Structural studies of the interaction of Crataeva tapia bark protein with heparin and other glycosaminoglycans

Abstract

CrataBL, a protein isolated from Crataeva tapia bark, which is both a serine protease inhibitor and a lectin, has been previously shown to exhibit a number of interesting biological properties, including anti-inflammatory, analgesic, antitumor, and insecticidal activities. Using a glycan array, we have now shown that only sulfated carbohydrates are effectively bound by CrataBL. Because this protein was recently shown to delay clot formation by impairing the intrinsic pathway of the coagulation cascade, we considered that its natural ligand might be heparin. Heparin is a glycosaminoglycan (GAG) that interacts with a number of proteins, including thrombin and antithrombin III, which have a critical, essential pharmacological role in regulating blood coagulation. We have thus employed surface plasmon resonance to improve our understanding of the binding interaction between the heparin polysaccharide and CrataBL. Kinetic analysis shows that CrataBL displays strong heparin binding affinity (KD = 49 nM). Competition studies using different size heparin-derived oligosaccharides showed that the binding of CrataBL to heparin is chain length-dependent. Full chain heparin with 40 saccharides or large oligosaccharides, having 16-18 saccharide residues, show strong binding affinity for CrataBL. Heparin-derived disaccharides through tetradecasaccharides show considerably lower binding affinity. Other highly sulfated GAGs, including chondroitin sulfate E and dermatan 4,6-disulfate, showed CrataBL binding affinity comparable to that of heparin. Less highly sulfated GAGs, heparan sulfate, chondroitin sulfate A and C, and dermatan sulfate displayed modest binding affinity as did chondroitin sulfate D. Studies using chemically modified heparin show that N-sulfo and 6-O-sulfo groups on heparin are essential for CrataBL-heparin interaction.

Figure 1

Structure of CrataBL. A. The amino acid sequence of CrataBL. Asterisks indicate glycosylation sites. B. A chain tracing (prepared with PyMol) showing the three-dimensional structure of a CrataBL monomer. Two glycosylated residues and the attached carbohydrates are shown in sticks.

Figure 2

Chemical structures of GAGs and heparin-derived oligosaccharides.

Figure 3

SPR sensorgrams of CrataBL-heparin interaction. Concentrations of CrataBL (from top to bottom): 250, 125, 63 and 32 nM, respectively. The black curves are the fitting curves using models from BIAevaluate 4.0.1.

Figure 4

Top: Sensorgrams of solution heparin oligosaccharides/surface heparin competition. CrataBL concentration was 125 nM, and concentrations of heparin oligosaccharides in solution were 1000 nM). Bottom: Bar graphs (based on triplicate experiments with standard deviation) of normalized CrataBL binding preference to surface heparin by competing with different size of heparin oligosaccharides in solution.

Figure 5

Top: Sensorgrams of solution GAGs/surface heparin competition. CrataBL concentration was 125 nM, and concentrations of GAGsin solution were 1000 nM). Bottom: Bar graphs (based on triplicate experiments with standard deviation) of CrataBL binding RU to surface heparin by competing with different GAGs.

Figure 6

Top: Sensorgrams of solution chemical modified heparin/surface heparin competition. CrataBL concentration was 125 nM, and concentrations of chemical modified heparin in solution were 1000nM). Bottom: Bar graphs (based on triplicate experiments with standard deviation) of CrataBL binding RU to surface heparin by competing with different chemical modified heparin in solution.

Figure 7

Proposed CrataBL binding GAG motif: eight repeating disaccharide units having two sulfo groups on a hexosamine residue linked to an uronic acid having a carboxyl group. Top: heparin dp 16 Middle: Dis-DS dp 16; Bottom: CS-E dp 16. Sugar symbols: Blue square, GlcNAc; Tan lower segment diamond, IdoA; Yellow square, GalNAc; Blue upper segment diamond, GlcA.