A Binding Site for Highly Sulfated Heparan Sulfate Is Identified in the N Terminus of the Circumsporozoite Protein

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 M 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.

Unexpected fold in the circumsporozoite protein target of malaria vaccines

“…2H). These results are consistent with mapping of HSPG binding to the conserved basic residues clustered around region I (16,17) (Fig. 1A).…”

“…The highly sulfated HSPGs of hepatocytes are proposed to mediate targeting of sporozoites to the liver and trigger sporozoite cell invasion (14,15). Heparin and HSPG binding sites have been identified in the CS ectodomain (11,16) and in conserved N-terminal region I and αTSR region II + peptides (12,13,17,18). The two basic residues in region II + , Arg-345, and Lys-347, are neither highly exposed nor present in a basic patch, and Arg-345 in the β2-strand interacts with Trp-331 in the π-cation bond described above and is also neutralized by a salt bridge to Glu-365 at the adjacent position in the β3-strand ( Fig.…”

Enhancing humoral responses to a malaria antigen with nanoparticle vaccines that expand T fh cells and promote germinal center induction

“…3B). ICMV vaccination also generated antibodies capable of recognizing the Region I domain, which may inhibit sporozoite invasion into hepatocytes by blocking receptor-ligand interactions during parasite entry (8). In contrast, the C-terminal fragment of VMP001 and the Region II domain, which share sequence homology to endogenous thrombospondin (20), were not recognized by antibodies elicited with either vaccine, suggesting lack of activation of self-reactive B cells.…”

“…We recently developed VMP001, a recombinant protein antigen composed of CSP core sequences derived from two widespread isolates of P. vivax (5,6). Although VMP001 mixed with either alum or Montanide elicits antigen-specific antibody responses (5,6), adjuvants capable of eliciting high-affinity antibodies against protective regions within CSP, which may lead to opsonization of sporozoites (7) or inhibition of their entry into hepatocytes (8), are of great interest.…”

Antigenic Diversity of the Plasmodium vivax Circumsporozoite Protein in Parasite Isolates of Western Colombia

“…The CR is flanked by conserved pre-repeat (5′NR) and post-repeat (3′NR) regions. 5 These flanking regions contain short, highly conserved sequences denoted as Region I (RI) and RII regions, 3 that are the binding domains for glycosaminoglycan heparin sulfate receptors, which are found on the surface of hepatocytes 6 and mosquito salivary glands. 7 The RI and RII domains appear to play important roles in parasite invasion to host cells, both in the mosquito and the vertebrate host.…”