Heparan sulfates (HS) are abundant and molecularly complex sugars of the extracellular space. They are involved in regulating cell-cell interactions, for example by modulating growth factor signaling or regulating the interaction between microbes and the human cells. HS functions are mediated by structural oligosaccharide motifs as a result of complex modifications of the sugars. Thus, understanding structure function relationships of the complex HS sugar molecules may open up new avenues of intervention, e.g. by interfering with growth factor signaling in malignancies or, in infectious diseases. A major problem in the study of HS function in the past has been the inability to visualize and analyze functional HS motifs in living animals and, to directly correlate HS structure with function in a defined cellular context in vivo. This application addresses these challenges by developing novel tools using C. elegans.
The first aim will develop a novel transgenic approach to visualize defined HS structures in living animals. To this end, animals transgenically expressing HS specific antibody::GFP fusions will be created. This will for the first time allow the analysis of HS patterns, i.e. the HS 'sugar landscape'and dynamics in time and space in a metazoan. This in vivo labeling approach will open the door for genetic analyses of the 'sugar landscape'in a whole animal and, will aid in identifying the genes that regulate HS expression and that respond to HS.
The second aim i s designed to directly correlate HS structure with function by using a combination of biochemical and transgenic approaches. To this end, tagged proteoglycan transgenes will be used to purify the HS core protein and, subsequently the attached sugars from defined cellular contexts. By cell-specific expressing the transgenes it will be possible to isolate and characterize cell-specific, core protein specific, and attachment site-specific HS sugars using established biochemical methods. By at the same using these transgenes to assay for rescue of an HS sugar dependent cell migration phenotype of a syndecan null mutant, it will be possible to directly correlate HS structure with function in vivo. In summary, this proposal will develop novel tools to visualize and analyze the function of defined HS structural motifs in vivo.
Sugars with complex modification patterns such as heparan sulfates are abundant in the extracellular space and involved in regulating many cell-cell signaling pathways involved in tumor formation. In addition, heparan sulfates are important for entry of HIV and Herpes viruses into cells and, cell-cell interactions between a range of microbes and the cells of the human host. We propose here to develop novel tools that allow us to study the biosynthesis of specific heparan sulfate structures as well as conduct detailed structure function analyses of heparan sulfate in a living organism. Understanding heparan sulfate structure function relationships may ultimately allow to interfere with deregulated cell-cell signaling during malignancies or host- microbe interactions.
