alpha-chymotrypsin and bis(sulfosuccinimidyl)suberate

Detergent resistant membranes (DRMs) of Plasmodium falciparum merozoites contain a large number of glycosylphosphatidylinositol (GPI)-anchored proteins that have been implicated in interactions between merozoites and red blood cells (RBCs). In this study, two cysteine-rich proteins anchored by GPI to merozoite DRMs (Pf92 and Pf113) were studied with the aim of identifying regions actively involved in RBC invasion. By means of binding assays, high-activity binding peptides (HABPs) with a large number of binding sites per RBC were identified in Pf92 and Pf113. The nature of the RBC surface receptors for these HABPs was explored using enzyme-treated RBCs and cross-linking assays. Invasion inhibition and immunofluorescence localization studies suggest that Pf92 and Pf113 are involved in RBC invasion and that their adhesion to RBCs is mediated by such HABPs. Additionally, polymorphism and circular dichroism studies support their inclusion in further studies to design components of an antimalarial vaccine.

Topics: Animals; Binding Sites; Chymotrypsin; Circular Dichroism; Cross-Linking Reagents; Cysteine; Detergents; Erythrocytes; Glycosylphosphatidylinositols; Host-Parasite Interactions; Immune Sera; Membrane Proteins; Merozoites; Models, Molecular; Neuraminidase; Peptides; Plasmodium falciparum; Polymorphism, Genetic; Protein Binding; Protein Structure, Secondary; Protozoan Proteins; Rabbits; Succinimides; Trypsin

Subunit interactions in the band 3 protein of the human red blood cell membrane have been examined by a combination of cross-linking, chemical labeling, and in situ proteolysis. In agreement with Staros (Staros, J. V. (1982) Biochemistry 21, 3950-3955), we find that the membrane-impermeant active ester bis(sulfosuccinimidyl) suberate (BSSS) cross-links band 3 in intact cells to a dimer, with no formation of higher oligomer. Combined cross-linking of the outer surface with BSSS and the cytoplasmic domain with Cu2+/o-phenanthroline does not produce significant covalent tetramer of band 3 (beyond that produced by Cu2+/o-phenanthroline alone). Therefore, the membrane domains and cytoplasmic domains of the same pair of subunits are cross-linked to each other. 4,4'-Diisothiocyanodihydrostilbene-2,2'-disulfonate (H2DIDS) is known to form a covalent cross-link between complementary chymotryptic fragments (Mr 60,000 and 35,000). Edman degradation of band 3 from H2DIDS/chymotrypsin-treated cells shows that the H2DIDS cross-link is between fragments of the same subunit. In contrast, BSSS forms both intramolecular and intermolecular cross-links between complementary chymotryptic fragments. No intermolecular cross-links between two 35,000-dalton or two 60,000-dalton fragments are detectable. We have localized one end of the BSSS intermolecular cross-link to within 4 residues of the exofacial chymotrypsin cleavage site. The polypeptide sequence on each side of the site suggests that hydrophobic membrane-crossing segments emerge at the cell surface near the site of intermolecular cross-linking. This is the first detailed information available on the regions of the band 3 primary structure near the interface between subunits.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Anion Exchange Protein 1, Erythrocyte; Chymotrypsin; Humans; Macromolecular Substances; Molecular Weight; Peptide Fragments; Protein Conformation; Stilbenes; Succinimides