Expression of a novel cell surface lipophosphoglycan-like glycoconjugate in Trypanosoma cruzi epimastigotes.

B.N Singh,J.J Lucas,D.H Beach,C.E Costello

doi:10.1016/s0021-9258(17)31743-x

Abstract

The lipophosphoglycan (LPG)-like glycoconjugate expressed on the cell surface of Trypanosoma cruzi epimastigotes was isolated, purified, and partially characterized. The glycoconjugate migrated as a homogeneous band (42 kDa) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Matrix-assisted laser desorption ionization mass spectral analysis of the native molecule indicated the presence of two major components whose molecular masses were about 18.4 and 22.5 kDa. The LPG could be metabolically labeled with [3H]galactose, [3H]mannose, [14C]glucose, or [3H]palmitic acid. Monosaccharide compositional analysis of the LPG indicated that galactose, glucosamine, and sialic acid predominate over mannose, galactosamine, and inositol. A peptide associated with the LPG molecule contained about 40 amino acid residues per inositol and had threonine as the predominant amino acid. The LPG showed strong binding to Ricinus communis agglutinin-1 and Tritium vulgare wheat germ agglutinin, indicating the presence of terminal beta 1,4-linked galactosyl residue(s) and N-acetylglucosamine, respectively. Lectin binding studies also suggested the presence of a terminal beta-galactose and GlcNAc in the glycan-inositol lipid core of LPG. Virtually all of the sialic acids appeared to be located in the saccharide portion of the molecule. Treatment of the LPG with phosphatidylinositol-specific phospholipase C liberated an alkylacylglycerol. Structural analysis of the alkylacylglycerol and its acidic methanolysis products by gas-liquid chromatography/mass spectrometry indicated that the glycerol substituents were primarily the C16 1-alkyl group and C16 2-acyl group. The ratio of inositol to 1-O-alkyl-2-O-acylglycerol was 1:1. Treatment of the glycoconjugate with nitrous acid released a major phospholipid product that migrated close to the phosphatidylinositol standard on thin layer chromatography. This result implied that phosphatidylinositol was glycosidically linked to the nonacetylated amino sugar. Furthermore, the LPG was found to contain phosphate and was labile to mild acid hydrolysis, strongly suggesting that the intact molecule is related to Leishmania LPG. The most striking and unique feature of T. cruzi LPG is the presence of large amounts of glucosamine and sialic acid as well as galactosamine. These results indicate that the glycoconjugate expressed on the T. cruzi cell surface is a new type of LPG-like molecule anchored on the cell surface via an alkylacylphosphatidylinositol.

Highlights

The LPG was by a GPI anchor(9).The infective metacyclic trypomastigote of found to contain phosphate and was labile to mild acid T cruzi expresses the 1G7 antigen, which is anchored t o hydrolysis, strongly suggesting that the intacmt olecule the parasite plasma membrane via a GPI membrane anchor is related to Leishmania LPG
Phenyl-Sepharose chromatography anolysis and PI-Phospholipase C-GLC and GCMS analyseosf of [3H]galactose-labeled LPG treated with PI-phospholipase C fatty acid methyl esters obtained after acid methanolysis of enzyme resulted in complete hydrolysis of LPG, as demon- LPG showed palmitic acid as the major fatty acid and stearic strated by quantitative elution of radioactivity from the column acid a s a minor fatty acid
Cell surface glycoconjugates of parasitic protozoans have been shown to play a key role in host-cell recognitiodinvasion and in parasite survival (1, 2)

Summary

Introduction

PI-Phospholipase C Release of the Lipid Component of Glycoconjugate-The LPG sample (["Hlpalmitate-labeledor unlabeled) was incubated in the presencoef 25 mM HEPES, pH7.4, containing 0.1%(w/v) sodium deoxycholate, and PI-phospholipase Cfrom B. thuringiensis for cruzi metabolically radiolabeled with ["Hlgalactose after solvent E extraction followed by Sepharose CL-4B gel filtration and octyl-Sepharose chromatography and methanol precipitation. The surface The major monosaccharides observed were galactose, glucosalabeled LPG had the sameelution characteristics on gel filtra- mine, mannose, sialic acid, and traces of galactosamine and tion and on octyl-Sepharose as nativeLPG (Fig. 2).

Results

Conclusion