Leishmania donovani: Cell-surface heparin receptors of promastigotes are recruited from an internal pool after trypsinization

Abstract

With the use of [ 3H]heparin, we recently demonstrated that Leishmania donovani promastigotes express a cell-surface receptor that is specific for the glycosaminoglycan heparin (Mukhopadhyay et al. (1989), The Biochemical Journal, 264, 517–525.). Treatment of the parasite with trypsin abolishes 75–90% of this [ 3H]heparin-binding activity. When trypsinized promastigotes were resuspended in fresh culture medium in the absence and presence of cycloheximide (10μ/ml), approximately 25–30% of the original heparin-binding capacity was restored within 1 hr, indicating that recruitment of receptors from an internal pool occurred without de novo protein synthesis. Scatchard analysis of the regenerated receptor revealed that the number of regenerated binding sites per cell was 2.3 × 10 5; these sites have a binding affinity of 6.7 × 10 −7 M. Like the native heparin receptors on the surface of freshly isolated cells, the receptors recruited after trypsinization are also highly specific for heparin, as a 25-fold excess of four other glycosaminoglycans displaced less than 10% of bound [ 3H]heparin from the trypsinized cells. The structural requirements of the ligand heparin, namely the number of monosaccharide units and degree of sulfation, were compared for both the native and regenerated receptor: for both receptors, oversulfated polysaccharide heparin fragments of at least six to eight sugar residues were most efficient at displacing [ 3H]heparin. The concentrations of oligosaccharide fragments required to displace 50% of [ 3H]heparin were 0.32 and 0.035 μ M for the hexa- and octasaccharides, respectively. Colloidal gold-labeled heparin was bound to promastigotes and visualized by electron microscopy. This analysis revealed that the heparin bound almost exclusively to the flagella of control cells (not subjected to trypsin) and those which had regenerated receptor after trypsinization. The physiological significance of this heparin-binding activity on the surface of promastigotes is discussed.