The Golgi is the organelle responsible for the posttranslational modification of proteins. Glycosylation, the most prominent posttranslational modification, results in the biosynthesis of glycoproteins and proteoglycans (PGs). Glycosylation is not template driven and it is hypothesized that highly ordered cellular glycosylation is regulated by the unique substrate specificities of the biosynthetic enzymes involved and the Golgi's compartmental structures housing the biosynthetic enzymes. The glycosaminoglycan (GAG) chains of PGs are among the most structurally complex and biologically important glycans. GAG structure, particularly heparan sulfate (HS), carries immense amounts of biological information. HS has been found to play an important role in fibroblast growth factor (FGF)-fibroblast growth factor receptor (FGFR) signaling complexes. The FGF signaling system is important in developmental biology and in cancer. Previous studies performed have shown that HS activates different FGF7FGFR complexes in a sequence dependent manner. These studies suggest that there is a discrete structure-activity relationship (SAR) for HS. The determination of HS-SAR, however, is extremely challenging, as there are no straightforward methods for the synthesis of HS having sufficient domain size and a wide variety of defined sequences. We have begun to address this challenge by designing an artificial Golgi organelle capable of the controlled synthesis of structurally defined, bioactive GAGs. This artificial organelle is based on a newly developed digital microfluidic platform designed to simulate the fluid membrane-based structure of the natural Golgi. This artificial Golgi has begun to provide the precise temporal and spatial control needed for the synthesis of GAGs of defined sequences. The artificial Golgi we have designed is amenable to computer automation and can be interfaced with on-line electrospray ionization-liquid chromatography/mass spectrometry analysis, facilitating high-throughput synthesis and analysis of GAGs. The five specific aims of this project are: 1. Investigate the biosynthesis of HS in a controlled and automated fashion using an artificial Golgi;2. Synthesize a limited combinatorial library of 1080 HS decasaccharide structural subtypes using the artificial Golgi;3. Screen this limited HS library for its ability to signal through FGF;4. Synthesize an HS decasaccharide library expanded around these hits to optimize bioactivity;and 5. Determine the specificity and potency of the HS decasaccharides to signal through the 126 possible FGFWFGFR signaling complexes and establish their SAR The outcome of these studies will be the development of a platform for the controlled synthesis of defined glycan libraries. In vitro high-throughput screening techniques will be established for investigating the biological activity of these glycans. Using these methodologies, we expect to identify potential therapeutic glycans, their sequences, and the specific enzymatic synthesis parameters required for their synthesis.
The proposed effort impacts human health by developing an artificial Golgi to synthesize a library of heparan sulfates and heparan sulfate oligosaccharides. This artificial Golgi uses a digital microfluidic platform for synthesis and screen activity on a cell microarray to develop a structure activity relationship for fibroblast growth factor signaling.
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