INVESTIGATOR: Somerville, C.R.
INSTITUTION: The Carnegie Institute of Washington
NON-TECHNICAL SUMMARY: 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 evident in the genomic DNA sequences of the higher plants for which whole genome sequences are available. 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 a novel assay method that may facilitate the assignment of function to most of the relevant proteins. The goal is to develop reagents and methods that will allow presentation of a large number of different oligosaccharide structures on a solid surface in a small area. In principle, this can be accomplished by using robots to print small amounts of material at high density on a suitably surface-modified slide (eg., a "glycochip") in much the same way that DNA microarrays are made. The glycans presented in this way are expected to serve as acceptors for glycosyltransferases, methylases and acetytransferases, and as substrates for enzymes such as glycosidases that remove sugars from glycans. In this pilot project, we will focus on only those oligosaccharides that can be derived from plant proteoglycans containing O-linked glycans. The oligosaccharides will be prepared by using pure polysaccharide hydrolytic enzymes to fragment naturally occurring O-linked glycans which will then be purified by chromatographic methods. The glycochips produced in this way will be tested for their ability to act as acceptors in enzyme assays for glycosyltransferase enzymes from plants.
OBJECTIVES: To develop high throughput methods and reagents that will facilitate the assignment of function to large numbers of glycosyltransferases and other glycan modifying enzymes.
APPROACH: (1) Development of a series of transgenic plants that express synthetic peptides that become O-glycosylated in various ways and which have regions of sequence that facilitate purification of the glycopeptides; (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 glycosyl transferases activities in protein extracts from plants.
KEYWORDS: Arabinogalactan, AGP, Extensin, Hydroxyproline-rich glycoprotein, Glycosyltransferase, Glycomodule, O-linked glycan, cell wall
Name: Somerville, C.R.
Phone: 650-325-1521 x203