Preparation of Arabinogalactan Glycoproteins from Plant Tissue

Abstract

This supplements an earlier protocol (Popper, 2011) for the extraction and assay of cell surface arabinogalactan proteins (AGPs). These highly glycosylated glycoproteins (~95% carbohydrate) contain numerous glycomodules with paired glucuronic acid residues that bind Ca2+ in a pH dependent manner (Lamport and Varnai, 2013). Classical AGPs comprise the bulk of cell surface glycoproteins and are thus integral components of a Ca2+ oscillator involved in a signalling pathway where calcium is a “universal signalling currency” analogous to ATP as the universal energy currency. The central role of these peripheral glycoproteins is thus reason enough for their further study. However, problems arise due to the extensive glycosylation and its apparent microheterogeneity generally assumed to preclude a simple reductionist approach.Here I describe a simple partial purification of classical AGPs based on their specific interaction with the β-D-glucosyl or galactosyl Yariv reagent, a synthetic diazo dye that precipitates AGPs as an insoluble complex in salt solutions at neutral pH. (The solubility of this complex in dilute alkali provides a rapid sensitive quantitative assay for AGPs.) Reduction of the Yariv diazo linkage releases soluble AGPs for further analysis. For example deglycosylation of AGPs in anhydrous hydrogen fluoride followed by column chromatography yields just a few major AGP polypeptides purified to homogeneity (Zhao et al., 2002). However, purification of individual AGP glycoproteins to homogeneity is rarely achieved (Darjania et al., 2002); not only do the closely related AGP glycosylation profiles vastly outweigh any contribution from the amino acid composition but the glycan polydispersity made isolation of a single molecular entity well-nigh impossible until AGPs genetically engineered with a hydrophobic green fluorescent protein tag allowed chromatographic purification (Zhao et al., 2002). New approaches to AGP fractionation into discrete classes is now also a distinct possibility based on their calcium content hitherto ignored!