Synthetic genes for glycoprotein design and the elucidation of hydroxyproline-O-glycosylation codes

Design of hydroxyproline (Hyp)-rich glycoproteins (HRGPs) offers an approac h for the structural and functional analysis of these wall components, whic h are broadly implicated in plant growth and development. HRGPs consist of multiple small repetitive "glycomodules" extensively O-glycosylated through the Hyp residues. The patterns of Hyp-O-glycosylation are putatively coded by the primary sequence as described by the Hyp contiguity hypothesis, whi ch predicts contiguous Hyp residues to be attachment sites of small arabino oligosaccharides (1-5 Ara residues/Hyp); while clustered, noncontiguous Hyp residues are sites of arabinogalactan polysaccharide attachment. As a test , we designed two simple HRGPs as fusion proteins with green fluorescent pr otein. The first was a repetitive Ser-Hyp motif that encoded only clustered noncontiguous Hyp residues, predicted polysaccharide addition sites, The r esulting glycoprotein had arabinogalactan polysaccharide O-linked to all Hy p residues. The second construct, based on the consensus sequence of a gum arabic HRGP, contained both arabinogalactan and arabinooligosaccharide addi tion sites and, as predicted, gave a product that contained both saccharide types. These results identify an O-glycosylation code of plants.