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• W2116715149 abstract "Heparan sulfate (HS) chains interact with various growth and differentiation factors and morphogens, and the most interactions occur on the specific regions of the chains with certain monosaccharide sequences and sulfation patterns. Here we generated a library of octasaccharides by semienzymatic methods by using recombinant HS 2-O-sulfotransferase and HS 6-O-sulfotransferase, and we have made a systematic investigation of the specific binding structures for various heparin-binding growth factors. An octasaccharide (Octa-I, ΔHexA-GlcNSO3-(HexA-GlcNSO3)3) was prepared by partial heparitinase digestion from completely desulfated N-resulfated heparin. 2-O- and 6-O-sulfated Octa-I were prepared by enzymatically transferring one to three 2-O-sulfate groups and one to three 6-O-sulfate groups per molecule, respectively, to Octa-I. Another octasaccharide containing 3 units of HexA(2SO4)-GlcNSO3(6SO4) was prepared also from heparin. This octasaccharide library was subjected to affinity chromatography for interactions with fibroblast growth factor (FGF)-2, -4, -7, -8, -10, and -18, hepatocyte growth factor, bone morphogenetic protein 6, and vascular endothelial growth factor, respectively. Based upon differences in the affinity to those octasaccharides, the growth factors could be classified roughly into five groups: group 1 needed 2-O-sulfate but not 6-O-sulfate (FGF-2); group 2 needed 6-O-sulfate but not 2-O-sulfate (FGF-10); group 3 had the affinity to both 2-O-sulfate and 6-O-sulfate but preferred 2-O-sulfate (FGF-18, hepatocyte growth factor); group 4 required both 2-O-sulfate and 6-O-sulfate (FGF-4, FGF-7); and group 5 hardly bound to any octasaccharides (FGF-8, bone morphogenetic protein 6, and vascular endothelial growth factor). The approach using the oligosaccharide library may be useful to define specific structures required for binding to various heparin-binding proteins. Octasaccharides with the high affinity to FGF-2 and FGF-10 had the activity to release them, respectively, from their complexes with HS. Thus, the library may provide new reagents to specifically regulate bindings of the growth factors to HS. Heparan sulfate (HS) chains interact with various growth and differentiation factors and morphogens, and the most interactions occur on the specific regions of the chains with certain monosaccharide sequences and sulfation patterns. Here we generated a library of octasaccharides by semienzymatic methods by using recombinant HS 2-O-sulfotransferase and HS 6-O-sulfotransferase, and we have made a systematic investigation of the specific binding structures for various heparin-binding growth factors. An octasaccharide (Octa-I, ΔHexA-GlcNSO3-(HexA-GlcNSO3)3) was prepared by partial heparitinase digestion from completely desulfated N-resulfated heparin. 2-O- and 6-O-sulfated Octa-I were prepared by enzymatically transferring one to three 2-O-sulfate groups and one to three 6-O-sulfate groups per molecule, respectively, to Octa-I. Another octasaccharide containing 3 units of HexA(2SO4)-GlcNSO3(6SO4) was prepared also from heparin. This octasaccharide library was subjected to affinity chromatography for interactions with fibroblast growth factor (FGF)-2, -4, -7, -8, -10, and -18, hepatocyte growth factor, bone morphogenetic protein 6, and vascular endothelial growth factor, respectively. Based upon differences in the affinity to those octasaccharides, the growth factors could be classified roughly into five groups: group 1 needed 2-O-sulfate but not 6-O-sulfate (FGF-2); group 2 needed 6-O-sulfate but not 2-O-sulfate (FGF-10); group 3 had the affinity to both 2-O-sulfate and 6-O-sulfate but preferred 2-O-sulfate (FGF-18, hepatocyte growth factor); group 4 required both 2-O-sulfate and 6-O-sulfate (FGF-4, FGF-7); and group 5 hardly bound to any octasaccharides (FGF-8, bone morphogenetic protein 6, and vascular endothelial growth factor). The approach using the oligosaccharide library may be useful to define specific structures required for binding to various heparin-binding proteins. Octasaccharides with the high affinity to FGF-2 and FGF-10 had the activity to release them, respectively, from their complexes with HS. Thus, the library may provide new reagents to specifically regulate bindings of the growth factors to HS. Heparan sulfate (HS) 1The abbreviations used are: HS, heparan sulfate; FGF, fibroblast growth factor; HGF, hepatocyte growth factor; VEGF, vascular endothelial growth factor; BMP, bone morphogenetic protein; GF, growth factor; HBGF, heparin-binding growth and differentiation factor; CD-SNS, completely desulfated, N-sulfated; 2ODS, 2-O-desulfated; 6ODS, 6-O-desulfated; GAG, glycosaminoglycan, IdoUA, l-iduronic acid; HexA, hexuronic acid; GlcNSO3, N-sulfoglucosamine; HS2ST, heparan sulfate 2-O-sulfotransferase; HS6ST, heparan sulfate 6-O-sulfotransferase; PBS, phosphate-buffered saline; BSA, bovine serum albumin; PAPS, adenosine 3′-phosphate,5′-phosphosulfate; ELISA, enzyme-linked immunosorbent assay; HPLC, high performance liquid chromatography; h, human; Octa, octasaccharide; SPR, surface plasmon resonance. exists ubiquitously as a component of proteoglycans on cell surfaces and in extracellular matrix and basement membranes and has divergent structures and functions (1Conrad H.E. Heparin-Binding Proteins. Academic Press, Inc., New York1998: 1-60Crossref Google Scholar, 2David G. FASEB J. 1993; 7: 1023-1030Crossref PubMed Scopus (374) Google Scholar, 3Yanagishita M. Hascall V.C. J. Biol. Chem. 1992; 267: 9451-9454Abstract Full Text PDF PubMed Google Scholar). HS chains are known to interact with a variety of proteins such as heparin-binding growth and differentiation factors (HBGFs), morphogens, extracellular matrix components, protease inhibitors, protease, lipoprotein lipase, and various pathogens (4Aviezer D. Hecht D. Safran M. Eisinger M. David G. Yayon A. Cell. 1994; 79: 1005-1013Abstract Full Text PDF PubMed Scopus (491) Google Scholar, 5Bernfield M. Gotte M. Park P.W. Reizes O. Fitzgerald M.L. Lincecum J. Zako M. Annu. Rev. Biochem. 1999; 68: 729-777Crossref PubMed Scopus (2323) Google Scholar, 6Folkman J. Klagsbrun M. Sasse J. Wadzinski M. Ingber D. Vlodavsky I. Am. J. Pathol. 1988; 130: 393-400PubMed Google Scholar, 7Kjellen L. Lindahl U. Annu. Rev. Cell Biol. 1991; 60: 443-475Google Scholar, 8Rapraeger A.C. Curr. Opin. Cell Biol. 1993; 5: 844-853Crossref PubMed Scopus (120) Google Scholar). These interactions have been shown to play a pivotal role in various patho-physiological phenomena as well as in tissue morphogenesis, as uncovered by recent genetic studies (9Nakato H. Kimata K. Biochim. Biophys. Acta. 2002; 1573: 312-318Crossref PubMed Scopus (131) Google Scholar, 10Grobe K. Ledin J. Ringvall M. Holmborn K. Forsberg E. Esko J.D. Kjellen L. Biochim. Biophys. Acta. 2002; 1573: 209-215Crossref PubMed Scopus (131) Google Scholar, 11HajMohammadi S. Enjyoji K. Princivalle M. Christi P. Lech M. Beeler D. Rayburn H. Schwartz J.J. Barzegar S. de Agostini A.I. Post M.J. Rosenberg R.D. Shworak N.W. J. Clin. Investig. 2003; 111: 989-999Crossref PubMed Scopus (140) Google Scholar, 12Bullock S.L. Fletcher J.M. Beddington R.S. Wilson V.A. Genes Dev. 1998; 12: 1894-1906Crossref PubMed Scopus (400) Google Scholar). In some of these phenomena, the interactions of HS with certain proteins have been shown in regions of the HS with specific monosaccharide sequences and sulfation patterns. Such functional domains are thought to be generated after the sequential modification steps during the biosynthesis of HS. In these modification steps, HS N-deacetylase/N-sulfotransferases (13Orellana A. Hirschberg C.B. Wei Z. Swiedler S.J. Ishihara M. J. Biol. Chem. 1994; 269: 2270-2276Abstract Full Text PDF PubMed Google Scholar, 14Eriksson I. Sandback D. Ek B. Lindahl U. Kjellen L. J. Biol. Chem. 1994; 269: 10438-10443Abstract Full Text PDF PubMed Google Scholar, 15Aikawa J. Esko J.D. J. Biol. Chem. 1999; 274: 2690-2695Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar, 16Aikawa J. Grobe K. Tsujimoto M. Esko J.D. J. Biol. Chem. 2001; 276: 5876-5882Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar), C5 epimerase (17Li J.-P. Hagner-McWhirter A. Kjellen L. Jaan Palgi M.J. Lindahl U. J. Biol. Chem. 1997; 272: 28158-28163Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar), HS 2-O-sulfotransferase (HS2ST) (18Kobayashi M. Habuchi H. Yoneda M. Habuchi O. Kimata K. J. Biol. Chem. 1997; 272: 13980-13985Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar), HS 6-O-sulfotransferases (HS6ST) (19Habuchi H. Kobayashi M. Kimata K. J. Biol. Chem. 1998; 273: 9208-9213Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 20Habuchi H. Tanaka M. Habuchi O. Yoshida K. Suzuki H. Ban K. Kimata K. J. Biol. Chem. 2000; 275: 2859-2868Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar), and HS 3-O-sulfotransferase (21Liu J. Shworak N.W. Sinay P. Schwartz J.J. Zhang L. Fritze L.M. Rosenberg R.D. J. Biol. Chem. 1999; 274: 5185-5192Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar, 22Shworak N.W. Liu J. Petros L.M. Zhang L. Kobayashi M. Copeland N.G. Jenkins N.A. Rosenberg R.D. Eriksson I. Sandback D. Ek B. Lindahl U. Kjellen L. J. Biol. Chem. 1999; 274: 5170-5184Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar) are involved. The expression patterns of the individual modification enzymes have been shown to differ from tissue to tissue, and as a result different functional structures of HS may be generated in the different tissues. Such structural diversity introduced to HS may result in the different response of each tissue to various heparin-binding proteins. So far, the sequences in HS that interact with FGF-1 or FGF-2 have been studied by biochemical and x-ray crystallographic analysis. It became apparent that the FGF-1-binding region was distinct from the minimal FGF-2-binding region (23Turnbull J.E. Fernig D.G. Ke Y. Wilkinson M.C. Gallagher J.T. J. Biol. Chem. 1992; 267: 10337-10341Abstract Full Text PDF PubMed Google Scholar, 24Habuchi H. Suzuki S. Saito T. Tamura T. Harada T. Yoshida K. Kimata K. Biochem. J. 1992; 285: 805-813Crossref PubMed Scopus (163) Google Scholar, 25Guimond S. Maccarana M. Olwin B.B. Lindahl U. Rapraeger A.C. J. Biol. Chem. 1993; 268: 23906-23914Abstract Full Text PDF PubMed Google Scholar, 26Ishihara M. Glycobiology. 1994; 4: 817-824Crossref PubMed Scopus (104) Google Scholar, 27Kreuger J. Salmivirta M. Sturiale L. Gimenez-Gallego G. Lindahl U. J. Biol. Chem. 2001; 276: 30744-30752Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar). In addition to the studies on FGF-1 and FGF-2, the HS sequences that mediate binding and/or activation of some HB-GFs have been reported in the systems including FGF-4 (23Turnbull J.E. Fernig D.G. Ke Y. Wilkinson M.C. Gallagher J.T. J. Biol. Chem. 1992; 267: 10337-10341Abstract Full Text PDF PubMed Google Scholar, 24Habuchi H. Suzuki S. Saito T. Tamura T. Harada T. Yoshida K. Kimata K. Biochem. J. 1992; 285: 805-813Crossref PubMed Scopus (163) Google Scholar), FGF-8b (28Loo B.M. Salmivirta M. J. Biol. Chem. 2002; 277: 32616-32623Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar), hepatocyte growth factor (HGF) (29Lyon M. Deakin J.A. Mizuno K. Nakamura T. Gallagher J.T. J. Biol. Chem. 1994; 269: 11216-11223Abstract Full Text PDF PubMed Google Scholar, 30Ashikari S. Habuchi H. Kimata K. J. Biol. Chem. 1995; 270: 29586-29593Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 31Deakin J.A. Lyon M. J. Cell Sci. 1999; 112: 1999-2009Crossref PubMed Google Scholar), and platelet-derived growth factor (32Feyzi E. Lustig F. Fager G. Spillmann D. Lindahl U. Salminirta M. J. Biol. Chem. 1997; 272: 5518-5524Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). These studies on the binding structures in HS appear to support the idea that each heparin-binding growth factor may recognize the respective unique structure. However, our knowledge about the heparin/HS structures involved in the interaction with a variety of HBGFs is still limited, and the greater part of the interactions between HS and the heparin-binding proteins remains to be studied. Furthermore, structural analyses of HS with binding activity for HBGFs often give different results when HS is isolated from different sources (29Lyon M. Deakin J.A. Mizuno K. Nakamura T. Gallagher J.T. J. Biol. Chem. 1994; 269: 11216-11223Abstract Full Text PDF PubMed Google Scholar, 30Ashikari S. Habuchi H. Kimata K. J. Biol. Chem. 1995; 270: 29586-29593Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). In the present study, we prepared an octasaccharide library consisting of well defined sulfated octasaccharides. The library comprised 2-O-sulfated or 6-O-sulfated octasaccharides generated from CDSNS-heparin-derived octasaccharide (Octa-I) by in vitro reactions with HS2ST or HS6ST. By using this library, we examined the structures that were specifically bound to the various HBGFs including FGF-2, FGF-4, FGF-7, FGF-8, FGF-10, FGF-18, HGF, BMP-6, and VEGF. Our results show that these HBGFs could be classified roughly into five groups on the basis of the difference in affinity with the oligosaccharides, and offer further evidence for specific interactions between heparin-binding growth factors and the corresponding domain structures in HS. Furthermore, for a physiological relevance of this study, we demonstrated specific release of FGF-10 and FGF-2 from HS by the addition of 6-O-sulfated Octa-I and 2-O-sulfated Octa-I, respectively. Materials—Completely desulfated, N-sulfated heparin (CDSNS-heparin), chondroitin 4-sulfate from whale cartilage, heparitinase I (Flavobacterium heparinum, EC 4.2.2.8), heparitinase II (F. heparinum, no number assigned), heparinase (F. heparinum, EC 4.2.2.7), 2-O-desulfated heparin (2ODS-heparin), 6-O-desulfated heparin (6ODS-heparin), and an unsaturated glycosaminoglycan disaccharide kit were obtained from Seikagaku Corp. (Tokyo, Japan). Heparin and unlabeled PAPS were purchased from Sigma. [35S]PAPS was purchased from PerkinElmer Life Sciences. [3H]NaBH4 (36 Ci/mmol) was purchased from Amersham Biosciences. Hiload Superdex 30 HR 16/60, fast desalting column HR 10/10, Mono Q HR 5/5 and PD-10 were from Amersham Biosciences. Senshu Pak Docosil was obtained from Senshu Scientific (Tokyo, Japan). Recombinant human FGF-7, FGF-10, and FGF-18 were provided by Amgen Inc. (Thousand Oaks, CA). Recombinant human FGF-2 was purchased from Progen Biotechnic GmbH (Heidelberg, Germany). Recombinant human FGF-8 was purchased from PeproTech (Rocky Hill, NJ). Recombinant human FGF-4 was purchased from R & D Systems (Minneapolis, MN). Recombinant human HGF was purchased from Genzyme-Techne (Minneapolis, MN). Recombinant bone morphogenetic protein 6 (BMP-6) was a gift from Creative Biomolecules Inc., Hopkinton, MA, courtesy of Dr. T. K. Sampath. Recombinant human VEGF165 was purchased from Diaclone Research (Cedex, France). Sensor chip SA was obtained from BIAcore AB (Uppsala, Sweden). Preparation of Octasaccharide Fractions from CDSNS-heparin and Heparin—One octasaccharide composed of HexA-GlcNSO3 (Octa-I) and another composed of HexA(2SO4)-GlcNSO3(6SO4) (Octa-II) were prepared from CDSNS-heparin and heparin, respectively, as follows. One hundred milligrams of CDSNS-heparin was digested with 0.1 unit of heparitinase I, which is known to preferentially cleave glucosaminidic linkages to nonsulfated HexA residues in heparin/HS, at 37 °C for 2 h. The unsaturated oligosaccharide products were separated by Superdex 30 chromatography based on size. Octasaccharide fractions were pooled and lyophilized. The lyophilized materials were applied to a Mono Q column. The Mono Q column was developed by using a linear gradient from 0.2 to 1.2 m NaCl in 50 mm glycine-HCl, pH 3.0. The fractions eluted around 0.34 to 0.38 m NaCl were pooled and desalted by PD-10 column chromatography. The purified octasaccharide thus obtained was designated Octa-I. About 250 nmol (as HexA) of Octa-I was obtained. One hundred milligrams of heparin was digested with 0.1 unit of heparinase, which is known to cleave preferentially glucosaminidic linkages to 2-O-sulfated IdoUA in heparin/HS, at 37 °C for 2 h. Octa-II was purified from the heparin digests by the methods described above except that the fractions eluted over 1.0 m NaCl were pooled in the Mono Q chromatography. About 2.4 μmol (as HexA) of Octa-II was obtained. Aliquots of Octa-I and Octa-II were reduced with [3H]NaBH4 as described by Shively and Conrad (33Shively J.E. Conrad H.E. Biochemistry. 1976; 15: 3932-3942Crossref PubMed Scopus (666) Google Scholar). The specific activities of 3H-labeled Octa-I and Octa-II were 1.5 × 104 and 1.2 × 104 dpm/nmol, respectively. Preparation of O-Sulfated CDSNS-Heparin Octasaccharide with Recombinant HS2ST and HS6ST-1—The recombinant hHS2ST and hHS6ST-1 were prepared and purified as described previously (19Habuchi H. Kobayashi M. Kimata K. J. Biol. Chem. 1998; 273: 9208-9213Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). Briefly, FLAG-CMV2-hHS2ST or hHS6ST-1 was transfected into COS-7 cells. After 72 h, the recombinant fusion proteins were extracted from the cell layer with 10 mm Tris-HCl, pH 7.2, 0.5% (v/v) Triton X-100, 0.15 m NaCl, 20% glycerol, 10 mm MgCl2, and 2 mm CaCl2 and purified with an anti-FLAG M2 antibody-conjugated affinity column. 2-O-Sulfated Octa-I and 6-O-sulfated Octa-I were prepared as follows. For 2-O-sulfation of Octa-I, the reaction mixture contained, in a final volume of 50 μl, 1.0 μmol of acetate buffer, pH 5.5, 3.75 μg of protamine chloride, 0.5 nmol of Octa-I, 1 μCi/2 nmol of [35S]PAPS, and 2.7 units of hHS2ST. For 6-O-sulfation, the reaction mixture (50 μl) contained 2.5 μmol of imidazole-HCl, pH 6.8, 3.75 μg of protamine chloride, 0.5 nmol of Octa-I, 2 nmol of [35S]PAPS (1 μCi), and 5.5 units of hHS6ST-1. After incubation at 37 °C overnight, the reactions were stopped by heating at 100 °C for 1 min. 2-O-35S-Sulfated Octa-I and 6-O-35S-sulfated Octa-I were precipitated with 3 volumes of ethanol containing 1.3% potassium acetate and 0.5 mm EDTA in the presence of the carrier chondroitin 4-sulfate (0.1 μmol as glucuronic acid). The precipitates were dissolved in a small volume of distilled water and subjected to Mono Q chromatography. The Mono Q chromatography was developed with a linear gradient from 0.2 to 0.8 m in 50 mm glycine-HCl, pH 3.0. Preparation of Growth and Differentiation Factor (HBGF)-conjugated Sepharose Gel—The growth and differentiation factor-conjugated Sepharose gel was prepared as described previously (30Ashikari S. Habuchi H. Kimata K. J. Biol. Chem. 1995; 270: 29586-29593Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). Briefly, each of FGF-2 (50 μg), FGF-4 (50 μg), FGF-7 (100 μg), FGF-8 (50 μg), FGF-10 (100 μg), FGF-18 (100 μg), HGF (100 μg), BMP-6 (100 μg), and VEGF (50 μg) was coupled to 0.3 ml of CNBr-activated Sepharose 4B gel according to the method recommended by the manufacturer. Acetylated heparin (10 μg) was added to the coupling reaction mixture to protect the heparin/heparan sulfate-binding site of these growth factors. HBGF Affinity Chromatography of Various Octasaccharides—0.1 nmol of 35S-labeled octasaccharide was dissolved in 0.5 ml of PBST, 0.9 mm CaCl2, and 0.2 mg/ml chondroitin 4-sulfate (Binding buffer) and applied to a syringe column of GF-Sepharose (0.3 ml) equilibrated with Binding buffer at 4 °C. The column was shaken gently for 1 h and then washed with 2.5 ml of PBS containing 0.9 mm CaCl2 and 0.05% Tween 20 (PBST(+)). The column was eluted stepwise with 1 ml of 0.20, 0.25, 0.30, 0.35, 0.40, 0.45, and 0.50 m NaCl in PBST(+) and 2 ml of 2 m NaCl in PBS containing 0.05% Tween 20 (PBST) (Elution buffer). In some cases, the octasaccharides were eluted with 2 ml of Elution buffer after the wash with 2.5 ml of PBST(+). The elution profiles were monitored by measuring the radioactivity in a liquid scintillation counter (30Ashikari S. Habuchi H. Kimata K. J. Biol. Chem. 1995; 270: 29586-29593Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). Surface Plasmon Resonance Analysis—Real time analysis of the interaction of growth factors and heparin/modified heparin was performed with a BIAcore 2000 SPR biosensor. Streptavidin-conjugated Sensor Chip SA was used to immobilize various glycosaminoglycans. Glycosaminoglycans were biotinylated according to the method recommended by the manufacturer. Two hundred micrograms of heparin, 2ODS-heparin, 6ODS-heparin, or chondroitin 4-sulfate was incubated with 74 μg of NHS-LS-Biotin (Pierce) in 100 μl of 50 mm sodium bicarbonate buffer, pH 8.5, for 30 min at room temperature. Biotinylated glycosaminoglycans were precipitated with 2.5 volumes of 95% (v/v) ethanol containing 1.3% (w/v) potassium acetate, and the process was repeated three times. In order to immobilize GAGs on the sensor chip SA, 0.5–2 μg/ml biotinylated GAGs in PBST were injected at a flow rate of 5 μl/min. The injection of biotinylated GAGs produced 10–200 response units of immobilized GAG on the biosensor surface. The amount of bound material on the biosensor chips was measured in arbitrary response units. All measurements were carried out at room temperature, and refractive index errors due to bulk solvent effects were corrected by subtracting away responses on the non-coated sensor chip for GF concentrations used. Each GF stock solutions were diluted with PBST. Various concentrations of growth factors were injected across the GAG-coated surface at a flow rate of 5 μl/min. The steady state binding level was monitored for 300 s. Sensorgrams were evaluated using BIAevaluation software. Dissociation constants (KD) for binding could be extracted from the dependence of steady state binding levels on GF concentration. In a steady state, this model calculates KD from a plot of Req against C according to the equation, KD = Req(Rmax – Req)/C, where Req is the steady state response level for the growth factor; Rmax is the maximal capacity of the sensor chip to bind growth factors expressed in response units; and C is the molar concentration of growth factor. HBGF-releasing Activity of Octasaccharides from HS—The releasing activity was measured by ELISA as described previously with a minor modification (30Ashikari S. Habuchi H. Kimata K. J. Biol. Chem. 1995; 270: 29586-29593Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). A 96-well streptavidin-coated plate (Thermo Lab-systems, Finland) was coated with 0.1 nmol (as hexuronic acid) of biotinylated pig aorta HS for 1 h at room temperature. Wells were washed three times with 200 μl of PBS and then blocked with 200 μl of PBS containing 10 mg/ml BSA for 1 h with gentle shaking. Wells were washed three times with PBS. Then 100 μl of Binding buffer (see above) containing 40 ng/ml digoxigenin-conjugated HBGF and 10 mg/ml BSA was added into each well. After 1 h at room temperature, unbound digoxigenin-conjugated HBGF was removed by three washes with PBST(+). Then 100 μl of Binding buffer containing 10 mg/ml BSA and 1 pmol to 1 nmol of each octasaccharide were added into the wells. After 1 h at room temperature, wells were washed, and then alkaline phosphatase-conjugated Fab fragments of anti-digoxigenin antibody (1:1000 dilution) were added. After 1 h at room temperature, unbound Fab fragments were removed by three washes with PBST, and the alkaline phosphatase substrate (1 mg/ml p-nitrophenyl phosphate in 1 m diethanolamine, pH 9.8) was added into each well. The enzyme activity in each well was measured by using a microplate reader. The experiments were independently repeated three times, and statistical analyses were performed using Student's t test. The criterion of significance was shown by p values. Compositional Analysis of Octasaccharides—About 0.3 nmol of octasaccharides was digested with a mixture of 1 milliunit of heparitinase I, 0.1 milliunit of heparitinase II, and 1 milliunit of heparinase in 50 μl of 50 mm Tris-HCl, pH 7.2, 1 mm CaCl2, and 5 μg of BSA at 37 °C for 1 h. Unsaturated disaccharide products were analyzed by fluorometric postcolumn high performance liquid chromatography (HPLC) as reported previously (35Toyoda H. Kinoshita-Toyoda A. Selleck S.B. J. Biol. Chem. 2000; 275: 2269-2275Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar). Preparation of an Octasaccharide Library—Octa-I, an oligosaccharide composed of HexA-GlcNSO3, and Octa-II, an oligosaccharide composed of HexA(2SO4)-GlcNSO3(6SO4), were prepared from CDSNS-heparin and heparin, respectively, as described under “Experimental Procedures.” The structures of these oligosaccharides were confirmed by digestion with a mixture of heparitinase and heparinase followed by HPLC analysis as described under “Experimental Procedures.” As shown in Table I, ΔHexA-GlcNSO3 was exclusively obtained from Octa-I, indicating that the structure of Octa-I was ΔHexA-GlcNSO3-(HexA-GlcNSO3)3. On the other hand, 3 mol of HexA(2SO4)-GlcNSO3(6SO4), 0.5 mol of HexA-GlcNSO3(6SO4), and 0.5 mol of other disaccharide components were released from 1 mol of Octa-II. Octa-II was thus a mixture containing 3 units of HexA(2SO4)-GlcNSO3(6SO4) per molecule.Table IDisaccharide compositions of octasaccharidesOctasaccharide libraryOcta-IOcta-II2S-12S-22S-36S-16S-26S-3units/octasaccharideDisaccharide component HexA-GlcNAcND0.1NDNDNDNDNDND HexA-GlcNS4.00.12.91.91.12.91.91.2 HexA-GlcNAc(6S)ND0.1NDNDNDNDNDND HexA(2S)-GlcNSND0.31.12.12.9NDNDND HexA-GlcNS(6S)ND0.5NDNDND1.12.12.8 HexA(2S)-GlcNS(6S)ND2.9NDNDNDNDNDNDYield (%)4016638208 Open table in a new tab 2-O-Sulfated and 6-O-sulfated Octa-I were prepared by incubating Octa-I with the recombinant HS2ST and HS6ST-1, respectively, together with PAPS as described under “Experimental Procedures.” The in vitro sulfated products were separated with Mono Q chromatography (Fig. 1). Both 2-O-sulfated Octa-I and 6-O-sulfated Octa-I were separated into three peaks: 2S-1, 2S-2, and 2S-3 for 2-O-sulfated Octa-I; and 6S-1, 6S-2, and 6S-3 for 6-O-sulfated Octa-I. All of these peaks were eluted at higher NaCl concentration than Octa-I and at lower NaCl concentration than Octa-II. Each peak was pooled separately. To examine the structure of these products, aliquots of these sulfated octasaccharides were digested extensively with the mixture of heparitinase and heparinase and subjected to HPLC as described under “Experimental Procedures.” From the disaccharide compositions shown in Table I, it is evident that 2S-1, 2S-2, and 2S-3 contained 1, 2, and 3 units, respectively, of the HexA(2SO4)-GlcNSO3 component; and 6S-1, 6S-2, and 6S-3 have 1, 2, and 3 units, respectively, of HexA-GlcNSO3(6SO4) component. Under the maximum reaction conditions used, more than 60% of Octa-I was sulfated by each sulfotransferase. The yields of these sulfated Octa-I were decreased as the content of 2-O-sulfated or 6-O-sulfate units was increased (Table I). Binding Abilities of Octa-I and Octa-II to Various HBGFs— Before determining the binding activity of oligosaccharides, we examined the binding ability and capacity of the growth and differentiation factor-conjugated Sepharose 4B affinity columns using 3H-labeled heparin (2 nmol as HexA). Every affinity column conjugated with FGF-2, FGF-4, FGF-7, FGF-8, FGF-10, FGF-18, HGF, VEGF, or BMP-6 bound more than 80% of the applied 3H-labeled heparin (data not shown). To examine the binding activity of the oligosaccharide libraries synthesized enzymatically, we first applied [3H]NaBH4-reduced Octa-II (0.5 nmol as octasaccharide) to the growth factor-conjugated columns. The amounts of Octa-II bound to various growth factor-conjugated columns are shown in Fig. 2. FGF-2, FGF-4, FGF-18, and HGF bound Octa-II strongly. FGF-10 and FGF-7 also bound Octa-II slightly weaker than FGF-2. In contrast, FGF-8, BMP-6, and VEGF hardly bound Octa-II (less than 15% of the applied). These results indicate that FGF-2, FGF-4, FGF-7, FGF-10, FGF-18, and HGF have high affinity to Octa-II as observed for heparin, but FGF-8, BMP-6, and VEGF have very weak or no affinity to Octa-II, although these proteins showed high affinity to heparin. The results suggest that oligosaccharides longer than octasaccharides may be required for the binding of FGF-8, BMP-6, or VEGF. Octa-II contains both O-sulfate and N-sulfate. To address which sulfate groups are necessary for the binding to these growth factors, we examined the binding of Octa-I which is devoid of O-sulfate but has N-sulfate. As shown in Fig. 2, Octa-I hardly bound at all to the growth factor-conjugated affinity columns (open bars in Fig. 2). These results clearly indicate that the O-sulfate groups in Octa-II are essential for the binding to these growth factors. Affinity of the Octasaccharide Library to Various HBGFs— Octa-II is composed of 3 units of HexA(2SO4)-GlcNSO3(6SO4). To determine whether either or both of the 2-O-sulfate and 6-O-sulfate groups interact with the growth factors, we examined the binding of 2-O-sulfated or 6-O-sulfated Octa-I to the growth factor-conjugated columns that could retain Octa-II. In Fig. 3, the binding of 2S-3 and 6S-3 to FGF-2, FGF-4, FGF-7, FGF-10, FGF-18, or HGF-conjugated columns is shown. FGF-2 bound 2S-3 strongly but did not bind 6S-3. These results are consistent with studies showing that the presence of 1 unit of the HexA(2SO4)-GlcNSO3 component in oligosaccharides is sufficient for the binding to FGF-2 (26Ishihara M. Glycobiology. 1994; 4: 817-824Crossref PubMed Scopus (104) Google Scholar, 27Kreuger J. Salmivirta M. Sturiale L. Gimenez-Gallego G. Lindahl U. J. Biol. Chem. 2001; 276: 30744-30752Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar). In contr" @default.
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• W2116715149 title "Characterization of Growth Factor-binding Structures in Heparin/Heparan Sulfate Using an Octasaccharide Library" @default.
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• W2116715149 doi "https://doi.org/10.1074/jbc.m313523200" @default.
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