Ether-Type Moieties in the Lipid Part of Glycoinositolphospholipids of Acanthamoeba rhysodes

Magdalena A. Karas 0 Ryszard Russa 0 0 M. A. Karas (&) R. Russa Department of Genetics and Microbiology, Maria Curie-Skodowska University , Akademicka 19, 20-033 Lublin, Poland Ether lipids were identified among components liberated with HF and nitrous acid deamination from Acanthamoeba rhysodes whole cells and its membrane glycoinositolphospholipids (GIPL). Liberated ether glycerols were converted to various derivatives that served characterization thereof. These included TMS and isopropylidene derivatives, oxidation with sodium periodate to aldehyde followed by reduction with NaBH4 to alcohol, and reaction of the alcohol with acetic anhydrite to form acetate derivatives. Periodate sensitivity demonstrated that the alkyl side chains were linked to the sn-1 position of glycerol. Combined information from TLC, GC-MS analysis, MALDI-TOF spectrometry, and chemical degradation experiments indicated the presence of ether-linked saturated normal and branched hydrocarbons with a length of C20-23 in the phospholipid fraction, C20-24 in free GPI, and C21-23 in the LPG polymer. The distribution of particular classes of alkylglycerols was similar for phospholipid and GPI fractions, and amounted to 2.62 % (0.04-0.28) 1-O-eicosanyl-sn-glycerol, 16.66 % (0.321.1) 1-O-uncosanyl-sn-glycerol, 9.18 % (0.33-1.37) anteiso-1-O-docosanyl-sn-glycerol, 47.56 % (0.32-2.14) 1-O-docosanyl-sn-glycerol, 20.56 % (0.58-1.67) anteiso1-O-tricosanyl-sn-glycerol, and 2.34 % (0.12-0.63) 1-Otricosanyl-sn-glycerol. For LPG preparation, the most abundant were anteiso-1-O-tricosanyl-sn-glycerol (57.26 %) and 1-O-docosanyl-sn-glycerol (30.12 %). The data from TLC and GC-MS analysis showed that ether lipids from phospholipids probably represent the lyso-alkylglycerol type, while those derived from GIPL are alkylacylglycerol moieties. - Thin layer chromatography Trimethylsilyl group 1-O-Alkyl-sn-glycerols usually form the backbone of complex ether-linked glycerolipids, including biologically active lipids e.g. PAF (platelet-activating factor) and plasmalogens [1]. They were also identified in parasite Leishmania lipophosphoglycan and glycoinositol phospholipids as 1-alkyl-2-acylglycerol and lyso-alkyl phosphatidylinositol backbones. These ether-type lipid moieties were unusual since they contained predominantly 18:0, 22:0, 24:0, or 26:0 alkyl chains. An increase in the content of 24:0 and 26:0 alkyls was observed with elongation of the carbohydrate chain and appeared almost exclusively in lipophosphoglycan [2, 3]. The existence of long alkyl chains in ether lipids is a rare feature. In general, alkyl chains contain an even number of carbons ranging from C14 to C22 (both saturated or monounsaturated), and saturated or monounsaturated O-alkyl chains of 16 and 18 carbon atoms are the most prevalent. Odd-numbered polyunsaturated and branched chains are only minor components [1]. It has been demonstrated that synthetic alkylacylglycerols corresponding to lipid parts of Leishmania donovani GPI [4], those derived from products of Leishmania lipophosphoglycan hydrolysis as 1-O-alkyl-2-lyso-glycerol [5] as well as the major surface molecules (glycoinositolphospholipids and lipophosphoglycan) isolated from the parasite cells and bearing ether lipids, are antagonists of protein kinase C (PKC) in vitro [4, 5]. Taken together, it was proposed that ether lipid moieties and not the carbohydrate domain could modulate the signalling pathways. Short-chain alkylglycerols have also been shown to increase permeability of tight junctions in the bloodbrain barrier (BBB) [6] and facilitate transport of some drugs [7]. Successful treatment of many brain disorders seems to be impossible because of very limited penetrations of drugs across the BBB, but the demonstrated property of alkylglycerols suggests that they possess several potent pharmacological activities. Glycoinositolphospholipids (GIPL) are heteropolymers in which the sugar portion is coupled to the lipid moiety via an intermediate inositol phosphate. In turn, glycosylphosphatidylinositol (GPI) anchors are a class of GIPL which carry a conservative core structure Mana1-2Mana1 6Mana1-4GlcNH2a1-6myo-inositol-1-PO4-lipid. The lipid moiety in the GPI structure can vary in the nature and the core glycan can have side-chain modifications such as ethanolamine phosphate, mannose, galactose, or sialic acid [8]. The chemical composition of the aliphatic residues is dependent on the organism and the stage in its life cycle. In a majority of eukaryotes, GPI are responsible for binding with a wide group of plasma membrane proteins. In protozoan organisms, they exist in the membrane as free or carry a carbohydrate moiety, e.g. in the lipophosphoglycan of Leishmania and lipophosphonoglycan (LPG) of Acanthamoeba. In Leishmania they are based on a type-2 GPI core, Mana1-3Mana1-4GlcNH2a1 6 PtdIns [9]. Acanthamoeba spp. belongs to parasitic protozoa responsible mainly for granulomatous amoebic encephalitis (GAE) and acanthamoeba keratitis (AK). The lipid content and isolation, purification, and partial characterization of LPG from plasma membranes of Acanthamoeba species have been described previously. Basic chemical analyses have contributed to establishing the composition of 77 % of LPG by weight containing: 26 % neutral sugars (Glc, Man, Gal, Xyl), 3.3 % amino sugars (GalN, GlcN), 8 % inositol, 10 % aminophosphonates (AEP, 1-OH AEP), 3.2 % acid-hydrolyzable phosphate, 14 % fatty acids, and 13 % long chain fatty acids [1012]. In those reports, the described lipid backbone of LPG was the ceramide-type. In the present study, we pre-analyzed lipids liberated with HF from whole cells of Acanthamoeba rhysodes but focused on these derived from isolated GIPL (LPG and free GPI). Among lipids released from whole cells, we identified lysoalkylglycerols with saturated normal and branched hydrocarbons with a length of C2023 as the side chain, which probably came from phospholipids. In the GPI fraction, alkyls with a length of C2124 occurred, and the distribution of the particular classes was similar to that obtained from phospholipids. In contrast, only C2123 AKG were identified in the LPG preparation. The most abundant among the ether lipids discovered in A. rhysodes were 1-O-docosanylsn-glycerol in phospholipid and GPI fractions and anteiso1-O-tricosanyl-sn-glycerol in LPG, respectively. To our knowledge, this is the first report of this kind of ether lipids in Acanthamoeba. Materials and Methods Microorganisms and Culture Conditions Acanthamoeba rhysodes (Hartmannella rhysodes) Changstrain was obtained from the culture collection of Poznan University, Poland. The endocytobiont free amoebae were grown axenically in 300-mL Erlenmeyer flasks containing 100 mL of PYG (peptone yeast glucose) medium, pH 6.6. The chemical composition of the PYG medium was essentially the same as that described by Band [13] and consisted of 15 g proteose peptone 3 (Difco), 5 g yeast extract (Difco), 10 g glucose, (...truncated)