| Hydroxyproline-rich glycoproteins (HRGPs) play important roles in all aspects of plant growth and development, including defense responses. Generally, HRGPs are highly O-glycosylated through the hydroxyproline (Hyp) residues which mean carbohydrate helps define the interactive molecular surface and therefore the function of HRGPs. The Hyp contiguity hypothesis predicts that contiguous Hyp residues are sites of HRGP arabinosylation while clustered non-contiguous Hyp residues are all attachment sites of arabinogalactan hetero-polysaccharides that characterize the arabinogalactan-proteins (AGPs). Early tests of the hypothesis using synthetic genes encoding only clustered non-contiguous Hyp in the sequence (Ser-Hyp)n or contiguous Hyp in the Hyp series (Ser-Hyp-Hyp)n and (Ser-Hyp-Hyp-Hyp-Hyp)n confirmed that arabinogalactan polysaccharide was added only to non-contiguous Hyp while arabinosylation occurred only on blocks of contiguous Hyp. Here the tests were extended to the codes which direct arabinogalactan-polysaccharide addition to Hyp by building genes encoding the repetitive sequences: (Ala-Pro) n, (Thr-Pro)n, and (Val-Pro)n and expressing them in Bright Yellow 2 (BY2) tobacco cells as enhanced green fluorescence protein (EGFP) fusion proteins. All of the Pro residues in the (Ala-Pro)n fusion protein were hydroxylated and, consistent with the hypothesis, every Hyp residue was glycosylated with arabinogalactan polysaccharide. In contrast, a significant amount of Pro remained un-hydroxylated in the (Thr-Pro) n, and (Val-Pro)n fusion proteins. Furthermore, although many of the Hyp residues were glycosylated with arabinogalactan polysaccharide, some Hyp remained un-glycosylated or were arabinosylated. These results suggest that amino acid side chains influence the extent of proline hydroxylation and subsequent Hyp glycosylation and may explain why non-contiguous Hyp in extensins are either un-glycosylated or arabinosylated. Two arabinogalactan polysaccharides (AHP-1 and AHP-2) were isolated following base hydrolysis of the (Ala-Pro)n-EGFP fusion protein and their structures were elucidated by NMR analysis. AHP-1 and AHP-2 were named as 4-Hyp-4-O-{beta-D-Gal p-(1→3)-beta-D-Galp-(1→6)-beta-D-Gal p-(1→3) [(beta-D-GlcA-(1→6))-(alpha-L-Araf -(1→3)-alpha-L-Araf-(1→3))]-beta-D-Gal p-(1→3)-[(alpha-L-Rhap-(1→4)-beta-D-GlcA-(1→6))(alpha-L-Ara f-(1→5)-alpha-L-Araf-(1→3)-alpha-L-Ara f-(1→3))]-beta-D-galactopyranoside} and 4-Hyp-4-O-(beta-D-Gal p-(1→3)-beta-D-Galp-(1→3)-beta-D-Gal p-(1→6)-beta-D-Galp-(1→3)-[(alpha-L-Rha p-(1→4)-beta-D-GlcA-(1→6))(alpha-L-Araf-(1→3))]-beta-D-Gal p-(1→3)-[(alpha-L-Rhap-(1→4)-beta-D-GlcA-(1→6))(alpha-L-Ara f-(1→3))]-beta-D-galactopyranoside}. These structures confirmed the linkage between Hyp and galactose and provided novel sequence information in the arabinogalactan polysaccharide component of arabinogalactan-proteins. |
