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, anti-tumor, and insecticidal activities. Using a glycan array we have now shown that only sulfated carbohydrates are effectively bound by CrataBL. Since this protein was recently shown to delay clot formation by impairing the intrinsic pathway of 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 better understand 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 to 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 comparable CrataBL binding affinity to 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.

Crystal Structure of Crataeva tapia Bark Protein (CrataBL) and Its Effect in Human Prostate Cancer Cell Lines

“…CrataBL activates caspase-3 in DU145 and PC3 cells ( Figure 11 ), similarly to what Jow et al [63] found on leukemia cells treated with beauvericin, a cyclic hexadepsipeptide. Zhang et al [64] showed that CrataBL strongly binds a number of sulfated carbohydrates whereas binding of other carbohydrates is insignificant, concluding that charge-charge interactions are responsible for lectin properties of this protein. In Figure 12 we show the positively charged surface regions in CrataBL, confirming their basic character.…”

The 2.8 Å Electron Microscopy Structure of Adeno-Associated Virus-DJ Bound by a Heparinoid Pentasaccharide

“…However, the 2-N-sulfo group of the middle pyranose ring and the 6-O-sulfo group of the fifth ring are both in proximity to the arginines previously implicated in binding, Arg 587 and Arg 590 , respectively 4, 8, 10, 39. SPR using heparinoid libraries and glycan array binding studies have both indicated that sulfate groups at 2-N and 6-O are more critical than at 2-O 19, 44. The four modeled fondaparinux sulfates, close to these arginines, superimpose within 2–4 Å of sulfates that were resolved in the SOS complex 9 .…”

A review of bioactive plant polysaccharides: Biological activities, functionalization, and biomedical applications

“…Anticoagulant activity of the heparin highly relies on its specific structure sequence, within which the binding site of an important protein (antithrombin III) is crucial for heparin to prevent the generation of a fibrin clot which is formed by the action of thrombin (SchedinWeiss, Richard, Hjelm, & Olson, 2008). Understanding of this structure-activity relationship of heparin offers the opportunity to develop drugs with higher specificity and better regulation of coagulation and to explore other therapeutic applications such as infection, inflammation, cancer and wound-healing treatment (Linhardt, 2003;Rajangam et al, 2006;Sakiyama-Elbert, 2014;Zhang et al, 2013a;Zhang, Wardwell, & Bader, 2013b). CS/DS chains are composed variably sulfated N-acetylgalactosamine and glucuronic acid or its epimeride iduronic acid disaccharides repeating units (Silbert & Sugumaran, 2002;Takegawa et al, 2011).…”