Abstract
Circumsporozoite protein (CSP) coats the malarial sporozoite and functions to target the liver for infection, which is the first step to developing malaria. An important tissue ligand for CSP is the glycosaminoglycan heparan sulfate (HS) found on the surface of hepatocytes and in the basement membrane of the space of Disse. To better understand this efficient targeting process, we set out to identify and characterize the HS binding site(s) of CSP. We synthesized a series of peptides corresponding to five regions of Plasmodium falciparum CSP containing basic residues, a common requirement of HS binding sites, and screened them for heparin and HS binding activity. Only one of these peptides (Pf 2), which contains a motif we have named region I-plus, demonstrated both high affinity heparin/HS binding activity and the ability to block the binding of recombinant CSP to heparin-Sepharose 4B. Analysis by isothermal titration calorimetry revealed that region I-plus has a binding constant of K(d) = 5.0 microm and a stoichiometry of n = 7.8 binding sites/heparin chain. Heparin binding was dependent on the amino acid sequence of region I-plus, and the binding sites on heparin/HS are contained within a decasaccharide. Furthermore, HS oligosaccharides rich in sulfate and iduronic acid content (heparin-like) are required for efficient binding. Because liver HS is exceptionally high in both these components relative to the HS of other organs, the HS structural requirements for efficient region I-plus/HS binding are consistent with this peptide sequence functioning to target sporozoites to the liver for attachment to hepatocytes. Finally, the region I-plus heparin/HS binding site was also discovered for two other species that infect humans, Plasmodium malariae and Plasmodium vivax, further supporting the existence of a HS binding domain in the N-terminal portion of CSP.
Highlights
Four species of the protozoan genus Plasmodium (P. falciparum, P. vivax, P. ovale, and P. malariae) cause malaria, the most devastating parasitic disease in humans
A Heparin/heparan sulfate (HS) Binding Site Identified in the N terminus of the Circumsporozoite protein (CSP) for P. falciparum—Malarial parasite CSPs, including that of P. falciparum, are approximately 400 amino acids in length organized into three domains (Fig. 1A): the N-terminal domain, which contains a conserved pentapeptide region I; a highly repetitive central domain; and a C-terminal domain containing region II, a sequence similar to the conserved adhesion sequence, TSR
To get more detailed sequence and binding kinetics information regarding the HS binding site(s), we set out to investigate the heparin binding activity of region II-plus and a series of peptides corresponding to different regions of CSP containing basic residues, a known requirement of heparin binding sites [9, 10, 13]
Summary
The initial rapid and specific invasion of hepatocytes is mediated by the interaction between the circumsporozoite protein (CSP), which coats the surface of the sporozoite [4], and the glycosaminoglycan (GAG), heparan sulfate (HS), found on the surface of the hepatocytes [5,6,7] and in the extracellular matrix in the space of Disse [8] Numerous proteins such as growth factors, serine protease inhibitors, extracellular matrix proteins, lipoproteins, and lipolytic enzymes are known to associate with HS as part of their normal function [9, 10], and many pathogenic organisms including Plasmodium have evolved proteins to target HS for attachment and invasion of specific host cells [11]. HS is generally less extensively modified with Ͻ50% IdoA and containing a more varied sulfation pattern with regions of high sulfation associated with high IdoA content and regions of little or no sulfation associated with high GlcA content
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