Functional Genomics and Proteomics Research

Glycosylation, function and development

In developing embryos, signals sent between cells involve molecules that play regulatory roles in programming cell fate, cell adhesion and cell migration properties. Many of these molecules are posttranslationally modified with glycan chains. Recent studies have shown that the posttranslational modification process is critical in regulating cell function and in orchestrating the gene expression patterns in groups of cells or in boundaries or compartments of tissues--important events in the formation of organ systems and anatomical structures.
     Our aims are to use a genome-wide approach to identify and study the functional components that are part of the posttranslational machinery or molecular targets of the posttranslational machinery. To develop and prove this technology we are beginning by targeting all glycosyltransferases and their putative protein substrates in a pilot scale study. (about 300 glycosyltransferases and thousands of glycosylated protein substrates). We are using the following approaches and goals:

Gene discovery and biochemical function

• bioinformatics (sequence motif modeling) to identify all glycosyltransferase encoded by the C. elegans genome"
• functional proteomics to express and characterize all glycosyltransferases in an active state
• chip-based assays of glycosyltransferase enzyme-specificity for non-peptide substrates
• high-throughput expression of unmodified proteins, targeted by glycosyltransferases
• single-chain antibodies for expression pattern mapping the glycosylation machinery

Biology, development and disease significance

• C. elegans is used as a simple model system to identify glycosyltransferase genes and generate screens to define their importance in development and organ formation and function

• RNA interference to rapidly knock-down expression of all glycosyltransferase genes

• 4-D microscopy to examine the loss-of-function phenotype associate with each glycosyltransferase, as a function of space and time during embryogenesis

• GFP-tagged cells in live transgenic animals to examine loss-of-function and model disease phenotypes associate with cell-fate programs and cell signaling events during development

• expression of single chain antibodies in C. elegans to inhibit glycosyltransferases during development in precise tissue and stage-specific patterns

Structure and function

• crystallography of glycosyltransferase will be performed through existing collaborations with international experts in this field

     This combined approach will integrate structural biology, biochemistry and biology to understand mechanisms in which glycosylation is important in fundamental processes during development and disease. The technologies that are being developed and the molecular reagents created through this research program will make possible a multitude of in-depth studies on many unexplored aspects of complex carbohydrates and will provide a model approach for future studies on other posttranslational processes.