Fractionation and Structural Characterization of Arabinogalactan-Proteins from the Cell Wall of Rose Cells

Abstract

Arabinogalactan-proteins (ACPs) have been purified from Paul's Scarlet rose (Rosa sp.) cell walls. As estimated by gel permeation chromatography, the apparent molecular masses of the two major cell-wall ACP fractions were 130 and 242 kD. Since the 130-kD ACP had a ratio of arabinose/glucuronic acid that was 12 times higher than that of the 242-kD ACP, the fractions were named cell-wall ACPl (CW-ACP1) and glucuronogalactan-protein (CCP), respectively. CW-AGP1 and CCP contained predominantly farabinofuranosyl residues; Ninked, 6-linked, and 3,6-branched galactopyranosyl residues; and 4-linked and t-glucuronopyranosyl residues. The 'H-nuclear magnetic resonance spectra of CW-ACP1 and CCP showed that the arabinofuranosyl and galactopyranosyl residues were predominantly in a-and p-anomeric configuration, respectively, and that GCP contained a few O-acetyl residues. The protein moieties of CW-ACP1 and CCP were both rich in hydroxyproline and alanine but differed in the percentage of various amino acids, including hydroxyproline, alanine, serine, and glycine. Cell-wall AGPs bound to (/3-D-glucosyl), Yariv phenylglycoside, but the stoichiometry of binding was about 6 times greater in CCP than in other Rosa ACPs. CCP seems to be peculiar to the cell wall, since no similar molecule was found in the culture medium.

Oxidative cross‐linking of plasma membrane arabinogalactan proteins

“…Protein blots probed with the AGP-specific ligand 13-glucosyl Yariv reagent and two mAbs that recognize AGPs (MAC207 and JIM8) revealed two plasma membrane AGP bands at approximately 82 and 97 kDa, as shown in Figure l(a). The relatively weak Yariv staining is consistent with the staining of other AGPs from both the plasma membrane (Komalavilas et aL, 1991) and the cell wall (Serpe and Nothnagel, 1995;Smallwood et aL, 1996).…”

“…This is probably due to the lower sensitivity of this staining method together with the lower abundance of this complex. Alternatively, the 210 kDa complex could have relatively few binding sites for the Yariv reagent, as indicated for other plasma membrane (Komalavilas et al, 1991) and cell wall (Serpe and Nothnagel, 1995;Smallwood eta/., 1996) AGPs. In addition to AGP cross-linking following wounding by excision, there seems to be developmental control over AGP cross-linking.…”

Arabinogalactan-proteins: structure, expression and function

“…Thus, the model that emerges is that AGPs containing a GPI anchor are associated with the plasma membrane and are cleaved so as to release the AGP to and beyond the cell wall. Immunolocalizations as well as biochemical isolations of identical, or nearly identical AGPs (family members), in multiple surface locations (e. g. cell wall and culture media; plasma membrane, cell wall and culture media) provide evidence for multiple surface locations for a given AGP and are consistent with the above processing model for GPI-anchored AGPs [5, 10,77,78]. Nonetheless, the possibility of exclusively targeting certain AGPs to specific cell surface locations cannot be excluded, and the contribution of particular sequence determinants in the carbohydrate or protein moieties to such a process remains to be investigated.…”

Presence of a Glycosylphosphatidylinositol Lipid Anchor on Rose Arabinogalactan Proteins

“…The GC-flame ionization detector and GC-MSD equipment and procedures were as described (17,18). Results of sugar analysis by GCflame ionization detector were occasionally checked and confirmed by high-performance anion-exchange chromatography with pulsed amperometric detection (19).…”

“…The model system for this work has been suspension-cultured cells of "Paul's Scarlet" rose (Rosa sp.). Through biochemical purification and chemical structural analysis, PM-AGPs bound to the plasma membrane, CW-AGPs bound to the cell wall, and soluble CM-AGPs of the cell wall space/culture medium have been characterized in this model system (17)(18)(19). Two major AGPs, plus other minor forms, were found at each of these three sites.…”