Complete Structure of the Glycan of Lipopeptidophosphoglycan from Trypanosoma cruxi Epimastigotes”

The lipopeptidophosphoglycan is the major cell surface glycoconjugate of the epimastigote forms of the parasitic protozoan Trypanosoma cruxi. A detailed partial structure for this molecule has been reported (Previato, J. O., Gorin, P. A. J., Mazurek, M., Xavier, M. T., Fournet, B., Wieruszesk, J. M., and Mendonca- Previato, L. (1990) J. Biol. Chem. 266, 2618-2626). In this study, we complete the primary structure as-signments and describe the microheterogeneity found in the lipopeptidophosphoglycan glycan, using a com-bination of ‘H and 31P NMR, fast atom bombardment mass spectrometry, methylation linkage analysis, and exoglycosidase sequencing. The lipopeptidophosphoglycan is a glycosylated inositol-phosphoceramide with striking homology to glycosylphosphatidylinositol membrane anchors found attached to a wide variety of plasma membrane proteins throughout the eukaryotes. The parasitic protozoan Trypanosoma cruzi is the causative agent of Chagas’ disease (South American trypanosomiasis). The generation of inositol-1,2-cyclic phosphate is characteristic of the action of bacterial PI-specific phospholipase C enzymes, and subsequent opening of the cyclic phosphate can lead to an additional mixture of inositol 1-phosphate and inositol 2-phosphate. In this case, only one inositol-monophosphate signal is seen in addition to the inositol 1,2-cyclic phosphate in the "'P NMR spectrum. Analysis of a mixture of unsubstituted inositol 1-phosphate and inositol 2-phos- phate indicated that the two forms give rise to well resolved signals at 5.28 and 6.06 ppm, respectively. The results indicate that, under the conditions used in this paper, B. thuringiensis PI-specific phospholipase C can generate both the 1,2-cyclic phosphate and the 1- phosphate from the LPPG substrate. This result is in agreement with the observations made for B. cereus PI-specific phospholipase C (Volwerk et al., 1990) that showed that this enzyme has an intrinsic, regio-specific, cyclic phosphodiesterase activity.

In this study, we complete the primary structure assignments and describe the microheterogeneity found in the lipopeptidophosphoglycan glycan, using a combination of 'H and 31P NMR, fast atom bombardment mass spectrometry, methylation linkage analysis, and exoglycosidase sequencing. The lipopeptidophosphoglycan is a glycosylated inositol-phosphoceramide with striking homology to glycosylphosphatidylinositol membrane anchors found attached to a wide variety of plasma membrane proteins throughout the eukaryotes.
The parasitic protozoan Trypanosoma cruzi is the causative agent of Chagas' disease (South American trypanosomiasis). The organism undergoes a complex life cycle between a wide variety of mammalian hosts and biting insect vectors (reduviid bugs). In the infected mammal, the parasite exists as intracellular dividing amastigote forms in tissues such as smooth and cardiac muscle. These forms give rise to extracellular nondividing bloodstream trypomastigote forms that spread the infection. The ingestion of trypomastigotes by the insect vector results in the differentiation of the parasite to the dividing epimastigote form, which efficiently colonizes the insect midgut. Migration of parasites to the insect hindgut results in their differentiation to metacyclic trypomastigote forms that are adapted for transmission to a mammalian host via fecal contamination of fresh wounds or mucus membranes.
The lipopeptidophosphoglycan (LPPG)' is the most abun- dant glycoconjugate of the epimastigote form of the parasite; a typical yield of LPPG is about 100 mg extracted from 2 X 10" cells . Assuming a molecular weight of around 1890 (Previato et al. (1990) and this study) this suggests a minimum copy number of around 1.5 X IO7 LPPG molecules/epimastigote. The function of LPPG is unknown, but it appears to be restricted to the epimastigote forms of the parasite (Zingales et al., 1982).
The LPPG fraction contains mannose, galactofuranose, 2aminoethylphosphonate (AEP), myo-inositol, phosphate, long chain bases, and fatty acids, together with traces of glucose and amino acids (Lederkremer et al., 1978;Ferguson et al., 1981Ferguson et al., ,1985Lederkremer et al., 1985). The lipid component is an inositol-phosphoceramide containing mainly palmitoylsphinganine, palmitoylsphingosine, and lignoceroylsphinganine. The ceramide can be released by phosphatidylinositol (PI)-specific phospholipase C and the glycan chain is attached to the inositol ring via a glycosidic linkage to a non-Nacetylated glucosamine (GlcN) residue ). These two latter features indicate that it is closely related to the glycosylphosphatidylinositol (GPI) membrane anchors common to many cell surface glycoproteins throughout the eukaryotes (reviewed recently by Thomas et al. (1990); Cross, 1990).
A detailed structure of the LPPG glycan was recently reported by Previato et al. (1990). In this paper, we confirm these structural features and complete the glycan structure by supplying (i) the nature of the glycosidic linkage between glucosamine and myo-inositol, (ii) the precise location of both galactofuranose residues, and (iii) the degree of heterogeneity in the glycan structure. The LPPG structure is discussed in the context of general and parasite-specific GPI metabolism.

DISCUSSION^
The analysis of the LPPG glycan moiety, generated by PIspecific phospholipase C cleavage of the ceramide lipid, presented some difficult problems. Attempts to fractionate the different glycan species by Dionex carbohydrate high pressure liquid chromatography produced a confusing array of peaks (data not shown) due to the presence of three major glycan species and compounded by the heterogeneity of the inositol phosphorylation state. Deamination and reduction was used to introduce a labeled 2,5-anhydromannitol terminus and to remove the inositol phosphate. However, Dionex high pres-Portions of this paper (including "Materials and Methods," "Results,'' Figs. 1-5, and Tables 1 and 2) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are included in the microfilm edition of the Journal that is available from Waverly Press. sure liquid chromatography of this fraction also produced multiple products (data not shown) due to the simultaneous deamination of the AEP group to a mixture of ethanolphosphonate, phosphate, and other unidentified products. One useful piece of information that did arise from analysis of the deaminated material was the detection of the 2b-anhydromannitol-6-ethanolphosphonate derivative by gas chromatograhy-MS, confirming the NMR assignment of GlcNH2-6-AEP in the original material. Due to the complications described above, the PI-specific phospholipase C-generated head groups were examined as a mixture for most of the subsequent analyses.
The structures shown in Fig. 6 are consistent with the NMR, FAB-MS, linkage composition, and exoglycosidase sequencing data and suggest that the LPPG fraction contains three major glycan structures. The largest of these, Structure 1, contains 4 Man and 2 Galf residues and constitutes about 65% of the molecules. This structure is comparable with the one described by Previato et al. (1990). However, in this case, we were able to assign (i) the GlcNH,-myo-inositol linkage as GlcNH2a1-6myo-inositol from the NMR nuclear Overhauser effect spectroscopy spectrum of fraction A and methylation analysis of fraction D, (ii) the location of the terminal Galf residues exclusively to the nonreducing terminal and subterminal mannose residues by FAB-MS analysis of fraction C and the NMR COSY spectrum of fraction A, and (iii) the existence of the two smaller species. The existence of the two smaller species was defined by the FAB-MS spectrum of fraction B, and their structures were inferred from the methylation analysis of fraction A and the detection of a Man3-as well as Man,-containing mannan cores in fraction E. Assuming that all three LPPG species represent members of a common biosynthetic series, three independent or concomitant pathways to the major product (Structure 1) can be envisaged (Fig. 7). Pulse-chase experiments will be necessary to investigate these possibilities.
The structure of the simplest known GPI protein anchor (from Leishmania major promastigote surface protease (Schneider et al., 1990)) is shown for comparison. EtN, ethanolamine. GPI family since they contain the structural motif Manal-4GlcNH2al-6myo-inositol-l-P04-lipid. Indeed, they are very closely related to GPI protein-anchor structures (see Fig. 6), although LPPG represents the first example of a defined GPI core structure linked to a ceramide. The presence of AEP has not been found in any other fully defined GPI family members, but it has been described in the glycosylated inositolphosphoceramide of Acanthamoeba castellani (Dearborn et al., 1976) and the glycolipids of various water animals such as sea hare (Hayashi, 1990). In the latter example, the AEP is found linked to the 6-position of Gal residues. Interestingly, the GPI anchor of the T. cruzi G-strain metacyclic trypomastigote 1G7-antigen glycoprotein is essentially identical with LPPG, except that it does not contain Galf and utilizes a glycerolipid rather than a ceramide.:' The function of LPPG is unknown. However, it is noteworthy that the related kinetoplastid parasites of the Leishmania species also express GPI-related glycophospholipids (lipophosphoglycans and glycoinositolphospholipids) in equally high copy number, around 1-2 x 10' molecules/cell, in the insect-dwelling promastigote forms (McConville et al. (1990a(McConville et al. ( , 1990b and references therein). Indeed, it is likely that both T. cruzi epimastigotes and Leishmania promastigotes present a particularly carbohydrate-rich surface to their environment. This might serve a protective function in the harsh conditions of the digestive tract of their insect vectors. Whether or not LPPG is involved in the adhesion of parasites to the insect gut epithelia, as suggested for Leishmania lipophosphoglycan (Davies et al., 1990), remains to be determined. In any case, the expression of LPPG by T. cruzi epimastigotes seems to represent another example of the adaptation of the ubiquitous GPI protein-anchor biosynthetic pathway in kinetoplastid parasites.