Abstract
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 alpha2b-(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-Araf-(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.
Highlights
Ala-Hyp-polysaccharide-1 isolated earlier from (Ala-Hyp)51GFP expressed in tobacco cells represented the first complete structure of a small Hyp-AG (15) characteristic of type II arabi
We proposed that ϳ15-residue repetitive blocks of decorated -(1–3) trigalactosyl subunits connected by -1,6 linkages create the larger arabinogalactan polysaccharides of AGPs, consistent with the small blocks separated by periodate-sensitive residues suggested earlier (16, 17)
The Structures of Interferon Hyp-polysaccharide-1, interferon Hyp-polysaccharide-2, and Ala-Hyp-polysaccharide-2 were determined based on earlier composition analyses and on the chemical shifts (15, 26) observed in one-dimensional 1H NMR spectra and two-dimensional correlation spectroscopy (COSY), total correlation spectroscopy (TOCSY), heteronuclear single quantum coherence (HSQC), and heteronuclear multiple bond coherence (HMBC) NMR spectra as follows: Primary Structure of Interferon Hyp-polysaccharide-1—Sizefractionated base hydrolysates of Interferon␣2-(Ser-Hyp)[20] yielded a single peak containing sugar and Hyp residues, described earlier (26)
Summary
Hydroxyproline; AGP, arabinogalactanprotein; AG, arabinogalactan; GFP, green fluorescent protein; COSY, homonuclear correlation spectroscopy; TOCSY, total correlation spectroscopy; NOE, nuclear Overhauser effect; NOESY, NOE spectroscopy; HSQC, heteronuclear single quantum coherence; HMBC, heteronuclear multiple bond coherence. We proposed that ϳ15-residue repetitive blocks of decorated -(1–3) trigalactosyl subunits connected by -1,6 linkages create the larger arabinogalactan polysaccharides of AGPs, consistent with the small blocks separated by periodate-sensitive residues suggested earlier (16, 17). We identified the fundamental similarities between these Hyp-arabinogalactans and determined if the non-glycosylated domains (interferon ␣2 versus GFP) or AGP motifs (Ser-Hyp versus Ala-Hyp repeats) influenced the glycan structure. The six-residue galactan backbone of these new Hyp-AGs consisted of two -1,3-linked galactosyl trisaccharides connected by a -1,6 linkage. Such “decorated” ϳ15-residue trisaccharide subunits likely constitute the fundamental building blocks of type II arabinogalactan polysaccharides; they are far less complex than commonly supposed (4, 19). The NMR analyses and molecular modeling of the glycans revealed major conformers that include a moderately compact folded structure
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