Direct Measurement of the Interactions of Glycosaminoglycans and a Heparin Decasaccharide with the Malaria Circumsporozoite Protein

Abstract

Circumsporozoite (CS) protein is a predominant surface antigen of malaria sporozoites, the infective form of the parasite, and has been used for making anti-malaria vaccines. For the first time we have examined the interaction of CS protein with various glycosaminoglycans in real time using surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC). Heparin was the best binder among the glycosaminoglycans tested and bound to CS protein with nanomolar affinity. Using purified and structurally defined small heparin oligosaccharides, we identified a decasaccharide to be the minimum sized CS protein-binding sequence. In an indirect competition assay, this decasaccharide blocked the CS protein interaction with HepG2 cells with an ID(50) of less than 60 nM. The decasaccharide has a structure commonly found in hepatic heparan sulfate, and the same sequence has recently been shown to bind specifically to apolipoprotein E. Examination of porcine liver heparan sulfate in this indirect competition assay showed that it and heparin were the only glycosaminoglycans that could effectively block CS protein interaction with HepG2 cells in culture. These data support the hypothesis that the invasion of liver cells by the parasite shares a common mechanism with the hepatic uptake of lipoprotein remnants from the blood.

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

“…Because low molecular weight heparin (M r ϭ 3000) was able to compete for region-I-plus/heparin binding, it appears that the minimum oligosaccharide required for binding is probably a decasaccharide or less. This again is in agreement with the observation that heparin decasaccharide but not tetrasaccharides could effectively inhibit recombinant CSP binding to HepG2 cells (34). The minimal binding sequence for fibroblast growth factor-2 and antithrombin is a pentasaccharide (10).…”

“…GAG binding activity of CSP is specific for heparin and highly sulfated HS species (34). Binding assays using isothermal titration calorimetry have demonstrated that heparin had the highest affinity for CSPs (K d ϭ 0.16 M and a stoichiometry of n ϭ 3.75/heparin chain) over HS, CS, and DS.…”

Isolation and characterization of heparan sulfate from various murine tissues

“…Our examination of porcine, bovine, and human liver HS [8,35] show that it is rich in highly sulfated sequences and that these are responsible for ApoE interaction [34]. Furthermore, these highly charged liver HS chains also appear to act as receptors for pathogens such as dengue virus [36] and malaria circumsporozoite [37]. It is interesting to note that mice, also sensitive to malaria, have a similar highly sulfated liver HS [38].…”

Dromedary glycosaminoglycans: Molecular characterization of camel lung and liver heparan sulfate

“…The disaccharide composition of camel liver HS, however, is strikingly different from that of other liver HS samples examined in our laboratory, with low levels of both unsulfated (ΔUA-GlcNAc) and trisulfated (ΔUA2S-GlcNS6S) disaccharides. Of particular note is the exceedingly low level of trisulfated disaccharide, since a octasaccharide sequence comprised of this repeating disaccharide corresponds to the liver receptor for apoE (Dong et al, 2001) as well as a receptor for the circumsporozoite form of the malaria parasite (Rathore et al, 2001). This peculiar structural feature of camel HS may play a role in maintaining the lower level of camel LDL compared to the LDL levels reported in other mammals (Nazifi et al, 2000).…”

“…The amount of perlecan is very low in normal liver, but increases dramatically in liver fibrosis (Kovalszky et al, 1988). Liver is also a target for a number of pathogens and liver HS has been demonstrated in several cases to play a pivotal role in infectivity (Chen et al, 1997;Rathore et al, 2001;Barth et al, 2003). Liver HSPGs at the endothelial level act as critical receptors for apoE and is involved in lipid metabolism (Bocksch et al, 2001;Dong et al, 2001;Bazin et al, 2002).…”