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Genomic Science Program

2006 Awardee

Development of a Proteoglycan Chip for Plant Glycomics

INVESTIGATOR: Somerville, C.R.

INSTITUTION: The Carnegie Institute ofWashington

NON-TECHNICAL SUMMARY: In order todevelop plants for use as optimal feedstocks for biofuelsproduction from cellulosic biomass, it will be necessary tounderstand how the polysaccharides that comprise themajority of plant biomass are made and deposited in cellwalls. More than a thousand genes for proteins that may beinvolved in synthesis and assembly of plant cell walls areevident in the genomic DNA sequences of the higher plantsfor which whole genome sequences are available. In orderto assign functions to such a large number of genes,high-throughput assay methods will be required. Thisproject concerns the development of a novel assay methodthat may facilitate the assignment of function to most ofthe relevant proteins. The goal is to develop reagents andmethods that will allow presentation of a large number ofdifferent oligosaccharide structures on a solid surface ina small area. In principle, this can be accomplished byusing robots to print small amounts of material at highdensity on a suitably surface-modified slide (eg., a "glycochip") inmuch the same way that DNA microarrays are made. The glycans presentedin this way are expected toserve as acceptors for glycosyltransferases, methylases andacetytransferases, and as substrates for enzymes such asglycosidases that remove sugars from glycans. In thispilot project, we will focus on only those oligosaccharidesthat can be derived from plant proteoglycans containingO-linked glycans. The oligosaccharides will be prepared byusing pure polysaccharide hydrolytic enzymes to fragmentnaturally occurring O-linked glycans which will then bepurified by chromatographic methods. The glycochipsproduced in this way will be tested for their ability toact as acceptors in enzyme assays for glycosyltransferaseenzymes from plants.

OBJECTIVES: To develop high throughputmethods and reagents that will facilitate the assignment offunction to large numbers of glycosyltransferases and otherglycan modifying enzymes.

APPROACH: (1) Development of a series oftransgenic plants that express synthetic peptides thatbecome O-glycosylated in various ways and which haveregions of sequence that facilitate purification of theglycopeptides; (2) purification of glycopeptides fromtransgenic plants; (3) determination of the structure ofthe glycans; (4) sequential cleavage of the glycans on eachof the glycopeptides to produce a series of partialglycans; (5) production of glycochips by robotic spottingof the various glycopeptides onto chemically modifiedsurfaces; (6) development of mass spectrometric methods formeasuring the mass of glycopeptides in a microformat; (7)use of the glycochips to assay for glycosyltransferasesactivities in protein extracts from plants.

KEYWORDS: Arabinogalactan, AGP, Extensin,Hydroxyproline-rich glycoprotein, Glycosyltransferase,Glycomodule, O-linked glycan, cell wall,

PROJECT CONTACT:

Name: Somerville, C.R.
Phone: 650-325-1521 x203
Fax: 650-325-6857
Email:crs@stanford.edu

Funding Announcement DE-FOA-0001865:
Systems Biology of Bioenergy-Relevant Microbes to Enable Production of Next-Generation Biofuels and Bioproducts.
Pre-App required 01/08/2018, 5 PM Eastern [12/17]

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