A lipopeptidophosphoglycan from Trypanosoma cruzi (epimastigota). Isolation, purification and carbohydrate composition

Abstract

A lipopeptidophosphoglycan was extracted from epimastigote forms of Trypanosoma cruzi by phenol (44%) treatment of sonicated cells. The substance was purified from other glycoproteins and nucleic acid as follows: ethanol fractionation. Bio-Gell P-150 column chromatography in the presence of 0.1% sodium dodecyl sulfate, extraction with chloroform/methanol/water (10 : 10 : 3) and precipitation of the pure compound by methanol. The substance migrated as a single band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis stained with periodic acid-Schiff and Coomassie blue. In the absence of sodium dodecyl sulfate very little or no migration was observef aggregates. In such gels a Sudan Black positive reaction coincident with the periodic acid-Schiff positive banc was obtained. Neutral sugars (60%, by phenol-sulfuric acid assay) were analysed by paper chromatography and gal-liquid chromatography. The following ratio was found: mannose : galactose : glucose = 35 : 22 : 1. Glucosamine, identified by paper chromatography, was colorimetrically estimated (0.8%). Sialic acid was not detected. Analysis by the biuret method gave 9.5% protein. All phosphorus present (2%) was released by hydrolysis, thus apparently excluding the possibility of an alkyl phosphonic acid as a structural component. Fatty acids were detected by thin layer chromatography in a hexane extract of the acid hydrolysate. Gas-liquid chromatography of the esterified mixture showed that the main component had the same retention time as palmitic acid methyl ester. The infrared spectrum was consistent with the general structure and indicated the presence of alpha-glycopyranosyl linkages. Low concentrations of the lipopeptidophosphoglycan were able to inhibit the concanavalin A-induced agglutination of epimastigotes.