Research Interests
Protein-Carbohydrate Interactions: Structure - Function Relationship of Glycosyltransferases.
The complex carbohydrate structures that are synthesized by various isomeric linkages between sugar residues, have an enormous potential for encoding biological information. Although there is no single unifying function assigned to these structurally varied molecules, they clearly serve as cellular recognition markers as part of glycoproteins and glycolipids at the cell surface, taking part in cell-cell interactions, bacterial and viral adhesions. The assembly of these complex carbohydrates requires the concerted action of a large number of Golgi resident glycosyltransferases. This family of enzymes catalyzes the transfer of a single sugar residue to a specific sugar acceptor to form oligosaccharides of different structures and specificities. A knowledge of the three dimensional structure of these enzymes and the conformational preferences of their oligosaccharide ligands is important in understanding the biosynthesis and the function of the carbohydrate moiety of glycoproteins and glycolipids. This information is vital for designing highly specific substrate analogs. In view of this, our laboratory is focusing on the delineation of the structural domains of Golgi glycosyltransferases and the conformational preferences of their oligosaccharide ligands. Molecular cloning approach, first initiated in our laboratory, using b-1,4-galactosyltransferase and its modifier protein a-lactalbumin as a model system, has led to the characterization and grouping of the Golgi resident glycosyltransferases. Despite the differences in the primary sequences of these enzymes, they all share a common topology: an N-terminal cytoplasmic tail, a transmembrane domain, a stem region and a C-terminal catalytic domain facing the lumen. This suggests that the topology plays an important role in their Golgi targeting and to perform their catalytic function. Using recombinant DNA methods and site-directed mutagenesis, we have shown that the hydrophobic length of the transmembrane domain of glycosyltransferases, which on the average is shorter than the corresponding domains in the proteins targeted to the plasma membrane , is an important factor contributing to their Golgi retention. Furthermore, the major binding regions for sugar acceptor and sugar-nucleotide donor lie in the N- and C-terminal halves of the catalytic portion of the protein, respectively, and the two binding surfaces overlap at the catalytic site. The three dimensional structure of the recombinant glycosyltransferases is currently being determined by X-ray crystallography. Molecular dynamics simulations are being carried out on a supercomputer to derive the information about the accessible conformations of oligosaccharides, which is vital for understanding the interactions of carbohydrates with proteins in general, and glycosyltransferases in particular. The conformational data so far has rationalized several biochemical observations like, the binding affinities of ligands to asialoglycoprotein receptor, the rate of cleavage of a-1,2-linked mannoses by a-mannosidases, the action of b-1,4-galactosyltransferase on biantennary oligosaccharides, and the processing pathways of the precursor oligosaccharide during Asn-linked glycan biosynthesis.