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

Authors

  • Zhan Dong Zhao,

    1. Department of Chemistry and Biochemistry, Ohio University, Athens, OH 45710, USA,
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  • Li Tan,

    1. Department of Chemistry and Biochemistry, Ohio University, Athens, OH 45710, USA,
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  • Allan Marshall Showalter,

    1. Department of Environmental and Plant Biology, Ohio University, Athens, OH 45710, USA,
    2. Department of Molecular and Cellular Biology Program, Ohio University, Athens, OH 45710, USA,
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  • Derek Thomas Anthony Lamport,

    1. School of Biological Sciences, University of Sussex, Falmer, Brighton BN1 9QG, UK
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  • Marcia Jane Kieliszewski

    Corresponding author
    1. Department of Chemistry and Biochemistry, Ohio University, Athens, OH 45710, USA,
    2. Department of Molecular and Cellular Biology Program, Ohio University, Athens, OH 45710, USA,
      For correspondence (fax +1 740 597 1772; e-mail kielisze@helios.phy.ohiou.edu).
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For correspondence (fax +1 740 597 1772; e-mail kielisze@helios.phy.ohiou.edu).

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.

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