Reinforced Polyproline II Conformation in a Hydroxyproline-Rich Cell Wall Glycoprotein from Carrot Root

Abstract

The salt-extractable hydroxyproline-rich cell wall glycoprotein from carrot (Daucus carota L.) roots is composed of 35% (w/w) protein, 3% (w/w) galactose, and 62% (w/w) arabinose. The arabinose is attached to hydroxyproline as tetra-and trisaccharides. The circular dichroism of the glycoprotein shows that it is completely in the polyproline II conformation. After deglycosylation of the glycoprotein, the polyproline II conformation of the peptide backbone was lost. This indicates that the carbohydrate reinforces the polyproline II conformation.Hydroxyproline-containing insoluble cell wall glycoproteins (extensins) occur in a wide variety of plants. One of the main problems in studying these extensins has been the difficulty in extracting the proteins from the wall (see 22 for review). They are usually covalently linked in the wall, presumably by isodityrosine cross-links (7,14). Wounded carrots accumulate a hydroxyproline-rich cell wall glycoprotein (see 30 for review) that slowly becomes insolubilized (4, 7). A considerable amount of this protein not yet covalently linked to the wall can be extracted under nondenaturing conditions with salts (4, 31). The biosynthesis of this protein has been studied extensively (30) but a reliable amino acid composition was not available until 1980 (31). The amino acid composition is similar to that of the hydroxyproline-rich potato agglutinin (24) which is localized in the wall (25). The major amino acids found in the carrot glycoprotein are the same amino acids which preferentially accumulate in the cell walls of elongating pea and bean stems (19,34).The hydroxyproline-rich cell wall glycoprotein is thought to be a structural wall component (22). We studied the structure of the hydroxyproline-rich cell wall glycoprotein from carrot roots (which we propose to call 'extensin precursor' in concordance with Lamport's usage of the term).MATERIALS AND METHODS Plant Material. Carrot (Daucus carota L.) roots were obtained from a local merchant and stored at 5°C until needed. For incubation of carrot slices, the roots were washed, surface sterilized with 1% (w/w) NaClO for 10 min and sliced (about 1 mm thick in a food processor). The slices were put in racks in a plastic box and incubated for 5 d in moist air at room temperature.Cell Wall Isolation. The fresh or incubated tissue (about 1.5 kg) was homogenized in a blender in a solution containing 0.5% ' Supported by grants from the National Science Foundation (PCM 7923550 and PCM 8104516) and from the Monsanto Co. (an unrestricted postdoctoral fellowship grant).(v/v) Nonidet P-40 (Sigma) plus 2 mM Na2S205 and filtered through Miracloth (Calbiochem). The cell walls were extensively washed with 2 mm-Na2S205 and finally with 20 mM Tris-HCl buffer, pH 8.0, plus 2 mM Na2S20.Isolation of Hydroxyproline-Rich Cell Wall Glycoprotein. In the following fractionation procedures, we determined the salt concentration of the fractions with the refractometer, the protein content by light absorption at 280 nm, and the hydroxyproline content-aft...

Polyproline II Confirmation in the Protein Component of Arabinogalactan-Protein from Lolium multiflorum

“…However, the ellipticity at 200 nm does not increase to the values of -10,000 to -12,000 degreescm2*dmolV' reported for fully denatured proteins. The apparent resistance of the polyproline II conformation of complete denaturation at 75C has also been observed with the carrot cell wall glycoprotein and with synthetic poly(hydroxyproline) (17).…”

“…1). Each ofthese changes is observed during the transition from an extended polyproline II conformation to an unordered structure at elevated temperatures in the carrot cell wall glycoprotein (17), poly(glycyl-prolylalanine) (5) and collagen (14). However, the ellipticity at 200 nm does not increase to the values of -10,000 to -12,000 degreescm2*dmolV' reported for fully denatured proteins.…”

“…However, using the value of [O] -1 1,300 for denatured collagen as a reference for unordered structures (14) and a value of [0] -46,000 as a reference ellipticity for polyproline II conformation (17), it can be estimated that the minimum ellipticity of [0] -21,000 for the protein from arabinogalactan-protein (Fig. 1) corresponds to about 30% of the protein in the polyproline II conformation.…”

“…Thus, most denatured conformations have a minimum value for [0] of -10,000 to -12,000 degrees cm2.dmol' (9) while proteins containing a high proportion of polyproline II conformation show an ellipticity ofapproximately -45,000 to -50,000 degrees cm2. VAN (14,17).…”

“…The hydroxyproline-rich cell wall glycoprotein from carrot has been shown by circular dichrometry to exist in the polyproline II conformation (17), which consists of a lefthanded helix of three residues per turn and a rise for residue of 0.31 nm (15). Based on optical rotary dispersion measurements, it has been suggested that lectins of the Solanaceae also contain polyproline II conformation (1,7).…”