Functional Genomics and Proteomics Research
The free-living, transparent nematode
(roundworm), Caenorhabditis elegans.
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). Our objective is to identify every member of the glycosyltransferase superfamily, using motif modeling and searching strategies. Each of these glycosyltransferases will be cloned and targeted in a reverse genetic screen to identify those glycosyltransferases that are critical for development. We believe that C. elegans is best suited for a comprehensive genomics approach because it is a very simple organism, composed of about 1000 somatic cells, in which the complete cell lineage is known at single cell resolution.
Furthermore, C. elegans is amenable to genetic manipulation and rapid RNA interference screens. These features will allow us to screen each glycosyltransferase gene for a loss-of-function phenotype. Those glycosyltransferases that are critical of development will then be characterized biochemically and structurally so that we can work on the interface of biology and biochemistry to elucidate important novel mechanisms in development. 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.
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