Contiguous hydroxyproline residues direct hydroxyproline arabinosylation in Nicotiana tabacum

Hydroxyproline (Hyp) O-glycosylation characterizes the hydroxyproline-rich glycoprotein (HRGP) superfamily of the plant extracellular matrix. Hyp glyc osylation occurs in two modes: Arabinosylation adds short oligoarabinosides (Hyp-arabinosides) while galactosylation leads to the addition of larger a rabinogalactan polysaccharides (Hyp-polysaccharides). We hypothesize that s equence-dependent glycosylation of small peptide motifs results in glycomod ules. These small functional units in combination with other repetitive pep tide 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 endopl asmic reticulum/Golgi for post-translational proline hydroxylation and glyc osylation 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 yi elded 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, Th ese results plus circular dichroic spectra of the glycosylated and deglycos ylated (Ser-Pro(2))(n), (Ser-Pro(3))(n), and (Ser-Pro(4))(n) modules corrob orate the Hyp contiguity hypothesis and indicate that Hyp O-glycosylation i s indeed sequence-driven.