Plant O-Hydroxyproline Arabinogalactans Are Composed of Repeating Trigalactosyl Subunits with Short Bifurcated Side Chains

Classical arabinogalactan proteins partially defined by type II O-Hyp-linked arabinogalactans (Hyp-AGs) are structural components of the plant extracellular matrix. Recently we described the structure of a small Hyp-AG putatively based on repetitive trigalactosyl subunits and suggested that AGs are less complex and varied than generally supposed. Here we describe three additional AGs with similar subunits. The Hyp-AGs were isolated from two different arabinogalactan protein fusion glycoproteins expressed in tobacco cells; that is, a 22-residue Hyp-AG and a 20-residue Hyp-AG, both isolated from interferon alpha 2b-(Ser-Hyp)(20), and a 14-residue Hyp-AG isolated from (Ala-Hyp)(51)-green fluorescent protein. We used NMR spectroscopy to establish the molecular structure of these Hyp-AGs, which share common features: (i) a galactan main chain composed of two 1 -> 3 beta-linked trigalactosyl blocks linked by a beta-1 -> 6 bond; (ii) bifurcated side chains with Ara, Rha, GlcUA, and a Gal 6-linked to Gal-1 and Gal-2 of the main-chain trigalactosyl repeats; (iii) a common side chain structure composed of up to six residues, the largest consisting of an alpha-L-Rhap-(1 -> 5)-alpha-L-Araf-(1 -> 3)-alpha-L-Araf-(1 -> 3- unit and an alpha-L-Rhap-(1 -> 4)-beta-D-GlcUAp-(1 -> 6)-unit, both linked to Gal. The conformational ensemble obtained by using nuclear Overhauser effect data in structure calculations revealed a galactan main chain with a reverse turn involving the beta-1 -> 6 link between the trigalactosyl blocks, yielding a moderately compact structure stabilized by H-bonds.