Structural and Functional Characterization of Oversulfated Chondroitin Sulfate/Dermatan Sulfate Hybrid Chains from the Notochord of Hagfish
2004; Elsevier BV; Volume: 279; Issue: 49 Linguagem: Inglês
10.1074/jbc.m404746200
ISSN1083-351X
AutoresC. Nandini, Tadahisa Mikami, Mitsuhiro Ohta, Nobuyuki Itoh, Fumiko Akiyama-Nambu, Kazuyuki Sugahara,
Tópico(s)Dendrimers and Hyperbranched Polymers
ResumoOversulfated chondroitin sulfate (CS)/dermatan sulfate (DS) hybrid chains were purified from the notochord of hagfish. The chains (previously named CS-H for hagfish) have an average molecular mass of 18 kDa. Composition analysis using various chondroitinases demonstrated a variety of d-glucuronic acid (GlcUA)- and l-iduronic acid (IdoUA)-containing disaccharides variably sulfated with a higher proportion of GlcUA/IdoUA-GalNAc 4,6-O-disulfate, revealing complex CS/DS hybrid features. The hybrid chains showed neurite outgrowth-promoting activity of an axonic nature, which resembled the activity of squid cartilage CS-E and which was abolished fully by chondroitinase ABC digestion and partially by chondroitinase AC-I or B digestion, suggesting the involvement of both GlcUA and IdoUA in neuritogenic activity. Purified CS-H exhibited interactions in a BIAcore system with various heparin-binding proteins and neurotrophic factors (viz. fibroblast growth factor-2, -10, -16, and -18; midkine; pleiotrophin; heparin-binding epidermal growth factor-like growth factor; vascular endothelial growth factor; brain-derived neurotrophic factor; and glial cell line-derived neurotrophic factor), most of which are expressed in the brain, although fibroblast growth factor-1 and ciliary neurotrophic factor showed no binding. Kinetic analysis revealed high affinity binding of these growth factors and, for the first time, of the neurotrophic factors. Competitive inhibition revealed the involvement of both IdoUA and GlcUA in the binding of these growth factors, suggesting the importance of the hybrid nature of CS-H for the efficient binding of these growth factors. These findings, together with those from the recent analysis of brain CS/DS chains from neonatal mouse and embryonic pig (Bao, X., Nishimura, S., Mikami, T., Yamada, S., Itoh, N., and Sugahara, K. (2004) J. Biol. Chem. 279, 9765–9776), suggest physiological roles of the hybrid chains in the development of the brain. Oversulfated chondroitin sulfate (CS)/dermatan sulfate (DS) hybrid chains were purified from the notochord of hagfish. The chains (previously named CS-H for hagfish) have an average molecular mass of 18 kDa. Composition analysis using various chondroitinases demonstrated a variety of d-glucuronic acid (GlcUA)- and l-iduronic acid (IdoUA)-containing disaccharides variably sulfated with a higher proportion of GlcUA/IdoUA-GalNAc 4,6-O-disulfate, revealing complex CS/DS hybrid features. The hybrid chains showed neurite outgrowth-promoting activity of an axonic nature, which resembled the activity of squid cartilage CS-E and which was abolished fully by chondroitinase ABC digestion and partially by chondroitinase AC-I or B digestion, suggesting the involvement of both GlcUA and IdoUA in neuritogenic activity. Purified CS-H exhibited interactions in a BIAcore system with various heparin-binding proteins and neurotrophic factors (viz. fibroblast growth factor-2, -10, -16, and -18; midkine; pleiotrophin; heparin-binding epidermal growth factor-like growth factor; vascular endothelial growth factor; brain-derived neurotrophic factor; and glial cell line-derived neurotrophic factor), most of which are expressed in the brain, although fibroblast growth factor-1 and ciliary neurotrophic factor showed no binding. Kinetic analysis revealed high affinity binding of these growth factors and, for the first time, of the neurotrophic factors. Competitive inhibition revealed the involvement of both IdoUA and GlcUA in the binding of these growth factors, suggesting the importance of the hybrid nature of CS-H for the efficient binding of these growth factors. These findings, together with those from the recent analysis of brain CS/DS chains from neonatal mouse and embryonic pig (Bao, X., Nishimura, S., Mikami, T., Yamada, S., Itoh, N., and Sugahara, K. (2004) J. Biol. Chem. 279, 9765–9776), suggest physiological roles of the hybrid chains in the development of the brain. Pivotal functions of glycosaminoglycan (GAG) 1The abbreviations used are: GAG, glycosaminoglycan; PG, proteoglycan; HS, heparan sulfate; CS, chondroitin sulfate; DS, dermatan sulfate; GlcUA, d-glucuronic acid; IdoUA, l-iduronic acid; 2-AB, 2-aminobenzamide; DMMB, 1,9-dimethylmethylene blue; MK, midkine; FGF, fibroblast growth factor; PTN, pleiotrophin; VEGF, vascular endothelial growth factor; HB-EGF, heparin-binding epidermal growth factor-like growth factor; BDNF, brain-derived neurotrophic factor; GDNF, glial cell line-derived neurotrophic factor; CNTF, ciliary neurotrophic factor; MES, 2-(N-morpholino)ethanesulfonic acid; HPLC, high performance liquid chromatography; ΔHexUA, 4-deoxy-α-l-threo-hex-4-enepyranosyluronic acid; ΔDi-diSE, ΔHexUAα1–3GalNAc(4S,6S); ΔDi-4S, ΔHexUAα1–3GalNAc(4S); ΔDi-6S, ΔHexUAα1–3GalNAc(6S); ΔDi-0S, ΔHexUAα1–3GalNAc; ΔDi-triS, ΔHexUA(2S)α1–3GalNAc(4S,6S). side chains of proteoglycans (PGs), especially heparan sulfate (HS), have been implicated in biological processes such as cell adhesion, proliferation, and differentiation and tissue morphogenesis (1Perrimon N. Bernfield M. Nature. 2000; 404: 725-728Crossref PubMed Scopus (661) Google Scholar). Chondroitin sulfate (CS) and dermatan sulfate (DS) are also found ubiquitously in the extracellular matrices and at cell surfaces, being main components of cartilage and skin, respectively. CS consists of repeating disaccharide units of -4GlcUAβ1–3GalNAcβ1-, whereas DS is an isomeric form of CS and is formed from precursor CS through the action of glucuronyl C5 epimerase, thus consisting of disaccharide units of -4GlcUAβ1–3GalNAcβ1- and -4IdoUAα1–3GalNAcβ1- in varying proportions (2Silbert J.E. Sugumaran G. IUBMB Life. 2002; 54: 177-180Crossref PubMed Scopus (257) Google Scholar). These disaccharide units are modified during chain elongation by specific sulfotransferases at C-2 of GlcUA/IdoUA and/or C-4 and/or C-6 of GalNAc in various combinations, producing characteristic sulfation patterns critical for binding to various functional proteins displaying enormous structural diversity, comparable with that of HS, by embedding multiple overlapping functional sequences (3Sugahara K. Yamada S. Trends Glycosci. Glycotechnol. 2000; 12: 321-349Crossref Scopus (96) Google Scholar). Sulfation profiles of GAGs change during development (4Mark M.P. Baker J.R. Kimata K. Ruch J.V. Int. J. Dev. Biol. 1990; 34: 191-204PubMed Google Scholar, 5Kitagawa H. Tsutsumi K. Tone Y. Sugahara K. J. Biol. Chem. 1997; 272: 31377-31381Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar). Growing evidence indicates the involvement of CS and DS in the signaling of various heparin-binding growth factors and cytokines (6Trowbridge J.M. Gallo R.L. Glycobiology. 2002; 12: 117R-125RCrossref PubMed Google Scholar, 7Sugahara K. Mikami T. Uyama T. Mizuguchi S. Nomura K. Kitagawa H. Curr. Opin. Struct. Biol. 2003; 13: 612-620Crossref PubMed Scopus (595) Google Scholar, 8Bechard D. Gentina T. Delehedde M. Scherpereel A. Lyon M. Aumercier M. Vazeux R. Richet C. Degand P. Jude B. Janin A. Fernig D.G. Tonnel A.B. Lassalle P. J. Biol. Chem. 2001; 276: 48341-48349Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar). We and others have shown the importance of this class of molecule, from simple chondroitin involved in cell division of a nematode (9Mizuguchi S. Uyama T. Kitagawa H. Nomura K.H. Dejima K. Gengyo-Ando K. Mitani S. Sugahara K. Nomura K. Nature. 2003; 423: 443-448Crossref PubMed Scopus (219) Google Scholar) to differentially oversulfated CS-D and CS-E involved in neuroregulatory functions (10Clement A.M. Nadanaka S. Masayama K. Mandl C. Sugahara K. Faissner A. J. Biol. Chem. 1998; 273: 28444-28453Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar, 11Clement A.M. Sugahara K. Faissner A. Neurosci. Lett. 1999; 269: 125-128Crossref PubMed Scopus (119) Google Scholar, 12Ueoka C. Kaneda N. Okazaki I. Nadanaka S. Muramatsu T. Sugahara K. J. Biol. Chem. 2000; 275: 37407-37413Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar) and the binding of growth factors in mammalian systems (13Deepa S.S. Umehara Y. Higashiyama S. Itoh N. Sugahara K. J. Biol. Chem. 2002; 277: 43707-43716Abstract Full Text Full Text PDF PubMed Scopus (294) Google Scholar). While pursuing the critical structural elements in the CS variants, we became aware of vital contributions of IdoUA to biological activities. Neuritogenic activities were shown for various IdoUA-rich oversulfated DS chains from marine animals (14Hikino M. Mikami T. Faissner A. Vilela-Silva A.C. Pavão M.S. Sugahara K. J. Biol. Chem. 2003; 278: 43744-43754Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar), and not only neuritogenic but also growth factor binding properties were revealed for the CS/DS hybrid chains attached to phosphacan/DSD-1-PG from neonatal mouse brains (14Hikino M. Mikami T. Faissner A. Vilela-Silva A.C. Pavão M.S. Sugahara K. J. Biol. Chem. 2003; 278: 43744-43754Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, 15Maeda N. He J. Yajima Y. Mikami T. Sugahara K. Yabe T. J. Biol. Chem. 2003; 278: 35805-35811Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar) and for the free hybrid chains isolated from embryonic pig brains (16Bao X. Nishimura S. Mikami T. Yamada S. Itoh N. Sugahara K. J. Biol. Chem. 2004; 279: 9765-9776Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar), both of which contain an appreciable proportion of functional IdoUA. The neurite outgrowth-promoting activities of the CS/DS hybrid chains and oversulfated CS/DS chains are in contrast to the conventional concept that CS chains are intrinsic inhibitory components for axonal growth and path finding of various neurons (17Morgenstern D.A. Asher R.A. Fawcett J.W. Prog. Brain Res. 2002; 137: 313-332Crossref PubMed Scopus (383) Google Scholar, 18Ichijo H. Connect. Tissue. 2003; 35: 11-17Google Scholar). The seemingly discrepant observations are most likely attributable to the structural changes during development as demonstrated for CS/DS hybrid chains of embryonic and adult pig brains (16Bao X. Nishimura S. Mikami T. Yamada S. Itoh N. Sugahara K. J. Biol. Chem. 2004; 279: 9765-9776Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). Enzymatic removal of CS chains permits axonal regeneration after nigrostriatal tract axotomy and spinal cord injury (19Moon L.D. Asher R.A. Rhodes K.E. Fawcett J.W. Nat. Neurosci. 2001; 4: 465-466Crossref PubMed Scopus (498) Google Scholar, 20Bradbury E.J. Moon L.D. Popat R.J. King V.R. Bennett G.S. Patel P.N. Fawcett J.W. McMahon S.B. Nature. 2002; 416: 636-640Crossref PubMed Scopus (1907) Google Scholar) and reactivation of ocular dominance plasticity in the adult visual cortex (21Pizzorusso T. Medini P. Berardi N. Chierzi S. Fawcett J.W. Maffei L. Science. 2002; 298: 1248-1251Crossref PubMed Scopus (1257) Google Scholar) and is therefore a promising clinical strategy. We are searching for CS, DS, or hybrid chains with neuritogenic activities that have potential for therapeutic applications to neuronal diseases and injuries. Here, we characterized GAGs from hagfish notochord, originally termed CS-H for hagfish (22Anno K. Seno N. Mathews M.B. Yamagata T. Suzuki S. Biochim. Biophys. Acta. 1971; 237: 173-177Crossref PubMed Scopus (60) Google Scholar). Notochord is a connective tissue that supports the neural tube and that induces the formation of the central nervous system during the development of vertebrates. CS-H contains the characteristic oversulfated units IdoUAα1–3GalNAc(4S,6S) and IdoUA(2S)α1–3GalNAc(4S,6S), where 2S, 4S, and 6S represent sulfate at C-2, C-4, and C-6, respectively (22Anno K. Seno N. Mathews M.B. Yamagata T. Suzuki S. Biochim. Biophys. Acta. 1971; 237: 173-177Crossref PubMed Scopus (60) Google Scholar, 23Ueoka C. Nadanaka S. Seno N. Khoo K.-H. Sugahara K. Glycoconj. J. 1999; 16: 291-305Crossref PubMed Scopus (39) Google Scholar), hence regarded as DS, and exhibits axonic and dendritic neuritogenic activities for mouse hippocampal neurons (14Hikino M. Mikami T. Faissner A. Vilela-Silva A.C. Pavão M.S. Sugahara K. J. Biol. Chem. 2003; 278: 43744-43754Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). In view of the importance of the hybrid nature of the functional mammalian CS/DS chains in mouse (14Hikino M. Mikami T. Faissner A. Vilela-Silva A.C. Pavão M.S. Sugahara K. J. Biol. Chem. 2003; 278: 43744-43754Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, 15Maeda N. He J. Yajima Y. Mikami T. Sugahara K. Yabe T. J. Biol. Chem. 2003; 278: 35805-35811Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar) and pig (16Bao X. Nishimura S. Mikami T. Yamada S. Itoh N. Sugahara K. J. Biol. Chem. 2004; 279: 9765-9776Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar) brains, we searched for CS/DS hybrid chains in CS-H and demonstrated axonic neuritogenic and binding activities for a number of growth factors and a few major neurotrophic factors expressed in the brain. Materials—GAG lyases and unsaturated disaccharides derived from CS were purchased from Seikagaku Corp. (Tokyo, Japan). HS from bovine intestinal mucosa was purchased from Sigma. Actinase E was from Kaken Pharmaceutical Co. (Tokyo). 2-Aminobenzamide (2-AB) was purchased from Nacalai Tesque (Kyoto, Japan). Sodium cyanoborohydride (NaBH3CN) and 1,9-dimethylmethylene blue (DMMB) were from Aldrich. Prepacked disposable PD-10 columns containing Sephadex G-25 (medium) were obtained from Amersham Biosciences (Tokyo). Sep-Pak Accell™ Plus QMA anion-exchange cartridges were from Waters Corp. (Milford, MA). EZ-Link™ biotin-LC-hydrazide was obtained from Pierce. Recombinant human midkine (MK) expressed in Escherichia coli and recombinant human fibroblast growth factor (FGF)-1 (or acidic FGF) expressed in E. coli were from PeproTech EC Ltd. (London, United Kingdom). Recombinant human FGF-2 (basic FGF) expressed in E. coli was from Genzyme TECHNE (Minneapolis, MN). Recombinant human pleiotrophin (PTN) expressed in E. coli and recombinant human vascular endothelial growth factor-165 (VEGF165) expressed in insect cells were from RELIA Tech GmbH (Braunschweig, Germany). Recombinant human heparin-binding epidermal growth factor-like growth factor (HB-EGF) expressed in Sf21 insect cells, recombinant human brain-derived neurotrophic factor (BDNF), and recombinant human glial cell line-derived neurotrophic factor (GDNF) were obtained from R&D Systems. Recombinant human FGF-10 expressed in E. coli was provided by Takashi Katsumata (Sumitomo Pharmaceutical Research Center, Osaka, Japan). Recombinant rat FGF-16 and recombinant mouse FGF-18 (24Danilenko D.M. Montestruque S. Philo J.S. Li T. Hill D. Speakman J. Bahru M. Zhang M. Konishi M. Itoh N. Chirica M. Delaney J. Hernday N. Martin F. Hara S. Talvenheimo J. Narhi L.O. Arakawa T. Arch. Biochem. Biophys. 1999; 361: 34-46Crossref PubMed Scopus (16) Google Scholar) and recombinant rat ciliary neurotrophic factor (CNTF) (25Ohta K. Hara H. Hayashi K. Itoh N. Ohi T. Ohta M. Biochem. Mol. Biol. Int. 1995; 35: 283-290PubMed Google Scholar) were prepared as described previously. Extraction and Purification of CS-H—CS-H was isolated from the notochord of hagfish (Eptatretus burgeri) as detailed previously (22Anno K. Seno N. Mathews M.B. Yamagata T. Suzuki S. Biochim. Biophys. Acta. 1971; 237: 173-177Crossref PubMed Scopus (60) Google Scholar). Briefly, acetone-dried notochord was subjected to Pronase digestion. The proteins were removed by precipitation with 20% trichloroacetic acid, and GAGs were precipitated with 2 volumes of ethanol containing 2.5% calcium acetate and 0.25 m acetic acid. The GAG mixture was fractionated on a Dowex 1-Cl– column and eluted stepwise by increasing the NaCl concentration of the eluents. The fraction obtained by elution with 2.0 m NaCl was used for further characterization and subjected to treatment with freshly prepared nitrous acid (pH 1.5) according to the procedure of Shively and Conrad (26Shively J.E. Conrad H.E. Biochemistry. 1976; 15: 3932-3942Crossref PubMed Scopus (666) Google Scholar) to remove HS. After the treatment, nitrous acid was neutralized by addition of 0.5 m Na2CO3, and the resultant HS fragments were separated from CS-H by passing the treated sample through a Sephadex G-50 column (56 × 1 cm) with 50 mm pyridine acetate buffer (pH 5.0) as eluent at a flow rate of 0.6 ml/min. Finally, the CS-H preparation was freed of hydrophobic peptides by passing it through a C18 cartridge. Molecular Mass Determination—Molecular mass was determined using a Superdex 200 column (10 × 300 mm) calibrated with known molecular mass markers, including dextran preparations (average molecular masses of 65.5, 37.5, and 18.1 kDa), HS from bovine intestinal mucosa (average molecular mass of 7.5 kDa), and heparin from porcine intestinal mucosa (average molecular mass of 6 kDa) (27Yamada S. Okada Y. Ueno M. Iwata S. Deepa S.S. Nishimura S. Fujita M. Van Die I. Hirayabashi Y. Sugahara K. J. Biol. Chem. 2002; 277: 31877-31886Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). V0 and Vt were determined using dextran (molecular mass of 170–200 kDa) and NaCl, respectively. Dextrans were monitored by the orcinol method for neutral sugars (28Bruckner J. Biochem. J. 1955; 60: 200-205Crossref PubMed Scopus (108) Google Scholar). The purified CS-H preparation (13 μg as uronic acid) was loaded onto the column and eluted with 0.2 m ammonium acetate at a flow rate of 0.3 ml/min; the fractions were collected at 3-min intervals, evaporated to dryness, and reconstituted in 100 μl of water. An aliquot was taken for estimating GAG using DMMB according to the procedure of Chandrasekhar et al. (29Chandrasekhar S. Esterman M.A. Hoffman H.A. Anal. Biochem. 1987; 161: 103-108Crossref PubMed Scopus (251) Google Scholar), except that the absorbance was measured at 525 nm. Determination of the Digestibility of the Purified CS-H Preparation by Various Chondroitinases—The purified CS-H preparation (1.3 μg as uronic acid) was digested with 10 mIU of chondroitinase ABC (30Saito H. Yamagata T. Suzuki S. J. Biol. Chem. 1968; 243: 1536-1542Abstract Full Text PDF PubMed Google Scholar), 5 mIU of chondroitinase AC-I (31Yamada S. Sugahara K. Methods Mol. Biol. 2003; 213: 71-78PubMed Google Scholar), 5 mIU of chondroitinase AC-II, or 2 mIU of chondroitinase B (32Sugahara K. Ohkita Y. Shibata Y. Yoshida K. Ikegami A. J. Biol. Chem. 1995; 270: 7204-7212Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). After each enzymatic treatment, the digest was reconstituted in 20 or 50 μl of distilled water, and an aliquot was taken to estimate the resistant structure of CS-H by complexation with the metachromatic dye DMMB, which complexes with sulfated GAGs and long oligosaccharides, but not with short oligosaccharides. Briefly, 35 μl of 0.05 m acetate buffer (pH 6.8) and 200 μl of DMMB solution were added to a 5–10-μl aliquot of the above digest, and the absorbance was measured at 525 nm within 1 min (29Chandrasekhar S. Esterman M.A. Hoffman H.A. Anal. Biochem. 1987; 161: 103-108Crossref PubMed Scopus (251) Google Scholar). The loss of reactivity toward the dye was checked after each digestion, and the amount remaining was calculated based on the absorbance value using the calibration curve obtained with varying amounts of standard commercial DS (0.2–1.0 μg) derived from porcine skin. The amount of GAG before digestion was taken as 100%. Neurite Outgrowth Promotion Assay—The neurite outgrowth-promoting activity of the purified CS-H preparation was assayed as reported previously (14Hikino M. Mikami T. Faissner A. Vilela-Silva A.C. Pavão M.S. Sugahara K. J. Biol. Chem. 2003; 278: 43744-43754Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). Briefly, the CS-H preparation (0.7 μg as uronic acid) or an equivalent amount of its digest with chondroitinase ABC, B, or AC-I diluted with phosphate-buffered saline was coated on plastic coverslips, which had been precoated with poly-dl-ornithine at 37 °C overnight. Hippocampal neuronal cells established from embryonic day 18 rat brain were plated at a density of 10,000 cells/cm2 in Eagle's minimal essential medium containing N2 supplements (Invitrogen, Tokyo), 0.1 mm pyruvate, 0.1% (w/v) ovalbumin, 0.29% l-glutamine, 0.2% sodium hydrogen carbonate, and 5 mm HEPES. The cultures were maintained in a humidified atmosphere at 37 °C with 5% CO2 for 24 h, after which the cells were fixed using 4% (w/v) paraformaldehyde, and the neurites were visualized by immunochemical staining using antibodies directed against microtubule-associated protein-2 and neurofilament. The immunostained cells on each coverslip were scanned and digitalized with a ×20 objective lens on an Olympus BX 51 optical microscope equipped with an Olympus HC-300Z/OL digital camera. The photographs were analyzed using morphological analysis software (Mac SCOPE, Mitany Corp., Tokyo). The length of the longest neurite, with at least one process chosen at random being longer than the cell body, was determined for 100 cells. At least three independent experiments per parameter or condition were carried out. Biotinylation of the Purified CS-H Preparation and HS—Purified CS-H or HS was dissolved in 100 mm MES (pH 5.5) at a concentration of 2 mg/ml. To this solution were added 50 mm biotin-LC-hydrazide freshly dissolved in dimethylsulfoxide and 0.5 m 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (Pierce) dissolved in MES. The mixture was incubated overnight at room temperature with continuous shaking. Excess biotinylating reagents were removed by dialysis in Spectrapor molecular porous membrane tubing (molecular mass cutoff of 3.5 kDa; Spectrum Medical Industries, Inc., Laguna Hills, CA) against several changes of phosphate-buffered saline. Immobilization of Purified CS-H and HS on a Sensor Chip—A streptavidin-coated sensor chip (BIAcore AB) was conditioned using 1 m NaCl and 50 mm NaOH for 1 min three times according to the manufacturer's instructions. Biotinylated CS-H or HS (3.3 μg as uronic acid) in phosphate-buffered saline was perfused and allowed to interact with the sensor's surface for 3 min in phosphate-buffered saline. Immobilization was confirmed by the increase in response units. Interaction Analysis—An interaction analysis was carried out in the BIAcore J system using the CS-H-immobilized sensor chip. Growth factors (200 ng each) were checked individually for binding to immobilized CS-H in running buffer (10 mm HEPES-NaOH (pH 7.4), 0.15 m NaCl, 3 mm EDTA, and 0.005% Tween 20). The flow rate was kept at a medium pace of 30 μl/min according to the manufacturer's protocol. Each growth factor was allowed to interact with immobilized CS-H for 3 min, which constituted the association phase. Dissociation of the growth factor, constituting the dissociation phase, was allowed to go on for an additional 2 min, after which the sensor chip was regenerated by injecting 1 m NaCl for 2 min. Neurotrophic factors (100 ng each) were tested for binding to immobilized CS-H or HS at a high flow rate of 60 μl/min, and the time allowed for the association and dissociation phases was 3 and 2 min, respectively. To determine the kinetics of the binding of various growth factors to CS-H, each growth factor in varying concentrations was allowed to interact with immobilized CS-H. Kinetic parameters (ka, kd, and Kd) were determined by collectively fitting the overlaid sensorgrams locally using the 1:1 Languimuir binding model with mass transfer of the BIAevaluation 3.1 software. Interaction analyses of CS-H and HS with BDNF and GDNF for determination of kinetic parameters were carried out by global fitting of the sensorgrams obtained by employing high flow rates using the model mentioned above. High flow rates were used because of the high mass transfer effects observed at medium flow rates, due to which overlaid sensorgrams could not be fit with the 1:1 Languimuir binding model with mass transfer. Disaccharide Composition Analysis—A composition analysis of purified CS-H was carried out after digesting 0.5 μg (as uronic acid) with 10 mIU of chondroitinase ABC or 1.0 μg (as uronic acid) with 5 mIU of chondroitinase AC-I or 1 mIU of chondroitinase B for 1 h at 37 or 30°C (for chondroitinase B) as described above. The digested products were then derivatized with 2-AB (33Kinoshita A. Sugahara K. Anal. Biochem. 1999; 269: 367-378Crossref PubMed Scopus (190) Google Scholar). Excess 2-AB was removed by repeated extraction with a 1:1 (v/v) water/chloroform mixture, and the water phase was dried. The residue was then reconstituted in 400 μl of 16 mm NaH2PO4 solution, and a 200-μl aliquot was analyzed by anion-exchange HPLC on an amine-bound silica PA-03 column (33Kinoshita A. Sugahara K. Anal. Biochem. 1999; 269: 367-378Crossref PubMed Scopus (190) Google Scholar). Gel Filtration Analysis of the Chondroitinase Digests of the CS-H Preparation on a Superdex Peptide Column—The purified CS-H preparation (0.17 μg as uronic acid) was digested with chondroitinase ABC, AC-I, or B, and an equal amount was digested sequentially with chondroitinase B followed by chondroitinase AC-I as described above. The digests were individually labeled with the fluorophore 2-AB and processed as described above. Each digest was made up with 400 μl of 0.2 m ammonium bicarbonate containing 7% 1-propanol, and a 200-μl aliquot was analyzed by gel filtration on a Superdex peptide column using the above-mentioned solvent as eluent at a flow rate of 0.4 ml/min. Effects of Digestion of CS-H on Its Ability to Bind Growth Factors— The binding of various growth factors to immobilized CS-H was evaluated based on the ability of exogenously added CS-H or its digested products to competitively inhibit the binding. Purified CS-H (1.7 μg as uronic acid) was digested with chondroitinase ABC (10 mIU), AC-I (5.0 mIU), or B (2 mIU), and a 0.12-μg equivalent (as uronic acid) of each digest was dispensed into individual Eppendorf tubes and dried. Control tubes without CS-H were prepared in a similar way to normalize any nonspecific inhibition. Before injection, the tubes were reconstituted in running buffer and incubated with 50 ng of the growth factor to be tested for 10 min at room temperature. Injection was carried out for 3 min at a medium flow rate (30 μl/min), and the response during the association phase was compared with that exhibited by the growth factor only. Isolation and Purification of CS-H from Hagfish Notochord—In vertebrates, the notochordal primodium plays a central role in morphogenetic movement during gastrulation to induce the central nervous system. In cyclostomes, to which hagfish belong, the notochord is not atrophied during evolution and is available in a sufficient amount for preparing GAG chains, although PGs in notochord have not been well characterized in terms of the core proteins. CS-H from hagfish notochord was isolated as described (22Anno K. Seno N. Mathews M.B. Yamagata T. Suzuki S. Biochim. Biophys. Acta. 1971; 237: 173-177Crossref PubMed Scopus (60) Google Scholar). Briefly, it was obtained by Pronase digestion of the acetone-dried notochord, followed by trichloroacetic acid precipitation for removal of proteins and ethanol precipitation of GAGs. It was then fractionated on a Dowex 1-Cl– column by stepwise elution at varying concentrations of NaCl. A unique predominant disaccharide unit (IdoUAα1–3GalNAc(4S,6S)), representing 68% of all disaccharides, was previously revealed in the major fraction eluted at 3.0 m NaCl from a Dowex column, in addition to the minor units IdoUAα1–3GalNAc(6S) and IdoUAα1–3GalNAc(4S) (22Anno K. Seno N. Mathews M.B. Yamagata T. Suzuki S. Biochim. Biophys. Acta. 1971; 237: 173-177Crossref PubMed Scopus (60) Google Scholar), and was named the H or iE unit (where "i" stands for iduronic acid) (7Sugahara K. Mikami T. Uyama T. Mizuguchi S. Nomura K. Kitagawa H. Curr. Opin. Struct. Biol. 2003; 13: 612-620Crossref PubMed Scopus (595) Google Scholar). In this study, the 2 m NaCl-eluted fraction, representing ∼35% of total CS-H, was characterized as having less sulfated CS/DS hybrid chains. Because the preparation showed trace amounts of HS disaccharides upon HPLC analysis after heparitinase digestion (data not shown), it was purified further by subjecting it to nitrous acid treatment (26Shively J.E. Conrad H.E. Biochemistry. 1976; 15: 3932-3942Crossref PubMed Scopus (666) Google Scholar) to remove HS. Peptides were removed by passing through a Sep-Pak C18 cartridge with water as eluent. The purified final preparation gave a single band between pig skin CS-B and shark cartilage CS-C upon electrophoresis on a cellulose acetate membrane (data not shown), which disappeared completely after digestion with chondroitinase ABC, but only partially after digestion with chondroitinase AC-I or B, suggesting the presence of both CS and DS chains or CS/DS hybrid chains in the CS-H preparation. Determination of the Molecular Mass of CS-H—The molecular size of purified CS-H was determined by gel filtration HPLC using a calibrated Superdex 200 column (27Yamada S. Okada Y. Ueno M. Iwata S. Deepa S.S. Nishimura S. Fujita M. Van Die I. Hirayabashi Y. Sugahara K. J. Biol. Chem. 2002; 277: 31877-31886Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). The CS-H preparation eluted as a single fairly symmetrical peak when monitored using the dye DMMB (Fig. 1), giving an average molecular mass of ∼18 kDa. This is similar to the molecular masses of porcine skin DS (19 kDa) and porcine intestine DS (21 kDa), but slightly larger than the molecular mass of DS from eel skin (14 kDa) (34Sakai S. Kim W.S. Lee I.S. Kim Y.S. Nakamura A. Toida T. Imanari T. Carbohydr. Res. 2003; 338: 263-269Crossref PubMed Scopus (36) Google Scholar). The CS/DS Hybrid Natur
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