Defining substrate interactions with calreticulin: an isothermal titration calorimetric study.
Calreticulin (CRT) is a soluble, lectin chaperone found in the endoplasmic reticulum of eukaryotes. It binds the N-glycosylated polypeptides via the glycan intermediate Glc(1)Man(5-9)GlcNAc(2), present on the target glycoproteins. Earlier we have studied interactions of substrate with CRT by isothermal titration calorimetry (ITC) and molecular modeling, to establish that CRT recognizes the Glcalpha1-3 linkage and forms contacts with each saccharide moiety of the oligosaccharide Glcalpha1-3Manalpha1-2Manalpha1-2Man. We also delineated the amino acid residues in the sugar binding pocket of CRT that play a crucial role in sugar-CRT binding. Here, we have used mono-deoxy analogues of the trisaccharide unit Glcalpha1-3Manalpha1-2Man to determine the role of various hydroxyl groups of the sugar substrate in sugar-CRT interactions. Using the thermodynamic data obtained by ITC with these analogues we demonstrate that the 3-OH group of Glc1 plays an important role in sugar-CRT binding, whereas the 6-OH group does not. Also, the 4-OH, 6-OH of Man2 and 3-OH, 4-OH of Man3 in the trisaccharide are involved in binding, of which 6-OH of Man2 and 4-OH of Man3 have a more significant role to play. This study sheds light further on the interactions between the substrate sugar of glycoproteins and the lectin chaperone CRT.
Molecular Biophysics Unit, Indian Institute of Science, Bangalore, 560012, India.
Garima
G
Gupta
Emiliano
E
Gemma
Stefan
S
Oscarson
Avadhesha
A
Surolia
D001665:Binding Sites; D002151:Calorimetry; D037282:Calreticulin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002240:Carbohydrate Sequence; D006023:Glycoproteins / Q000737:chemistry / Q000378:metabolism; D008024:Ligands; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013816:Thermodynamics
D037282:Calreticulin; D006023:Glycoproteins; D008024:Ligands; D009844:Oligosaccharides; D011993:Recombinant Fusion Proteins