Leishmania adleri , a lizard parasite, expresses structurally similar glycoinositolphospholipids to mammalian Leishmania

Glycoblology vol. 7 no. 5 pp. 687-695, 1997

Leishmania adleri, a lizard parasite, expresses structurally similar glycoinositolphospholipids to mammalian Leishmania

J.O.Previato, CJones 1 , R-Wait2, F.Routier3, E.Saraiva and L.Mendonca-Previato4 Instituto de Microbiologia, Universidade Federal do Rio de Janeiro, 21 944 970, Cidade Universiuiria, Rio de Janeiro-RJ, Brazil, 'Laboratory for Molecular Structure, NIBSC, Potters Bar, Herts EN6 3QG, UK, ^ n t r e for Applied Microbiology and Research, Salisbury SP4 OJG, UK, and 3 Laboratoire de Chimie Biologique, UMR 111 du CNRS, USTL, 59 655 Villeneuve d'Ascq, France *To whom correspondence should be addressed

Key words: fast atom bombardment-mass spectrometry/ Leishmania adleriJnuclear magnetic resonance spectroscopy/ glycolipid/oligosaccharide structure

Introduction Parasites of the Trypanosomatidae family are responsible for many diseases of clinical and veterinary importance. Examples include Leishmania species, Trypanosoma cruzi, the etiological agent of Chagas's disease, and the members of the T.brucei complex which cause sleeping sickness in humans and nagana in cattle. The genus Leishmania contains a large number of pathogenic species and subspecies, which are responsible for an overlapping complex of visceral, cutaneous and mucocutaneO Oxford University Press

687

Downloaded from glycob.oxfordjournals.org by guest on July 13, 2011

Glycoinositolphospholipids (GIPLs) were isolated from promastigotes of the lizard parasites Leishmania adleri by phenol/water extraction. Phosphoinositol oligosaccharides were liberated by mild alkaline hydrolysis, purified by gel filtration and high pH anion exchange chromatography, and characterized by methylation analysis, fast atom bombardment mass spectrometry, and nuclear magnetic resonance spectroscopy. The four major compounds (I-IV) from L.adUri were linked to alkylacyl glycerol, and their glycan moieties had the following structures: Mana(l2)Mana(l-6)[ Mana(l-3)] Mana(l^t)GlcNa(l-6)Ins-lPO 4 (I), Galpa(l-6) G a l p a ( l - 3 ) G a ( / P ( l - 3 ) M a n a ( l 3 ) M a n a ( l - 4 ) G l c N a ( l - 6 ) I n s - l - P O 4 (II), G a l ^ a ( l 3)Gal/P(l-3)Mana(l-3)Mana(l-4)GlcNa(l-6)Ins-l-PO 4 (III), Mana(l-2)[EtNP(-6)]Mana(l-6)[ M a n a ( l - 3 ) ] Mana(l-4)GIcNa(l-6)Ins-l-PO 4 (IV). These compounds are analogous to the previously characterized GIPLs from New and Old World leishmania] parasites of mammals designated iM4 (identical to compound I), GIPLs 3 and 2 (identical to compounds II and III, respectively), and EPiM4 (identical to compound IV), which is consistent with a close phylogenetic relationship between lizard and mammalian Leishmania, However, in contrast to the mamma- Han parasites, the abundant surface glycoconjugate known as lipophosphoglycan was either absent or confined to the flagellar pocket region in L.adlert

ous clinical syndromes. These range from relatively mild selflimiting cutaneous forms caused by (among other species) Lmajor and Ltropica in the Old World and Lmexicana and LbrazMensis in South America, to visceral leishmaniasis (kala azar) caused by Ldonovani and Linfantum (Old World) and Lchagasi (New World), which, if untreated, results in extremely high mortality (Grimaldi and Tesh, 1993). Leishmaniases are endemic in 82 countries on four continents, with an estimated 12 million individuals infected (Desjeux, 1992). Although cases are concentrated in third-world countries, the disease is not exclusive to developing nations, and occurs, for example, throughout die Mediterranean basin. The number of Linfantum and HTV coinfections in southern Europe is increasing rapidly (Alvar, 1994). Leishmania species which parasitize non-human hosts have also been identified, including several parasites of Old World lizards. The promastigote stages of these lizard parasites are morphologically similar to those of mammalian Leishmania, and both groups are transmitted by blood-sucking sandflies. However, as amastigote forms are rarely observed, and multiplication within macrophages has not been unequivocally demonstrated in vivo, the taxonomic status of these organisms is ambiguous, and a new genus, the Sauroleishmania has been proposed to accommodate them (Saf janova, 1986; Lainson and Shaw, 1987). Since these organisms have been extensively used as experimental systems, it is important to clarify their relationship to the leishmanial parasites of mammals. The cell surfaces of Leishmania species which parasitize mammals are coated with glycosylphosphatidylinositol (GPI)anchored proteins and two classes of protein-free GPIcontaining lipids: the lipophosphoglycans (LPGs), and the lowmolecular mass glycoinositolphospholipids (GIPLs) (McConville and Ferguson, 1993). The structures of LPGs from five species of Leishmania (L.major, L.mexicana, Ldonovani, Ltropica and Laethiopica) have now been elucidated (McConville et al., 1995), and indicate the existence of significant intra- and inter-specific polymorphism, though die variability is confined to the capping oligosaccharides and the side-chains, since the phosphosaccharide repeats and die GPI core are apparently conserved. The GIPLs of Leishmania have been classified into three lineages on the basis of the linkage between the two mannose residues proximal to inositol (McConville and Ferguson, 1993). In type-1 GIPLs, the second mannose is (1-6) linked to the first, as in the GPI-protein anchors. Type-2 GIPLs differ in that this linkage is (1-3), as in the LPG anchor. Hybrid type GEPLs are branched structures with both (1-3) and (1-6) mannose residues linked to the first mannose. Lmajor promastigotes synthesize predominantly galactose-terminating GIPLs belonging to type-2 lineage, whereas L.mcxicana, Ldonovani, Ltropica, and Laethiopica express mannoseterminating materials from the type-1 and hybrid series. The function of these molecules is still unclear. However, because their structures vary between species and genera they

J.O.Previato et al

Table I. Alkylacylglycerol composition of Ladleri GIPLs* Ladleri GIPL Alkyl of alkylglycerol 22.0 24:0 26:0 Fatty acid 14:0 16:0 18:0

30.3 59.6 4.1 29.4 12.8 57.8

"The values are expressed in mol %.

nuclear magnetic resonance (NMR) spectroscopy and by fast atom bombardment mass spectrometry (FAB-MS). The FAB spectrum contained quasi-molecular ions at m/z 1070, 1193 and 1232. The 500 MHz NMR spectrum similarly suggested considerable glycan heterogeneity. The mixture was fractionated on a Carbopak PA 100 anion exchange column to give four fractions, designated Pl-oligosaccharides I, n, DI, and IV, which were present in a molar ratio of 2:2:1:3 (Figure 1). The : H NMR spectra of these fractions (Figure 2) suggested that they were relatively homogenous, except that some phosphate migration from inositol O-l to O-2 had occurred during the base treatment (Previato et al, 1992). Partial separation of the Ins-1- and the Ins-2-phosphorylated oligosaccharides during HPAE chromatography is likely to be the explanation for the peak splitting observed in the chromatogram (Figure 1). A similar phenomenon was previously reported in the context of HPAE fractionation of base-liberated Pl-oligosaccharides from the GIPLs of Leptomonas samueli (Previato et al., 1994).

Results Isolation and chemical composition of L.adleri glycoinositolphospholipids (GIPLs) A glycolipid fraction, soluble in chloroform/methanol/water (10:10:3) was recovered from the aqueous phase of a hot phenol water extract of Ladleri promastigotes. Compositional analysis revealed the presence of mannose (Man), galactose (Gal), glucosamine (GlcN), inositol (Ins), and phosphorus. The mixture of fatty acids and alkylglycerols listed in Table I was obtained on methanolysis of the GIPLs. The major fatty acids were octadecanoic, tetradecanoic, and hexadecanoic acids. The alkylglycerols were characterized by gas chromatography and mass spectrometry (GC/MS) of their trimethylsilyl (TMS) ethers. In all cases, a base peak was observed at m/z = 205, originating from cleavage between the glycerol C-l and C-2 with charge retention on the TMSO-CH-CH2-OTMS fragment (Myher et al., 1974). No molecular ions were observed in the electron impact spectra, the peaks of highest mass being M-15 species, which were observed at m/z 529, 557, and 585, corresponding to 1-O-docosanyl, -tetracosanyl, and -hexacosanylsn-glycerols, respectively (Table I). Other characteristic fragment ions were observed at M-90 and M-104. The composition of the lipid moiety of the GEPLs of Ladleri is thus similar to that previously reported for Ltarentolae (Routier et al, 1993). Isolation, analysis and fractionation by high-pH anion-exchange chromatography (HPAEC) of phosphoinositol (Pl)-oligosaccharides from L.adleri GIPLs The mixture of the of Pl-oligosaccharides obtained by treatment of the GIPL fraction with 1 M KOH was examined by 688

TIME (min) Fig. 1. High pH anion exchange chromatogram showing separation of the Pl-oligosaccharide fractions I-IV from Leishmania adleri.

Downloaded from glycob.oxfordjournals.org by guest on July 13, 2011

could provide useful phylogenetic markers in the trypanosomatidae family. In evolutionary terms, the GPI-anchored glycoproteins seem to precede the GIPLs. For example, the earliest-diverging trypanosomatid lineage is T.brucei, which is unable to synthesize GIPLs although its glycoproteins have typical GPI-anchors. T.cruzi is the first trypanosomatid lineage in which free GIPLs appear, and these compounds are closely related in structure to the GPI anchors of the surface glycoproteins of this organism (Previato et al, 1995; Carreira et al., 1996). However, the GIPLs of homoxenous and heteroxenous species, which have separated from the trypanosomatid lineage more recently, are increasingly divergent in structure from GPI-protein anchors (Previato et al., 1994; Routier et al., 1995). Comparative examination of GIPL and LPG structures may thus clarify the phylogenetic relationship between the leishmanial parasites of lizards and those of mammals. Ladleri is a lizard parasite that is intermediate in some respects between saurian and mammalian Leishmania. Like mammalian Leishmania (subgenus Leishmania), but in contrast to most other lizard Leishmania, it undergoes the sandfly vector stage of its developmental cycle in the anterior portion of the mid-gut (Heisch, 1958), and is able to establish transient cutaneous infections when experimentally inoculated into humans (Manson-Bahr and Heisch, 1961). In this study we have characterized four GIPLs purified from promastigotes of Ladleri, and we have also attempted to purify LPG from this organism. We show that the structures of the glycan and lipid domains of Ladleri GIPLs are similar to those described for mammalian leishmanias, implying a clear phylogenetic relationship between the lizard and mammalian parasites. However, in contrast to the mammalian parasites, LPG appeared to be absent from lizard leishmanias.

Glycoinositolphospholipids of Leishmania adleri

Chemical Shift (ppm)

Fig. 2. Partial 500 MHz 'H NMR spectra of the Pl-oligosaccharide fractions from Leishmania adleri. (A) Pl-oligosaccharide I. (B) Pl-oligosacchande IV. (C) Pl-oligosaccharide n. (D) Pl-oligosaccharide m.

Characterization of the galactose-containing Pl-oligosaccharides II and HI Positive ion FAB-MS of Pl-oligosaccharide El revealed a quasi-molecular ion at m/z 1070.4 compatible with the composition Hex4-GlcN-InsP (calculated [M+H]+ = 1070.3). Collision induced dissociation (CID) of this species resulted in a series of Y type fragment ions at m/z 908 (Y5), 746 (Y4), 584 (Y3), and 422 (Y2), suggesting an unbranched structure. Facile cleavage between GIcN and Ins resulted in an abundant B 5 ion at m/z 810. The quasi-molecular ion of Pl-oligosaccharide II was observed at m/z 1232, suggesting the presence of an additional hexose (Hex) residue compared to Pl-oligosaccharide HI. Fragment ions in the collision spectrum of this species at m/z 1070, 908, 746, and 584 likewise suggested a linear structure. These conclusions were confirmed by FAB-MS of permethylated samples. Quasi-molecular ions were observed at m/z 1596.9 (Pl-oligosaccharide II) and 1392.6 (Pl-oligosaccharide IE), corresponding to the addition of 26 and 23 methyl groups respectively, and indicating derivatization of all free hydroxyls, and di-N-methylation of GlcN. Abundant reducing-terminal containing fragment ions were observed, even in the absence of collisional activation, at m/z 1406 (1'5X6), 1378 (Y6), 1202 (I>SX5), 1174 (Y s ), 998 ( ' • % ) , 970 (Y4), providing further

evidence of an unbranched structure. In contrast to the spectra of the underivatized Pl-oligosaccharides, the intensities of the '•'X,, ring cleavage ions were more abundant than the corresponding Yn ions. Six major signals were observed in the anomeric region of the 'H NMR spectrum of Pl-oligosaccharide II (Figure 2C). These were assigned as the anomeric protons of two residues of ot-Man (5.173 and 5.270 ppm), two of a-Galp (4.983 and 5.067 ppm), Gal/" (5.199 ppm) (Previato et al, 1993), and GlcN (5.681 ppm). The P spectrum contained only a phosphomonoester resonance, and provided no evidence of phosphorus linked-substituents. Assignment of the *H spectrum of Ploligosaccharide II was achieved using 2D experiments (Table HI). The chemical shifts of the residue designated Galp(2) (Table II) were consistent with a terminal location (Jansson et al., 1989) and an nOe observed between its anomeric resonance (4.983 ppm) and one of the H-6 signals of the other a-Gal/? residue implied a terminal Galp(al-6)Gal(otl- structure. This conclusion was supported by the low-field position of the H-5 resonance of a-Galp(l) (4.323 ppm; triplet, 6 Hz), and by the detection of 2,3,4,6-tetra-0-methyl-l,5-di-C>-acetyl and 2,3,4-tri-O-methyl-l,5,6-tri-O-acetyl galactitol in the methylation analysis (Table IV). The anomeric resonance of the inner a-Gal^ residue exhibited nOes to the H-3 and H-4 of 689

Downloaded from glycob.oxfordjournals.org by guest on July 13, 2011

Chemical Shift (ppm)

J.O.Previato et al

Table IL Structure of Pl-oligosaccharides from Ladleri GIPLs Pl-obgosaccharide

Structure Mana(l-2)Mana(l-6) (4) (3) Mana(l-3) (2)

x

Mana(M)GlcNa(l-6)Ins PO4 /(I)

Galpa(l-6)Galpa(l-3)Gal/P(l-3)Mana(l-3)Mana(l-4)GlcNa(l-6)Ins PO4 (2) (1) (2) (1) 111

Gal/x*(l-3)Gal/P(l-3)Mana(l-3)Mana(l^)GlcNa(l-6)InsPO4 (2) (1) EtNP I Mana(l-2)Mana(l-6) (4) (3)

IV

, Mana(l-4)GlcNa(l-6)Ins PO4 (1) Mana(l-3) (2)

Characterization of the mannose-containing Pl-oligosaccharides I and IV A quasi-molecular ion was observed at m/z 1070 in the FAB spectrum of Pl-oligosaccharide I, suggesting the same compo-

sition as Pl-oligosaccharide IQ. The CID spectrum was qualitatively similar to that obtained from Pl-oligosaccharide HI, except for the absence of the signal at m/z 584, assigned as Y3 in Pl-oligosaccharides II and HI, suggesting a branched structure in which the proximal hexose is substituted with a hexose residue (Table II). FAB-MS of Pl-oligosaccharide IV revealed a quasimolecular ion at m/z 1193, consistent with the composition (Hex)4-GlcN-InsP together with an ethanolamine phosphate (EtNP) substituent. Collisional activation and analysis of the daughters of m/z 1193 by means of a linked scan at constant B/E showed that the EtNP substituent is apparently located on the penultimate Hex rather than GlcN, because of the mass increment of 285 between the ions assigned as Y 4 and Y3, and because the Y2 ion representing glycosidic cleavage at GlcN is observed at m/z 422 as in the other Pl-oligosaccharides, rather than at m/z 545 as would be expected if the GlcN was substituted with EtNP (Wait et al., 1994). The absence of a fragment ion at m/z 584 suggests the presence of a Hex branch in the same position as in Pl-oligosaccharide I. Analysis of ID and 2D ! H NMR spectra of Pl-oligosaccharide IV indicated the presence of four a-Man spin systems, one GlcN and one InsP. Resonances characteristics of EtNP

Table m . 'H NMR assignment for the L adleri Pl-oligosacchandes Pl-oligosaccharide I

H-l

H-2

H-3

Ins GlcN Man(l) Man(2) Man(3) Man(4) Pl-oligosaccharide Q Ins GlcN Man(l) Man(2) Gal^ GalplJ., Stierhof,Y.-D. and Overath.P. (1994) Surface antigens of Leishmania mexicana amastigotes: characterization of glycoinositol phospholipids and a macrophage-derived glycosphingolipid. J. CeUScL, 107,2471-2482. Received on December 23, 1996; accepted on January 30, 1997

Downloaded from glycob.oxfordjournals.org by guest on July 13, 2011

695