Tomato LeAGP‐1 arabinogalactan‐protein purified from transgenic tobacco corroborates the Hyp contiguity hypothesis

• Published in: The Plant journal : for cell and molecular biology 2002-08, Vol.31 (4), p.431-444
• Main Authors: Zhao, Zhan Dong, Tan, Li, Showalter, Allan Marshall, Lamport, Derek Thomas Anthony, Kieliszewski, Marcia Jane
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Amino Acids - metabolism; Analytical, structural and metabolic biochemistry; arabinogalactan‐protein; Base Sequence; Biological and medical sciences; Carbohydrate Metabolism; Cells, Cultured; Circular Dichroism; Fundamental and applied biological sciences. Psychology; Gene Expression Regulation, Plant; Glycoproteins; glycosyl phosphatidylinositol anchor; Glycosylation; Glycosylphosphatidylinositols - chemistry; Glycosylphosphatidylinositols - metabolism; green fluorescent protein; Green Fluorescent Proteins; Hydroxyproline - metabolism; hydroxyproline‐rich glycoprotein; Luminescent Proteins - genetics; Luminescent Proteins - metabolism; Membrane Glycoproteins - genetics; Membrane Glycoproteins - metabolism; Models, Molecular; Molecular Sequence Data; Mucoproteins - chemistry; Mucoproteins - genetics; Mucoproteins - isolation & purification; Nicotiana - genetics; O‐glycosylatoin; Plant Proteins - genetics; Plant Proteins - metabolism; Plants, Genetically Modified - metabolism; Protein Conformation; Proteins; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism; Solanum lycopersicum - genetics; Solanum lycopersicum - metabolism
• ISSN: 0960-7412; 1365-313X
• DOI: 10.1046/j.1365-313X.2002.01365.x

Summary Functional analysis of the hyperglycosylated arabinogalactan‐proteins (AGPs) attempts to relate biological roles to the molecular properties that result largely from O‐Hyp glycosylation putatively coded by the primary sequence. The Hyp contiguity hypothesis predicts contiguous Hyp residues as attachment sites for arabino‐oligosaccharides (arabinosides) and clustered, non‐contiguous Hyp residues as arabinogalactan polysaccharide sites. Although earlier tests of naturally occurring hydroxyproline‐rich glycoproteins (HRGPs) and HRGPs designed by synthetic genes were consistent with a sequence‐driven code, the predictive value of the hypothesis starting from the DNA sequences of known AGPs remained untested due to difficulties in purifying a single AGP for analysis. However, expression in tobacco (Nicotiana tabacum) of the major tomato (Lycopersicon esculentum) AGP, LeAGP‐1, as an enhanced green fluorescent protein fusion glycoprotein (EGFP)‐LeAGP‐1, increased its hydrophobicity sufficiently for chromatographic purification from other closely related endogenous AGPs. We also designed and purified two variants of LeAGP‐1 for future functional analysis: one lacking the putative glycosylphosphatidylinositol (GPI)‐anchor signal sequence; the other lacking a 12‐residue internal lysine‐rich region. Fluorescence microscopy of plasmolysed cells confirmed the location of LeAGP‐1 at the plasma membrane outer surface and in Hechtian threads. Hyp glycoside profiles of the fusion glycoproteins gave ratios of Hyp‐polysaccharides to Hyp‐arabinosides plus non‐glycosylated Hyp consistent with those predicted from DNA sequences by the Hyp contiguity hypothesis. These results demonstrate a route to the purification of AGPs and the use of the Hyp contiguity hypothesis for predicting the Hyp O‐glycosylation profile of an HRGP from its DNA sequence.