Biophysical analysis of the plant-specific GIPC sphingolipids reveals multiple modes of membrane regulation

The plant plasma membrane (PM) is an essential barrier between the cell and external environment, controlling signal perception transmission. It consists of asymmetrical lipid bilayer made up three different classes: sphingolipids, sterols, phospholipids. glycosyl inositol phosphoryl ceramides (GIPCs), representing to 40% total are assumed be almost exclusively in outer leaflet PM. However, their biological role properties poorly defined. In this study, we investigated GIPCs organization. Because not commercially available, developed a protocol extract isolate GIPC-enriched fractions from eudicots (cauliflower tobacco) monocots (leek rice). Lipidomic analysis confirmed presence trihydroxylated long chain bases 2-hydroxylated very long-chain fatty acids 26 carbon atoms. glycan head groups dicots were analyzed by gas chromatograph–mass spectrometry, revealing sugar moieties. Multiple biophysics tools, namely Langmuir monolayer, ?-Potential, light scattering, neutron reflectivity, solid state 2H-NMR, molecular modeling, used investigate physical GIPCs, as well interaction with free conjugated phytosterols. We showed that increase thickness electronegativity model membranes, interact differentially phytosterols species, regulate gel-to-fluid phase transition during temperature variations. These results unveil multiple roles played contains main classes lipids: phytosterols, phospholipids, all high level complexity, see (1Cacas J.L. Buré C. Grosjean K. Gerbeau-Pissot P. Lherminier J. Rombouts Y. Maes E. Bossard Gronnier Furt F. Fouillen L. Germain V. Bayer Cluzet S. Robert et al.Revisiting lipids tobacco: A focus on sphingolipids.Plant Physiol. 2016; 170: 367-384Crossref PubMed Scopus (99) Google Scholar, 2Yetukuri Ekroos Vidal-Puig A. Oreši? M. Informatics computational strategies for study lipids.Mol. Biosyst. 2008; 4: 121-127Crossref (163) Scholar). Sphingolipids part involved regulation cellular signaling, trafficking, growth, stress responses. Ubiquitous eukaryotes, they structurally animal, fungi, kingdoms (3Markham J.E. Lynch D.V. Napier J.A. Dunn T.M. Cahoon E.B. Plant sphingolipids: Function follows form.Curr. Opin. Biol. 2013; 16: 350-357Crossref (123) While some sphingolipid structures such sphingoid conserved both plants others specific fungi plants. sphingolipids highly studied involvement human health pathologies (4Hannun Y.A. Obeid L.M. metabolism physiology disease.Nat. Rev. Mol. Cell 2018; 19: 175-191Crossref (781) most abundant animal sphingomyelin (SM) gangliosides. absent, whereas other complex comprised bound sphingolipid. major subclass (GIPCs). discovered 1950s (5Carter H.E. Gigg R.H. Law J.H. Nakayama T. Weber Biochemistry sphingolipides. XI. Structure phytoglycolipide.J. Chem. 1958; 233: 1309-1314Abstract Full Text PDF structural diversity lies glycosylation hydroxylation, degree position saturation acid (FA) chain, base (LCB) (6Pata M.O. Hannun Ng C.K.Y. Decoding enigma sphinx.New Phytol. 2010; 185: 611-630Crossref (155) predominantly consist t18:0 or t18:1 LCB (trihydroxylated saturated monounsaturated) amidified (VLCFA) VLCFA (hVLCFA) form ceramide 7Buré Cacas Wang Gaudin Domergue Mongrand Schmitter J.M. Fast screening glycosylated tandem mass spectrometry.Rapid Commun. Mass Spectrom. 2011; 25: 3131-3145Crossref (67) GIPC group linked phosphate inositol, forming (IPC) backbone, which then further substituted broad polar heads 23 species algae largely unknown vary widely across taxa (8Cacas Maalouf J.P. Badoc Antajan Biochemical survey glycosylinositolphosphoceramides unravels diversity.Phytochemistry. 96: 191-200Crossref (54) classified into series, based (7Buré plants, characterized date have glucuronic (GlcA) first IPC, followed at least one more unit varying identity. For example, series defined monosaccharide addition GlcA-IPC 1960s, characterization structure Nicotiania tabacum (tobacco) was described (9Hsieh T.C. Lester R.L. Laine R.A. Glycophosphoceramides Purification novel tetrasaccharide derived tobacco leaf glycolipids.J. 1981; 256: 7747-7755Abstract 10Carter Kisic Countercurrent distribution inosol seeds.J. Lipid Res. 1969; 10: 356-362Abstract 11Carter Koob bean leaves (Phaseolus vulgaris).J. 363-369Abstract 12Kaul Characterization inositol-containing phosphosphingolipids leaves.Plant 1975; 55: 120-129Crossref 13Sperling Heinz Structural diversity, biosynthesis, genes functions.Biochim. Biophys. Acta. 2003; 1632: 1-15Crossref (215) 14Buré Branched inositolphosphosphingolipid revealed MS3 analysis.J. 51: 305-308Crossref (4) 15Mortimer J.C. Yu X. Albrecht Sicilia Huichalaf Ampuero D. Michaelson L.V. Murphy A.M. Matsunaga Kurz Stephens Baldwin Ishii A.P. al.Abnormal glycosphingolipid mannosylation triggers salicylic acid-mediated responses Arabidopsis.Plant Cell. 1881-1894Crossref (73) 16Fang Ishikawa Rennie E.A. Murawska G.M. Lao Yan Tsai A.Y. Baidoo E.E. Xu Keasling J.D. Demura Kawai-Yamada Scheller H.V. Mortimer Loss phosphorylceramide induces immune reduces cellulose content arabidopsis.Plant 28: 2991-3004Crossref (46) 17Luttgeharm K.D. Kimberlin A.N. R.E. Cerny Markham Sphingolipid strikingly pollen Arabidopsis compositional gene expression profiling.Phytochemistry. 2015; 115: 121-129Crossref (31) 18Tellier Maia-Grondard Schmitz-Afonso I. Faure Comparative sphingolipidomic reveals seeds oil.Phytochemistry. 2014; 103: 50-58Crossref (24) 19Nagano Fujiwara Fukao Kawano Shimamoto Plasma microdomains Rac1-RbohB/H-mediated immunity rice.Plant 1966-1983Crossref (74) 20Rennie Ebert B. Miles G.P. Christiansen K.M. Stonebloom Khatab H. Twell Petzold C.J. Adams P.D. Dupree Heazlewood Identification ?-glucuronosyltransferase function 26: 3314-3325Crossref (63) 21Ishikawa Fang Sechet Jing Moore W. GLUCOSAMINE INOSITOLPHOSPHORYLCERAMIDTRANSFERASE1 (GINT1) GlcNAc-containing glycosylinositol glycosyltransferase.Plant 177: 938-952Crossref (25) 22Mortimer Synthesis glycosylation.Trends Sci. 2020; 522-524Abstract (10) 23Kaiser H.J. Lingwood Levental Sampaio Kalvodova Rajendran Simons Order phases membranes.Proc. Natl. Acad. U. 2009; 106: 16645-16650Crossref (323) 24Levental Veatch S.L. continuing mystery rafts.J. 428: 4749-4764Crossref (181) 25Lingwood rafts membrane-organizing principle.Science. 327: 46-50Crossref (3169) 26Baumgart Hunt G. Farkas E.R. Webb W.W. Feigenson G.W. Fluorescence probe partitioning Lo/Ld membranes.Biochim. 2007; 1768: 2182-2194Crossref (382) 27Mannock D.A. Lewis R.N.A.H. McMullen T.P.W. McElhaney R.N. effect variations phospholipid sterol nature lipid-sterol interactions membranes.Chem. Phys. Lipids. 163: 403-448Crossref (113) 28Gerbeau-Pissot Der Thomas Anca I.A. Roche Perrier-Cornet Simon-Plas Modification organization cells elicited cryptogein.Plant 164: 273-286Crossref (29) 29Grosjean Beney Differential specificities phytosphingolipids phytosterols.J. 290: 5810-5825Abstract 30Beck J.G. Mathieu Loudet Buchoux Dufourc E.J. sterols “rafts”: better way thermal shocks.FASEB 21: 1714-1723Crossref (124) 31Furt Lefebvre Cullimore Bessoule J.J. rafts: Fluctuat nec mergitur.Plant Signal. Behav. 2: 508-511Crossref (15) 32Moreau Nyström Whitaker B.D. Winkler-Moser J.K. Baer D.J. Gebauer S.K. Hicks K.B. Phytosterols derivatives: distribution, metabolism, analysis, health-promoting uses.Prog. 70: 35-61Crossref (205) 33Sonnino Prinetti Gangliosides regulators functions.Adv. Exp. Med. 688: 165-184Crossref (48) 34Lingwood Binnington Róg Vattulainen Grzybek Coskun Ü. C.A. Cholesterol modulates glycolipid conformation receptor activity.Nat. 7: 260-262Crossref (165) 35Markham Jaworski Rapid measurement thaliana reversed-phase high-performance liquid chromatography coupled electrospray ionization 1304-1314Crossref (200) 36Mamode Cassim Gouguet Laurent N. Grison Boutté Key players function.Prog. 2019; 73: 1-27Crossref (105) 37Voxeur Fry S.C. Glycosylinositol phosphorylceramides Rosa cultures boron-bridged complexes rhamnogalacturonan II.Plant 79: 139-149Crossref (89) 38Kitazawa Tryfona Yoshimi Hayashi Kawauchi Antonov Tanaka Takahashi Kaneko Tsumuraya Kotake ?-galactosyl yariv reagent binds ?-1,3-galactan arabinogalactan proteins.Plant 161: 1117-1126Crossref (108) 39Deleu Crowet Nasir M.N. Lins Complementary biophysical tools specificity bioactive molecules membrane: review.Biochim. 1838: 3171-3190Crossref (115) 40Tjellström Hellgren L.I. Wieslander Å. Sandelius A.S. asymmetry membranes: Phosphate deficiency-induced replacement restricted cytosolic leaflet.FASEB 24: 1128-1138Crossref (55) 41Maget-Dana R. monolayer technique: potent tool studying interfacial antimicrobial membrane-lytic peptides 1999; 1462: 109-140Crossref (519) 42Marsh Lateral pressure 1996; 1286: 183-223Crossref (883) 43Róg Pasenkiewicz-Gierula Non-polar cholesterol phospholipids: dynamics simulation study.Biophys. 2004; 107: 151-164Crossref (45) 44Smondyrev Berkowitz M.L. Molecular dimyristoylphosphatidylcholine bilayers cholesterol, ergosterol, lanosterol.Biophys. 2001; 80: 1649-1658Abstract (119) 45Yeagle P.L. Martin R.B. Lala A.K. Lin H.K. Bloch effects lanosterol artificial 1977; 74: 4924-4926Crossref (107) 46Róg Karttunen Ordering its analogues.Biochim. 1788: 97-121Crossref (449) 47Kubsch Robinson Steinkühler Dimova Phase behavior charged vesicles under symmetric asymmetric solution conditions monitored fluorescence microscopy.J. Vis. 2017; https://doi.org/10.3791/56034Crossref (17) 48Jiang Z. Zhou Tao Yuan Liu Wu Xiang Niu Li Ye Byeon Xue Zhao al.Plant cell-surface sense salt trigger Ca2+ influx.Nature. 572: 341-346Crossref (206) 49Seelig Deuterium magnetic resonance: Theory application membranes.Q. 353-418Crossref (1071) 50Davis description conformation, order 2H-NMR.Biochim. 1983; 737: 117-171Crossref (924) 51Kang S.Y. Gutowsky H.S. Oldfield Spectroscopic studies specifically deuterium labeled systems. Nuclear resonance investigation protein-lipid Escherichia coli membranes.Biochemistry. 1979; 18: 3268-3272Crossref (38) 52Seneviratne Frech Furneaux Phases transitions P(EO)6LiSbF6.Electrochim. 48: 2221-2226Crossref (16) 53Simenel Coddeville Delepierre Latgé Fontaine Glycosylinositolphosphoceramides Aspergillus fumigatus.Glycobiology. 84-96Crossref 54Gutierrez A.L.S. Farage Melo Mohana-Borges R.S. Guerardel Wieruszeski Mendonça-Previato Previato J.O. glycoinositolphosphoryl mutant strains Cryptococcus neoformans.Glycobiology. 17: 1C-11CCrossref (18) 55Buré spectrometry.Anal. Bioanal. 406: 995-1010Crossref (49) 56Lenar?i? Albert Böhm Hodnik Pirc Zavec A.B. Podobnik Pahovnik Žagar Pruitt Greimel Yamaji-Hasegawa Kobayashi Zienkiewicz Gömann al.Eudicot plant-specific determine host selectivity microbial NLP cytolysins.Science. 358: 1431-1434Crossref (103) 57Lefebvre Hartmann M.A. Carde Sargueil-Boiron Rossignol Medicago truncatula root proteomic raft-associated redox system.Plant 144: 402-418Crossref (218) 58Mongrand Morel Laroche Claverol Bonneu Lessire higher cells: triton X-100-insoluble membrane.J. 279: 36277-36286Abstract (375) extraction method required hundreds kilograms material liters solvents. From reported still has best date: GlcNAc(?1?4)GlcA(?1?2)inositol-1-O-phosphorylceramide, Figure 1A. Additional moieties described, glucosamine (GlcN), N-acetyl-glucosamine (GlcNAc), arabinose (Ara), galactose (Gal), mannose (Man), may lead observed patterns seven sugars, so-called B noteworthy Kaul calculated ratio carbohydrate/LCB/inositol purified polyglycosylated contain 19 20 sugars (12Kaul Scholar), opens large field investigation. Polyglycosylated found Zea mays (corn) Erodium display branched (13Sperling species- tissue-specific. Arabidopsis, headgroup Man-GlcA-IPC predominant callus (15Mortimer array N-acetyl pentose units present (17Luttgeharm Amino-acylated N-acylated oil (18Tellier GlcN(Ac)-GlcA-IPC mainly rice monocots, core yet deciphered. GIPC’s responsible polarity GIPC, accounting insolubility traditional solvents, chloroform/methanol. Consequently, lost aqueous interface. although fundamental components PM model, been studied, because absence commercial preparations. Recent evidence demonstrated loss lethal (20Rennie Scholar) misglycosylation affects abiotic biotic responses, reviewed (22Mortimer This highlighted importance investigating understanding chemical these functions Lipids homogeneously distributed within bilayers. lateral might differential behaviors due (23Kaiser using approaches super resolution microscopy (24Levental domains liquid-ordered (Lo) formed phospholipids sterol, liquid-disordered unsaturated (25Lingwood Lo phases, conformational imposed acyl tails rigid ring cholesterol. increases packing, remain laterally mobile (27Mannock Sphingolipid-sterol recently important determinants (28Gerbeau-Pissot 50% depending organ (31Furt harboring wide including dominated ?-sitosterol, stigmasterol, campesterol (32Moreau play significant regulating ternary mixtures (sterol/sphingolipid/saturated phospholipid) less sensitivity compared systems mimicking (30Beck me