Design of hydroxyproline (Hyp)-rich glycoproteins (HRGPs) offers an approach for the structural and functional analysis of these wall components, which 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, which predicts contiguous Hyp residues to be attachment sites of small arabinooligosaccharides (1-5 Ara residues͞Hyp); 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 protein. The first was a repetitive Ser-Hyp motif that encoded only clustered noncontiguous Hyp residues, predicted polysaccharide addition sites. The resulting glycoprotein had arabinogalactan polysaccharide O-linked to all Hyp residues. The second construct, based on the consensus sequence of a gum arabic HRGP, contained both arabinogalactan and arabinooligosaccharide addition sites and, as predicted, gave a product that contained both saccharide types. These results identify an O-glycosylation code of plants.

Hydroxyproline (Hyp) O-glycosylation characterizes the hydroxyproline-rich glycoprotein (HRGP) superfamily of the plant extracellular matrix. Hyp glycosylation occurs in two modes: Arabinosylation adds short oligoarabinosides (Hyp-arabinosides) while galactosylation leads to the addition of larger arabinogalactan polysaccharides (Hyp-polysaccharides). We hypothesize that sequence-dependent glycosylation of small peptide motifs results in glycomodules. These small functional units in combination with other repetitive peptide modules define the properties of HRGPs. The Hyp contiguity hypothesis predicts arabinosylation of contiguous Hyp residues and galactosylation of clustered noncontiguous Hyp residues. To determine the minimum level of Hyp contiguity that directs arabinosylation, we designed a series of synthetic genes encoding repetitive (Ser-Pro 2 ) n , (Ser-Pro 3 ) n , and (Ser-Pro 4 ) n . A signal sequence targeted these endogenous substrates to the endoplasmic reticulum/Golgi for post-translational proline hydroxylation and glycosylation in transformed Nicotiana tabacum cells. The fusion glycoproteins also contained green fluorescence protein, facilitating their detection and isolation. The (Ser-Pro 2 ) n and (Ser-Hyp 4 ) n fusion glycoproteins yielded Hyp-arabinosides but no Hyp-polysaccharide. The motif (Ser-Pro 3 ) n was incompletely hydroxylated, yielding mixed contiguous/noncontiguous Hyp and a corresponding mixture of Hyparabinosides and Hyp-polysaccharides. These results plus circular dichroic spectra of the glycosylated and deglycosylated (Ser-Pro 2 ) n , (Ser-Pro 3 ) n , and (Ser-Pro 4 ) n modules corroborate the Hyp contiguity hypothesis and indicate that Hyp O-glycosylation is indeed sequence-driven. Hydroxyproline-rich glycoproteins (HRGPs)1 participate in the plant extracellular matrix as networks, exudates, and glycocalyx, comprising a superfamily that includes extensins (1), proline-rich proteins (PRPs) (2) and arabinogalactan-proteins (AGPs) (3). The three major families are distinguished by characteristic repetitive structural motifs: Ser-Hyp 4 in extensins, Pro-Hyp-Val-Tyr-Lys repeats and variants in PRPs, and XaaHyp-Xaa-Hyp repeats plus the presence of arabinogalactan polysaccharide in AGPs.The major post-translational modifications of HRGPs, proline hydroxylation and subsequent O-Hyp glycosylation, determine the properties of HRGPs to a greater or lesser extent. Carbohydrate accounts for as much as 95% of the hyperglycosylated AGPs and about 60% of extensins, thus forming the interactive molecular surface. In the lightly glycosylated PRPs, however, sugar may contribute as little as 1% of the mass.O-Hyp glycosylation occurs in two distinct modes, Hyp arabinosylation (4) and Hyp galactosylation (5), respectively. Hyp arabinosylation of virtually all HRGPs results in short (usually 1-4 residues), neutral, linear homooligosaccharides of L-arabinofuranose (Hyp-arabinosides). Hyp galactosylation, which is restricted to the AGPs, results in addition of much larger arabinogalactan heteropolysac...

Extensins are cell wall hydroxyproline-rich glycoproteins that form covalent networks putatively involving tyrosyl and lysyl residues in cross-links catalyzed by one or more extensin peroxidases. The precise cross-links remain to be chemically identified both as network components in muro and as enzymic products generated in vitro with native extensin monomers as substrates. However, some extensin monomers contain variations within their putative cross-linking motifs that complicate cross-link identification. Other simpler extensins are recalcitrant to isolation including the ubiquitous P3-type extensin whose major repetitive motif, Ser-(Hyp) 4 -Ser-Hyp-Ser-(Hyp) 4 -Tyr-Tyr-Tyr-Lys, is of particular interest, not least because its Tyr-Tyr-Tyr intramolecular isodityrosine cross-link motifs are also putative candidates for further intermolecular cross-linking to form di-isodityrosine. Therefore, we designed a set of extensin analogs encoding tandem repeats of the P3 motif, including Tyr 3 Phe and Lys 3 Leu variations. Expression of these P3 analogs in Nicotiana tabacum cells yielded glycoproteins with virtually all Pro residues hydroxylated and subsequently arabinosylated and with likely galactosylated Ser residues. This was consistent with earlier analyses of P3 glycopeptides isolated from cell wall digests and the predictions of the Hyp contiguity hypothesis. The tyrosine-rich P3 analogs also contained isodityrosine, formed in vivo. Significantly, these isodityrosine-containing analogs were further crosslinked in vitro by an extensin peroxidase to form the tetra-tyrosine intermolecular cross-link amino acid diisodityrosine. This is the first identification of an intermolecular cross-link amino acid in an extensin module and corroborates earlier suggestions that di-isodityrosine represents one mechanism for cross-linking extensins in muro.Hydroxyproline-rich glycoproteins (HRGPs), 1 which include the extensins, proline-rich proteins, and arabinogalactan proteins (AGPs), contribute to extracellular matrix architecture throughout the plant kingdom and the chlorophycean green algae (1-3). HRGPs are involved in all aspects of plant growth and development, including wall assembly during embryogenesis (4), and responses to biotic and abiotic stress that include mechanical stress (5), physical wounding (6), pathogenesis (7), and symbiosis (8 -10).HRGPs are extended macromolecules consisting of small repetitive peptide and glycopeptide motifs that form peptide modules and glycomodules of functional significance, as in "mix-and-match" mode they define the molecular properties of the overall macromolecule. The glycomodules result from a combination of post-translational modifications unique to plants, namely proline hydroxylation (11) and its subsequent glycosylation (12) that leads either to short arabino-oligosaccharide or larger arabinogalactan polysaccharide addition to the Hyp residues. A sequence-dependent O-Hyp glycosylation code directs the addition of oligosaccharide and polysaccharides (13), and it is likely that...

Hydroxyproline-rich glycoproteins (HRGPs) are ubiquitous architectural components of the growing plant cell wall, accounting for as much as 10-20% of the dry weight. HRGPs are implicated in all aspects of plant growth and development, including responses to stress. The HRGP superfamily contains three major groups which represent a continuum of peptide periodicity and hydroxyproline-O-glycosylation. These groups range from the highly periodic and lightly arabinosylated repetitive proline-rich proteins (PRPs), through the cross-linked extensins which are periodic and highly arabinosylated, to the arabinogalactan-proteins (AGPs) which are the most highly glycosylated and least periodic. The repetitive units are small, often only four- to six-residue-glycosylated modules viewed hypothetically as functional motifs, or glycomodules. The Hyp contiguity hypothesis predicts that Hyp arabinosylation increases with Hyp contiguity and that clustered noncontiguous Hyp residues are sites of arabinogalactan polysaccharide addition in the AGPs and gums. Recent results involving glycosylation site mapping of endogenous HRGPs and HRGP design using synthetic genes have corroborated the hypothesis. The uses of synthetic genes in HRGP glycosylation site mapping and structural/functional analysis are also discussed.