, Helenius and Surolia, Avadhesha (2003) Interactions of Substrate with Calreticulin, an Endoplasmic Reticulum Chaperone. In: Journal of Biological Chemistry, 278 (8). pp. 6194-6200.
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Calreticulin is a molecular chaperone found in the endoplasmic reticulum in eukaryotes, and its interaction with N-glycosylated polypeptides is mediated by the glycan Glc1Man7-9GlcNAc2 present on the target glycoproteins. Here, we report the thermodynamic parameters of its interaction with di-, tri-, and tetrasaccharide, which are truncated versions of the glucosylated arm of Glc1Man7-9GlcNAc2, determined by the quantitative technique of isothermal titration calorimetry. This method provides a direct estimate of the binding constants (Kb) and changes in enthalpy of binding (Delta Hb°) as well as the stoichiometry of the reaction. Unlike past speculations, these studies demonstrate unambiguously that calreticulin has only one site per molecule for binding its complementary glucosylated ligands. Although the binding of glucose by itself is not detectable, a binding constant of 4.19 × 104 M-1 at 279 K is obtained when glucose occurs in alpha -1,3 linkage to Manalpha Me as in Glcalpha 1-3Manalpha Me. The binding constant increases by 25-fold from di- to trisaccharide and doubles from tri- to tetrasaccharide, demonstrating that the entire Glcalpha 1-3Manalpha 1-2Manalpha 1-2Manalpha Me structure of the oligosaccharide is recognized by calreticulin. The thermodynamic parameters thus obtained were supported by modeling studies, which showed that increased number of hydrogen bonds and van der Waals interactions occur as the size of the oligosaccharide is increased. Also, several novel findings about the recognition of saccharide ligands by calreticulin vis á vis legume lectins, which have the same fold as this chaperone, are discussed.
|Item Type:||Journal Article|
|Additional Information:||Copyright belongs to The American Society for Biochemistry and Molecular Biology.|
|Department/Centre:||Division of Biological Sciences > Molecular Biophysics Unit|
|Date Deposited:||15 Oct 2007|
|Last Modified:||19 Sep 2010 04:39|
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