Structural Studies of the Interaction of Crataeva tapia Bark Protein with Heparin and Other Glycosaminoglycans

• Type of Material: Article
• Authors: Fuming Zhang, Rensselaer Polytechnic Institute; Benjamin Walcott, Rensselaer Polytechnic Institute; Dongwen Zhou, National Cancer Institute; Alla Gustchina, National Cancer Institute; Yi Lasanajak, Emory University; David Smith, Emory University; Rodrigo S. Ferreira, Universidade Federal de São Paulo; Maria Tereza S. Correia, Universidade Federal de Pernambuco; Patrícia M.G. Paiva, Universidade Federal de Pernambuco; Nicolai V. Bovin, Russian Academy of Sciences; Alexander Wlodawer, National Cancer Institute; Maria L.V. Oliva, Universidade Federal de São Paulo; Robert J. Linhardt, Rensselaer Polytechnic Institute
• Language: English
• Date: 2013-03-26
• Publisher: American Chemical Society
• Publication Version: Author Accepted Manuscript (After Peer Review)
• Copyright Statement: © 2013 American Chemical Society
• Final Published Version (URL): http://dx.doi.org/10.1021/bi400077b
• Title of Journal or Parent Work: Biochemistry
• ISSN: 0006-2960
• Volume: 52
• Issue: 12
• Start Page: 2148
• End Page: 2156
• Grant/Funding Information: This work was supported in part by grants from the National Institutes of Health: GM-38060 to R.J.L, in part by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research, and in part by grant Molecular and Cell Biology (Presidium RAS) to N.V.B.; The authors would like to acknowledge The Consortium for Functional Glycomics funded by the NIGMS GM62116 and GM98791 for services provided by the Glycan Array Synthesis Core (The Scripps Research Institute, LaJolla, CA) that produced the mammalian glycan microarray and the Protein-Glycan Interaction Core (Emory University School of Medicine, Atlanta, GA) that assisted with analysis of samples on the array.
• Keywords: BINDING LECTIN; HEPARIN/HEPARAN SULFATE; Biochemistry & Molecular Biology; Science & Technology; Life Sciences & Biomedicine
• Research Categories: Biology, Bioinformatics; Chemistry, Biochemistry

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 (K D = 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.