Hydroxyproline-rich glycoproteins (HRGPs) occur in the extracellular matrix of land plants and green algae. HRGPs contain from 2 to 95% of their dry weight as carbohydrate, predominantly as oligoarabinosides and/or as heteropolysaccharides which are O-linked to the hydroxyproline residues. A glycosylation code that determines the presence or absence and extent of arabinosylation at each hydroxyproline residue is likely, as each HRGP has a unique arabinosylation profile. Previously we noted a positive correlation between the contiguity of hydroxyproline residues and the extent of HRGP O-arabinosylation (Kieliszewski, M., deZacks, R., Leykam, J. F., and Lamport, D. T. A.(1992) Plant Physiol. 98, 919-926); most arabinosylated hydroxyproline residues and the longer arabinofuranoside chains occur in HRGPs where Hyp residues occur as blocks of tetrahydroxyproline, while those with little or no contiguous Hyp exhibit very little Hyp arabinosylation. In order to test this Hyp contiguity hypothesis, we have for the first time determined the arabinosylation site specifics of an HRGP, namely the proline and hydroxyproline-rich glycoprotein (PHRGP) isolated from Douglas fir (Pseudotsuga menziesii). Pronase digests of PHRGP yielded a major peptide and three glycopeptides whose structures were determined directly from the unfractionated, underivatized Pronase digest by tandem mass spectrometry using collisionally induced dissociation. We corroborated the peptide and glycopeptide structures by Edman degradation, neutral sugar analyses, hydroxyproline arabinoside profiles, and further mass spectrometric analyses after purification of the major peptide and glycopeptides by a combination of hydrophilic interaction and reverse phase column chromatography. Consistent with the Hyp contiguity hypothesis, the structural analyses indicate that while the sequence Ile-Pro-Pro-Hyp is never arabinosylated and Lys-Pro-Hyp-Val-Hyp is only occasionally monoarabinosylated at Hyp-5, the peptide containing contiguous Hyp, Lys-Pro-Hyp-Hyp-Val, is always arabinosylated at Hyp-3, mainly by a triarabinoside. We also obtained precise molecular masses for both intact and anhydrous hydrogen fluoride-deglycosylated PHRGPs (73.113 and 53.834 kDa) via matrix-assisted laser desorption/ionization time of flight mass spectrometry, representing the first HRGP to be analyzed by this method.

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