Forum 01/29/2004

“Generating a library of oligosaccharides to develop sugar-array technology and assess the specificity of carbohydrate binding proteins”

Ola Blizt, Core Manager and Director – Department of Molecular Biology, The Scripps Research Institute, La Jolla, CA

The expanding interest for carbohydrates and glycoconjugates in cell communication has led to an increased demand of these structures for biological studies. Complicated chemical strategies in carbohydrate synthesis are now more frequently replaced by regiospecific and stereospecific enzymes. The exploration of microbial resources and improved production of mammalian enzymes have introduced glycosyltransferases as an efficient complementary tool in carbohydrate synthesis. In this presentation, we introduce strategies to build up diverse series of glycans in a large-scale (up to 10 grams), which are being used in developing a library of carbohydrate compounds. Our work demonstrates that chemoenzymatic synthesis of complicated carbohydrates can reach a facile and practical level by employing a functional toolbox of glycosyltransferases. We have produced 24 different glycosyltransferases in bacterial, fungus, insect cell, and mammalian expression systems. Utilizing these enzymes has enabled us to synthesize various glycans such as; sialoside derivatives of N- and O-linked glycans, ganglioside mimics, blood group antigens, poly-N-acetyllactosamine and its corresponding fucosylated and/or sialylated compounds. We have chemically derivatized our compounds with a built-in neutral spacer that makes them suitable for practical manipulations such as matrix immobilization, biotinylation or polyacrylamide substitutions. The library of oligosaccharides with a window of more than 100 structures, are made available through the Consortium for Functional Glycomics for investigators both inside and outside the program [1]. The versatility together with diversity makes this library favorable of being used in our newly developed sugar-chip arrays technologies for high throughput studies of carbohydrate-protein interactions. These methodologies can further be employed in constructing non-natural analog molecules with therapeutic values or entitled to drug discovery. Identifications of novel carbohydrate ligands with high potency for the sialic acid immunoglobulin superfamily lectins (Siglec) are demonstrated and are expected to facilitate biological exploration of this family of membrane proteins.