Development of a proteoglycan chip for plant glycomics

ESTEVEZ, J. M. & C. SOMERVILLE

Maryland USA

Workshop; DOE Workshop; 2008

In order to develop plants for use as optimal feedstocks for biofuels production from cellulosic biomass, it will be necessary to understand how the polysaccharides that comprise the majority of plant biomass are made and deposited in cell walls. More than a thousand genes for proteins that may be involved in synthesis and assembly of plant cell walls are known. In order to assign functions to such a large number of genes, high-throughput assay methods will be required. This project concerns the development of the first generation of genetically encoded peptides (GEP) that acts as substrates for O-glycosylation in planta. The goal is to develop a plataform that will allow us to produce a large number of different oligosaccharide structures on a solid surface. The glycans presented in the GEP are expected to serve as acceptors for glycosyltransferases, and as substrates for enzymes such as glycosidases that would remove sugars from the original glycans. In this pilot project, we are focused only in those oligosaccharides that can be derived from plant proteoglycans containing O-linked Arabinogalactans. The oligosaccharides on the GEP are being purified and characterized. The next step in this project is to generate several different glycan structures on the GEP by using pure polysaccharide hydrolytic enzymes to fragment naturally occurring O-linked glycans. The glycochips produced in this way will be tested for their ability to act as acceptors in enzyme assays for glycosyltransferase enzymes from plants. APPROACH: (1) Development of a series of transgenic plants that express synthetic peptides that become O-glycosylated ; (2) purification of glycopeptides from transgenic plants; (3) determination of the structure of the glycans; (4) sequential cleavage of the glycans on each of the glycopeptides to produce a series of partial glycans; (5) production of glycochips by robotic spotting of the various glycopeptides onto chemically modified surfaces; (6) development of mass spectrometric methods for measuring the mass of glycopeptides in a microformat; (7) use of the glycochips to assay for glycosyltransferases activities in protein extracts from plants. KEYWORDS: Arabinogalactan, AGP, Extensin, Hydroxyproline-rich glycoprotein, Glycosyltransferase, Glycomodule, O-linked glycan, cell wall.