The lysine-rich arabinogalactan-protein subfamily in Arabidopsis: gene expression, glycoprotein purification and biochemical characterization.
AtAGP17, AtAGP18 and AtAGP19 are homologous genes encoding three putative glycosylphosphatidylinositol (GPI)-anchored classical arabinogalactan-proteins (AGPs) in Arabidopsis. They are distinguished from other AGPs by a short, C-terminal lysine-rich region. Organ-specific expression of these genes was revealed by Northern blot analysis. AtAGP17 was strongly expressed in leaves and stems, and weakly expressed in flowers and roots; AtAGP18 was strongly expressed in flowers, and moderately expressed in roots, stems and young leaves; and AtAGP19 was strongly expressed in stems, moderately expressed in flowers and roots, and weakly expressed in young leaves. One of these genes, AtAGP17, was expressed and purified as a green fluorescent protein (GFP) fusion protein in transgenic tobacco cells using hydrophobic interaction chromatography, size exclusion chromatography and reverse phase high-performance liquid chromatography. The fusion (glyco)protein produced a characteristic AGP 'smear' with a molecular mass of 80-150 kDa when detected by Western blot analysis. Glycosyl composition and linkage analyses of purified GFP-AtAGP17 showed that carbohydrate accounted for approximately 86% of the molecule, with arabinose and galactose as major, and rhamnose and glucuronic acid as minor glycosyl residues and with 1,3,6-galactose, 1,4-glucuronic acid, 1,3-galactose and terminal arabinose as major linkages. GFP-AtAGP17 was also precipitated by beta-Yariv reagent, further confirming that AtAGP17 is a bona fide AGP. Confocal fluorescence microscopy of plasmolysed, transformed cells indicated that AtAGP17 is localized on the plasma membrane and in Hechtian strands. Hydroxyproline (Hyp) glycoside profiles of GFP-AtAGP17 in conjunction with the deduced protein sequence also served to corroborate the Hyp contiguity hypothesis, which predicts contiguous Hyp residues as attachment sites for arabinosides and clustered, non-contiguous Hyp residues as attachment sites for arabinogalactan polysaccharides.
Sun
Wenxian
W
Department of Environmental and Plant Biology, Ohio University, Athens, OH 45701-2979, USA.
Xu
Jianfeng
J
Yang
Jie
J
Kieliszewski
Marcia J
MJ
Showalter
Allan M
AM
D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000737:chemistry* / Q000235:genetics*; D029681:Arabidopsis Proteins / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification; D001483:Base Sequence; D018744:DNA, Plant / Q000235:genetics; D015870:Gene Expression; D017343:Genes, Plant; D006909:Hydroxyproline / Q000737:chemistry; D008239:Lysine / Q000737:chemistry; D008969:Molecular Sequence Data; D015394:Molecular Structure; D008970:Molecular Weight; D009088:Mucoproteins / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification; D005810:Multigene Family; D010802:Phylogeny; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
D029681:Arabidopsis Proteins; D018744:DNA, Plant; D009088:Mucoproteins; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; C006619:arabinogalactan proteins; D008239:Lysine; D006909:Hydroxyproline