Derek T. A. Lamport
  • School of Life Sciences, University of Sussex, UK
Research fields
  • Biochemistry
Preparation of Arabinogalactan Glycoproteins from Plant Tissue
Author:  Derek T. A. Lamport, date: 10/05/2013, view: 11284, Q&A: 1
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!

Hydroxyproline Assay Using NaBr/NaOCl
Author:  Derek T. A. Lamport, date: 10/05/2013, view: 9936, Q&A: 0
Hydroxyproline (Hyp) is a major constituent of a relatively few proteins that are major structural components of the extracellular matrix and primary cell wall of animals and plants respectively. Significant amounts of the cyclic amino acids proline and hydroxyproline decrease polypeptide flexibility; thus proline/hydroxyproline-rich proteins are ideal scaffold components. Collagens typify animal tissues but extensins, arabinogalactan proteins (AGPs) and their close relatives, collectively referred to as hydroxyproline-rich glycoproteins (HRGPs), typify plants (Lamport et al., 2011). While collagens are minimally glycosylated generally via a galactosyl hydroxylysine linkage, plant HRGP glycosylation involves short neutral oligosaccharides (in extensins) or much larger acidic polysaccharide substituents (in AGPs) O-linked via the hydroxyproline hydroxyl group. Hydroxyproline assay is thus an integral part of their characterization and dominates the biochemical properties of these glycoproteins. The colourimetric assay described here quantifies free hydroxyproline (e.g. released by acid hydrolysis) based on Kivirikko and Liesmma (1959) with hypobromite as an oxidant but modified by avoiding the use of hazardous liquid bromine. A number of oxidants have been used over the years, Vogel (1961, page 395) explains the preference for hypobromite as follows: “Hypochlorites tend to react slowly with reducing agents. Hypobromites although rather unstable when prepared directly from bromine and alkali, often react more rapidly; it is therefore advantageous to produce hypobromite in situ by adding an excess of bromide to the sample of hypochlorite:” OCl- + Br- → OBr- + Cl- “By this means the relative stability of hypochlorite is combined with the more effective oxidizing properties of hypobromite.
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