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Glycosylinositol phosphorylceramides from Rosa cell cultures are boron-bridged in the plasma membrane and form complexes with rhamnogalacturonan II.

Voxeur A, Fry SC - Plant J. (2014)

Bottom Line: Using high-voltage paper electrophoresis, we showed that addition of GIPCs decreased the electrophoretic mobility of radiolabelled RG-II, suggesting formation of a GIPC-B-RG-II complex.We conclude that B plays a structural role in the plasma membrane.Finally, our results suggest a role for GIPCs in the RG-II dimerization process.

View Article: PubMed Central - PubMed

Affiliation: The Edinburgh Cell Wall Group, Institute of Molecular Plant Sciences, The University of Edinburgh, Edinburgh, EH9 3JH, UK.

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Mass spectrometric and thin-layer chromatographic analysis of Rosa glycosylinositol phosphorylceramides (GIPCs). (a) ESI-MS analysis of GIPC extract from Rosa cell culture. The spectrum was acquired in the negative ion mode. Abbreviations: Hex, hexose residue (probably α-mannose); HexA, hexuronic acid residue (probably α-glucuronic acid); Pent, pentose residue; Ins, myo-inositol; P, phosphate; Cer, phytoceramide. Inset: proposed structure of the predominant GIPC species; phytoceramide moiety in grey box. (b) ESI-MS/MS (collision-induced dissociation spectrum) analysis of the predominant Hex-HexA-Ins-P-Cer peak seen in (a) as the [M-2H]2− ion at m/z 630. Nitrogen was used as collision gas in a Q-TRAP instrument, with the collision energy set to −40 eV. The standard nomenclature for glycolipid fragmentation has been applied (Costello and Vath, 1990; Levery et al., 2001). Inset: proposed identity of the ion at m/z = 438.4, indicating an h24:0 ceramide moiety. (c, d) Thin-layer chromatography (TLC) of GIPC extract. Lipids were chromatographed in CHCl3/CH3OH/4 m NH4OH (9:7:2, by vol.) with 0.2 m ammonium acetate (Kaul and Lester, 1978) and located by orcinol reagent (c) or periodic acid–Schiff staining (d). Lipid bands are labelled: 1, Hex-HexA-Ins-P-Cer; 2, (Hex)2-HexA-Ins-P-Cer; 3, Pent-(Hex)2-HexA-Ins-P-Cer.
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fig01: Mass spectrometric and thin-layer chromatographic analysis of Rosa glycosylinositol phosphorylceramides (GIPCs). (a) ESI-MS analysis of GIPC extract from Rosa cell culture. The spectrum was acquired in the negative ion mode. Abbreviations: Hex, hexose residue (probably α-mannose); HexA, hexuronic acid residue (probably α-glucuronic acid); Pent, pentose residue; Ins, myo-inositol; P, phosphate; Cer, phytoceramide. Inset: proposed structure of the predominant GIPC species; phytoceramide moiety in grey box. (b) ESI-MS/MS (collision-induced dissociation spectrum) analysis of the predominant Hex-HexA-Ins-P-Cer peak seen in (a) as the [M-2H]2− ion at m/z 630. Nitrogen was used as collision gas in a Q-TRAP instrument, with the collision energy set to −40 eV. The standard nomenclature for glycolipid fragmentation has been applied (Costello and Vath, 1990; Levery et al., 2001). Inset: proposed identity of the ion at m/z = 438.4, indicating an h24:0 ceramide moiety. (c, d) Thin-layer chromatography (TLC) of GIPC extract. Lipids were chromatographed in CHCl3/CH3OH/4 m NH4OH (9:7:2, by vol.) with 0.2 m ammonium acetate (Kaul and Lester, 1978) and located by orcinol reagent (c) or periodic acid–Schiff staining (d). Lipid bands are labelled: 1, Hex-HexA-Ins-P-Cer; 2, (Hex)2-HexA-Ins-P-Cer; 3, Pent-(Hex)2-HexA-Ins-P-Cer.

Mentions: In order to characterize GIPCs from a rose cell culture, we extracted and analysed them according to a protocol described previously (Buré et al., 2011). Mass spectrometric analyses of GIPCs revealed five clusters of compounds (Figure1a). The most intense peak was found in the first cluster at m/z 629.9 ([M-2H]2− ion) corresponding to a GIPC with one hexuronic acid residue, one hexose residue and a t18:1 h24:0 ceramide moiety (where t18:1 indicates a trihydroxylated long-chain base with 18 C atoms and one C=C bond, and h24:0 indicates a monohydroxylated fatty acid with 24 C atoms and no C=C bonds) and containing one 13C atom (out of the 60). The other peaks of this cluster were mainly attributed to mono-hexosylated GIPCs composed of long-chain base t18:0 and t18:1 and fatty acid chains h22:0 to h28:0. Ions of doubly charged species corresponding to the other clusters were assigned to dihexosylated GIPC (m/z = 711.2), and the same containing up to three pentoses and thus having mainly a t18:1 h24:0 ceramide moiety (m/z = 777.5, 843.5, 909.5). These structures were further confirmed by MS fragmentation analysis (Figure1b). The hexose and hexuronic acid present in the major GIPC were not identified; however, they are most likely to be α-d-mannose and α-d-glucuronic acid respectively because the same Rosa culture releases into its culture medium a GIPC-derived fragment which has been characterized as α-d-mannopyranosyl-(1→4)-α-d-glucuronopyranosyl-(1→2)-myo-inositol (Smith and Fry, 1999; Smith et al., 1999).


Glycosylinositol phosphorylceramides from Rosa cell cultures are boron-bridged in the plasma membrane and form complexes with rhamnogalacturonan II.

Voxeur A, Fry SC - Plant J. (2014)

Mass spectrometric and thin-layer chromatographic analysis of Rosa glycosylinositol phosphorylceramides (GIPCs). (a) ESI-MS analysis of GIPC extract from Rosa cell culture. The spectrum was acquired in the negative ion mode. Abbreviations: Hex, hexose residue (probably α-mannose); HexA, hexuronic acid residue (probably α-glucuronic acid); Pent, pentose residue; Ins, myo-inositol; P, phosphate; Cer, phytoceramide. Inset: proposed structure of the predominant GIPC species; phytoceramide moiety in grey box. (b) ESI-MS/MS (collision-induced dissociation spectrum) analysis of the predominant Hex-HexA-Ins-P-Cer peak seen in (a) as the [M-2H]2− ion at m/z 630. Nitrogen was used as collision gas in a Q-TRAP instrument, with the collision energy set to −40 eV. The standard nomenclature for glycolipid fragmentation has been applied (Costello and Vath, 1990; Levery et al., 2001). Inset: proposed identity of the ion at m/z = 438.4, indicating an h24:0 ceramide moiety. (c, d) Thin-layer chromatography (TLC) of GIPC extract. Lipids were chromatographed in CHCl3/CH3OH/4 m NH4OH (9:7:2, by vol.) with 0.2 m ammonium acetate (Kaul and Lester, 1978) and located by orcinol reagent (c) or periodic acid–Schiff staining (d). Lipid bands are labelled: 1, Hex-HexA-Ins-P-Cer; 2, (Hex)2-HexA-Ins-P-Cer; 3, Pent-(Hex)2-HexA-Ins-P-Cer.
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fig01: Mass spectrometric and thin-layer chromatographic analysis of Rosa glycosylinositol phosphorylceramides (GIPCs). (a) ESI-MS analysis of GIPC extract from Rosa cell culture. The spectrum was acquired in the negative ion mode. Abbreviations: Hex, hexose residue (probably α-mannose); HexA, hexuronic acid residue (probably α-glucuronic acid); Pent, pentose residue; Ins, myo-inositol; P, phosphate; Cer, phytoceramide. Inset: proposed structure of the predominant GIPC species; phytoceramide moiety in grey box. (b) ESI-MS/MS (collision-induced dissociation spectrum) analysis of the predominant Hex-HexA-Ins-P-Cer peak seen in (a) as the [M-2H]2− ion at m/z 630. Nitrogen was used as collision gas in a Q-TRAP instrument, with the collision energy set to −40 eV. The standard nomenclature for glycolipid fragmentation has been applied (Costello and Vath, 1990; Levery et al., 2001). Inset: proposed identity of the ion at m/z = 438.4, indicating an h24:0 ceramide moiety. (c, d) Thin-layer chromatography (TLC) of GIPC extract. Lipids were chromatographed in CHCl3/CH3OH/4 m NH4OH (9:7:2, by vol.) with 0.2 m ammonium acetate (Kaul and Lester, 1978) and located by orcinol reagent (c) or periodic acid–Schiff staining (d). Lipid bands are labelled: 1, Hex-HexA-Ins-P-Cer; 2, (Hex)2-HexA-Ins-P-Cer; 3, Pent-(Hex)2-HexA-Ins-P-Cer.
Mentions: In order to characterize GIPCs from a rose cell culture, we extracted and analysed them according to a protocol described previously (Buré et al., 2011). Mass spectrometric analyses of GIPCs revealed five clusters of compounds (Figure1a). The most intense peak was found in the first cluster at m/z 629.9 ([M-2H]2− ion) corresponding to a GIPC with one hexuronic acid residue, one hexose residue and a t18:1 h24:0 ceramide moiety (where t18:1 indicates a trihydroxylated long-chain base with 18 C atoms and one C=C bond, and h24:0 indicates a monohydroxylated fatty acid with 24 C atoms and no C=C bonds) and containing one 13C atom (out of the 60). The other peaks of this cluster were mainly attributed to mono-hexosylated GIPCs composed of long-chain base t18:0 and t18:1 and fatty acid chains h22:0 to h28:0. Ions of doubly charged species corresponding to the other clusters were assigned to dihexosylated GIPC (m/z = 711.2), and the same containing up to three pentoses and thus having mainly a t18:1 h24:0 ceramide moiety (m/z = 777.5, 843.5, 909.5). These structures were further confirmed by MS fragmentation analysis (Figure1b). The hexose and hexuronic acid present in the major GIPC were not identified; however, they are most likely to be α-d-mannose and α-d-glucuronic acid respectively because the same Rosa culture releases into its culture medium a GIPC-derived fragment which has been characterized as α-d-mannopyranosyl-(1→4)-α-d-glucuronopyranosyl-(1→2)-myo-inositol (Smith and Fry, 1999; Smith et al., 1999).

Bottom Line: Using high-voltage paper electrophoresis, we showed that addition of GIPCs decreased the electrophoretic mobility of radiolabelled RG-II, suggesting formation of a GIPC-B-RG-II complex.We conclude that B plays a structural role in the plasma membrane.Finally, our results suggest a role for GIPCs in the RG-II dimerization process.

View Article: PubMed Central - PubMed

Affiliation: The Edinburgh Cell Wall Group, Institute of Molecular Plant Sciences, The University of Edinburgh, Edinburgh, EH9 3JH, UK.

Show MeSH
Related in: MedlinePlus