Interactions of substrate with calreticulin, an endoplasmic reticulum chaperone.
Calreticulin is a molecular chaperone found in the endoplasmic reticulum in eukaryotes, and its interaction with N-glycosylated polypeptides is mediated by the glycan Glc(1)Man(7-9)GlcNAc(2) 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 Glc(1)Man(7-9)GlcNAc(2), determined by the quantitative technique of isothermal titration calorimetry. This method provides a direct estimate of the binding constants (K(b)) and changes in enthalpy of binding (Delta H(b) degrees ) 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 x 10(4) m(-1) at 279 K is obtained when glucose occurs in alpha-1,3 linkage to Man alpha Me as in Glc alpha 1-3Man alpha Me. The binding constant increases by 25-fold from di- to trisaccharide and doubles from tri- to tetrasaccharide, demonstrating that the entire Glc alpha 1-3Man alpha 1-2Man alpha 1-2Man alpha 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.
Kapoor
Mili
M
Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560012, India.
Srinivas
Honnappa
H
Kandiah
Eaazhisai
E
Gemma
Emiliano
E
Ellgaard
Lars
L
Oscarson
Stefan
S
Helenius
Ari
A
Surolia
Avadhesha
A
D000595:Amino Acid Sequence; D002151:Calorimetry / Q000379:methods; D037282:Calreticulin / Q000737:chemistry* / Q000378:metabolism*; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D003001:Cloning, Molecular; D016384:Consensus Sequence; D004721:Endoplasmic Reticulum / Q000378:metabolism; D019277:Entropy; D008958:Models, Molecular; D018832:Molecular Chaperones / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry* / Q000378:metabolism*; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013816:Thermodynamics
D037282:Calreticulin; D018832:Molecular Chaperones; D009844:Oligosaccharides; D011994:Recombinant Proteins