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Interactions Between Nattokinase and Heparin/Gags

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Interactions Between Nattokinase and Heparin/Gags Glycoconj J (2015) 32:695–702 DOI 10.1007/s10719-015-9620-8 ORIGINAL ARTICLE Interactions between nattokinase and heparin/GAGs 1 2,5 1,2,3,4 Fuming Zhang & Jianhua Zhang & Robert J. Linhardt Received: 9 July 2015 /Revised: 30 August 2015 /Accepted: 4 September 2015 /Published online: 28 September 2015 # Springer Science+Business Media New York 2015 Abstract Nattokinase (NK) is a serine protease extracted examine NK interacting with heparin as well as other from a traditional Japanese food called natto. Due to its strong glycosaminoglycans (GAGs). These studies showed that fibrinolytic and thrombolytic activity, NK is regarded as NK is a heparin binding protein with an affinity of avaluabledietarysupplementornutraceuticalforthe ~250 nM. Examination with differently sized heparin oral thrombolytic therapy. In addition, NK has been in- oligosaccharides indicated that the interaction between vestigated for some other medical applications including NK and heparin is chain-length dependent and the min- treatment of hypertension, Alzheimer’sdisease,and imum size for heparin binding is a hexasaccharide. vitreoretinal disorders. The most widely used clinical Studies using chemically modified heparin showed the anticoagulants are heparin and low molecular weight 6-O-sulfo as well as the N-sulfo groups but not the 2-O- heparins. The interactions between heparin and proteins sulfo groups within heparin, are essential for heparin’s modulate diverse patho-physiological processes and hep- interaction with NK. Other GAGs (including HS, DS, arin modifies the activity of serine proteases. Indeed, and CSE) displayed modest binding affinity to NK. heparin plays important roles in almost all of NK’spo- NK also interfered with other heparin-protein interac- tential therapeutically applications. The current report tions, including heparin’sinteractionwithantithrombin relies on surface plasmon resonance spectroscopy to and fibroblast growth factors. Keywords Heparin . Nattokinase . Binding . Surface * Jianhua Zhang plasmon resonance [email protected] * Robert J. Linhardt [email protected] Abbreviations 1 Department of Chemical and Biological Engineering, Center for GAG Glycosaminoglycan Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic NK Nattokinase SPR, surface plasmon resonance Institute, Troy, NY 12180, USA 2 HS Heparan sulfate Department of Chemistry and Chemical Biology, Rensselaer CSA Chondroitin sulfate A Polytechnic Institute, Troy, NY 12180, USA 3 DS Dermatan sulfate Department of Biomedical Engineering, Center for Biotechnology CSC Chondroitin sulfate C and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY 12180, USA CSD Chondroitin sulfate D CSE Chondroitin sulfate E 4 Department of Biology, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, SA Streptavidin Troy, NY 12180, USA FC Flow-cell 5 Department of Food Science and Technology, Shanghai Jiao Tong RU Resonance unit University, Shanghai 200240, People’sRepublicofChina dp Degree of polymerization. 696 Glycoconj J (2015) 32:695–702 Introduction proteins through various mechanisms, including localiza- tion of proteins in the ECM, regulation of enzymatic ac- Nattokinase (NK) is a protein that is extracted from a popular tivities, ligands binding to receptors, and protein protection Japanese food called natto, a vegetable cheese-like food, against proteolysis [13]. Heparin or HS proteoglycans are which is boiled soybeans fermented with Bacillus subtilis also essential cofactors for the interaction of fibroblast natto. Natto has been used as a folk remedy for diseases of growth factors (FGFs) with their receptors (FGFRs) [14, the heart and cardiovascular diseases for hundreds of years 15]. The goal of this study is to analyze molecular inter- [1]. Modern studies on NK demonstrated that it was not a actions of heparin/GAGs with NK. A BIAcore 3000, kinase, but rather a subtilisin-like serine protease, and that this employed for this study, relies on surface plasmon reso- activity was responsible for NK’sfibrinolyticeffects[2]. NK nance (SPR) for the direct quantitation, in real-time, of is regarded as a valuable dietary supplement and/or a nutra- molecular interactions. ceutical since it is sufficiently stable in the gastrointestinal tract to make this enzyme a potentially useful agent for the oral thrombolytic therapy [3]. In vitro and in vivo studies have Materials and methods demonstrated the potent pro-fibrinolytic effect of the enzyme [4–7]. An open-label, self-controlled clinical trial showed that Materials on oral administration NK could be considered a nutraceutical for the prevention of cardiovascular disease, decreasing plas- Nattokinase was purchased from Thermo Fisher ma levels of fibrinogen, factor VII, and factor VIII [7]. There Scientific Inc. (Waltham, MA). Porcine mucosal heparin are other potential applications for NK including treatment of (~16 kDa) as well as porcine mucosal HS were from hypertension, Alzheimer’s disease, and vitreoretinal disorders Celsus Laboratories, (Cincinnati, OH); porcine rib carti- [3, 8–11]. A randomized, double-blind, placebo-controlled lage chondroitin sulfate type A (~20 kDa), porcine mu- study, to examine how nattokinase supplementation impacted cosal chondroitin sulfate type B (also known as systolic and diastolic blood pressure in pre-hypertension sub- dermatan sulfate, ~30 kDa) and shark cartilage chon- jects, showed NK supplementation reduced both. After ab- droitin sulfate type C (~20 kDa) were from Sigma sorption through the intestine NK retained its protease activity Chemical (St. Louis, MO); whale cartilage chondroitin suggesting it could decrease blood pressure through cleavage sulfate type D (~20 kDa) and squid cartilage chondroi- of plasma fibrinogen. These NK-degradation products, pre- tin sulfate type E (20 kDa) were from Seikagaku vent the elevation of plasma angiotensin II level to suppress (Tokyo, Japan). Controlled partial heparin lyase I depo- hypertension [8]. It was reported that NK can assist toxic lymerization of beef lung heparin (Sigma) with subse- amyloid fibril catabolism, and that such fibrils can in- quent size-fractionation [17]wasusedtopreparehepa- clude insulin fibrils in diabetes, fibrils in Alzheimer’s rin tetrasaccharide (dp4), heparin hexasaccharide (dp6), Disease, and fibrils prion disease [10]. Based on the heparin octasaccharide (dp8), heparin decasaccharide (dp10), preliminary results of amyloid-degrading ability of NK, heparin dodecasaccharide (dp12), heparin tetradecasaccharide they suggested it might be useful in the treatment of (dp14), heparin hexadecasaccharide (dp16) and heparin amyloid-related diseases. However, it is not clear how octadecasaccharide (dp18). The 2-O-desulfonated IdoA hep- NK or NK fragments can enter the circulation when arin (~13 kDa) and the N-desulfonated heparin (~14 kDa) taken orally. Many of the basic biochemistry mechanisms were prepared as previously reported [16]. The 6-O- behind all the promising medical applications of NK are still desulfonated heparin (~13 kDa) [17]wasagiftfrom reminded to be elucidated. Professor L. Wang of the University of Georgia. Human anti- Heparan sulfate (HS) and heparin are members of the thrombin III (AT III) was from Haematologic Technologies glycosaminoglycan (GAG) family. These are anionic, poly- (Essex Junction, Vermont). Fibroblast growth factor-1 and -2 disperse, linear polysaccharides that are often highly sul- (FGF1 and FGF2) were generously provided by Amgen fated. HS, found on the external cell membrane and also (Thousand Oaks, CA). GE Healthcare Bio-Sciences AB in the extracellular matrix (ECM), can interact with many (Uppsala, Sweden) was the source of the SA sensor chips. A of protein ligands [12–14]. Many physiological and patho- BIAcore 3000, running under BIAcore 3000 control and physiological processes can be modulated through protein- Version 4.0.1 BIAevaluation software, was used for SPR heparin/HS interactions including: blood coagulation, cell measurements. growth and differentiation, host defense, viral infection, lipid transport and metabolism, cell-to-cell and cell-to- Heparin biochip preparation matrix signaling, inflammation, Alzheimer’s disease and cancer [12]. HS-protein interactions have been demonstrat- Biotin-heparin conjugate was synthesized through the reac- ed to critically modulate the biological activity of the tion of long-chain sulfo-N-hydroxysuccinimide biotin Glycoconj J (2015) 32:695–702 697 Fig. 1 Chemical structures of HOOC OSO - OH 3 heparin, heparin-derived O O OH O oligosaccharides and other GAGs O HOOC O HO O HO OH O HO - O3SHN CH3COHN O O O Heparan sulfate - O3SO Heparin (major sequence) (major sequence) - - OSO3 OSO3 - HOOC OH O OSO3 O O HO HO O O O HO -O SHN 3 OH - -O - O2C NHSO3 O3SO Heparin oligosaccharides n dp4, n = 1... dp18, n = 8 - O3SO OH R6 OR4 O O HOOC OOC OH O O O O O H O O O NHCOCH3 HO NHCOCH3 2 O OR HO Dermatan sulfate Chondroitin sulfates A,C,D,E 4 - 2,6 6 - 2,4 A, R =SO3 , R =H; C, R =SO3 ,R =H; 2,6 - 4 4,6 - 2 D, R =SO3 ; R =H; E, R =SO3 , R =H (Thermo Fisher, Rockford, IL) with the unsubstituted amino injected over heparin chip at 30 μL/min. The dissocia- groups of the polysaccharide chain’s glucosamine residues tion and the regeneration, as described above, were per- based on published methods [18–20]. The biotin-heparin con- formed after each run. A control experiment (protein jugate was then bound to a streptavidin chip. A 20-μl only with no heparin or heparin oligosaccharides) was solution
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