Tomato LeAGP-1 arabinogalactan-protein purified from transgenic tobacco corroborates the Hyp contiguity hypothesis.
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.
Department of Chemistry and Biochemistry, Ohio University, Athens, OH 45710, USA.
Zhan Dong
ZD
Zhao
Li
L
Tan
Allan Marshall
AM
Showalter
Derek Thomas Anthony
DT
Lamport
Marcia Jane
MJ
Kieliszewski
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism; D001483:Base Sequence; D050260:Carbohydrate Metabolism; D002478:Cells, Cultured; D002942:Circular Dichroism; D018506:Gene Expression Regulation, Plant; D006031:Glycosylation; D017261:Glycosylphosphatidylinositols / Q000737:chemistry / Q000378:metabolism*; D049452:Green Fluorescent Proteins; D006909:Hydroxyproline / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D018551:Solanum lycopersicum / Q000235:genetics / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009088:Mucoproteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism; D030821:Plants, Genetically Modified / Q000378:metabolism*; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014026:Nicotiana / Q000235:genetics*
D000596:Amino Acids; D017261:Glycosylphosphatidylinositols; D008164:Luminescent Proteins; D008562:Membrane Glycoproteins; D009088:Mucoproteins; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; C006619:arabinogalactan proteins; D049452:Green Fluorescent Proteins; D006909:Hydroxyproline