Leishmania donovani: Surface membrane carbohydrates of promastigotes

Abstract

Cloned promastigote forms of Leishmania donovani, a human protozoan pathogen, were agglutinated specifically with the plant lectins concanavalin A (Con A), fucose binding protein, phytohemagglutinin-M and -P, soybean agglutinin (SBA), and wheat germ agglutinin (WGA). Cell agglutination with lectins was inhibited, and reversed in the presence of specific lectin-binding monosaccharides. Promastigotes also were agglutinated by viable influenza virions. Neuraminidasetreated cells were not agglutinated by the virions. Cells were agglutinated randomly with all lectins and the influenza virions suggesting a uniform distribution in the promastigote cell surface of the lectin and influenza virion-binding saccharide ligands. Both living and glutaraldehyde-fixed cells gave similar agglutination results with the various lectins and influenza virus. Promastigotes were also agglutinated with low concentrations of cationized ferritin indicative of a negative surface charge. Noncationized ferritin did not induce cell agglutination. Living L. donovani promastigotes were also specifically adsorbed onto the surface of Con A-Sepharose beads but not onto heat-denatured Con A-Sepharose or Sepharose 4B. No cell adsorption to Con A-Sepharose was observed in controls treated with α-methyl- D-mannoside. The lectin-binding kinetics of the cell surface α- D-mannose receptor ligand was assessed using 3H-labeIed Con A. At saturation, 2.78–4.86 × 10 −4 μmole of [ 3H]Con A were bound per 10 8 promastigotes with a mean of 3.73 × 10 −4 μmole/10 8 cells. Membrane receptor sites sterically available for binding [ 3H]Con A were calculated from kinetic data, and ranged from 1.67–2.93 × 10 6 sites/cell with a mean of 2.24 × 10 6 ligand sites/promastigote. Cell-surface-bound Con A was visualized at the ultrastructure level with an iron-dextran (Fe-Dex) conjugate. The dense Fe-Dex particles were distributed randomly on the pellicular and flagellar membrane outer lamina. Con A bound at the cell surface was also visualized using horseradish peroxidase (HRPO) and diaminobenzidine (DAB) coupled reactions. Dense Con A-HRPO-DAB reaction product was deposited uniformly on the cell pellicular and flagellar membranes. SBA and WGA were chemically conjugated to HRPO and these were used in DAB coupled reactions with the L. donovani promastigotes. The fine structure cell surface localization and distribution of these HRPO-conjugated lectins was similar to that obtained with the Con A-HRPO-DAB preparations. Influenza virions visualized at the fine structure level were bound randomly to the outer lamina of the cell pellicular and flagellar membranes. At the ultrastructure level, cationized ferritin was uniformly distributed over the entire cell surface. The cumulative results demonstrate that ligands similar or identical to α- d-mannose, N-acetylgalactosamine, N-acetylglucosamine, α- l-fucose, and an N-acetylated neuraminic acid are constituents of the L. donovani pellicular and flagellar membranes. These carbohydrates probably represent terminal moieties of cell membrane structural and functional glycoproteins. Further, the polyanionic carbohydrates impart a net negative charge to the L. donovani surface membrane.