Chondroitin Sulfate/Dermatan Sulfate Hybrid Chains from Embryonic Pig Brain, Which Contain a Higher Proportion of L-Iduronic Acid than Those from Adult Pig Brain, Exhibit Neuritogenic and Growth Factor Binding Activitiesстатья из журнала
Аннотация: We have shown that over-sulfated chondroitin sulfate/dermatan sulfate (CS/DS) chains from various marine organisms exhibit growth factor binding activities and neurite outgrowth-promoting activities in embryonic mouse hippocampal neurons in vitro. In this study we demonstrated that CS/DS hybrid chains purified from embryonic pig brain displayed marked neuritogenic activity and growth factor binding activities toward fibroblast growth factor 2 (FGF2), FGF10, FGF18, pleiotrophin, and midkine, all of which exhibit neuroregulatory activities in the brain. In contrast, the CS/DS preparation from adult pig brain showed considerably less activity to bind these growth factors and no neuritogenic activity. Structural analysis indicated that the average size of the CS/DS chains was similar (40 kDa) between these two preparations, but the disaccharide compositions differed considerably, with a significant proportion of l-iduronic acid (IdoUA)-containing disaccharides (8∼9%) in the CS/DS chains from embryos but not in those from adults (<1%). Interestingly, both neurite outgrowth-promoting activity and growth factor binding activities of the CS/DS chains from embryos were abolished by digestion not only with chondroitinase ABC but also with chondroitinase B, suggesting that the IdoUA-containing motifs are essential for these activities. These findings imply that the temporal expression of CS/DS hybrid structures containing both GlcUA and IdoUA and binding activities toward various growth factors play important roles in neurogenesis in the early stages of the development of the brain. We have shown that over-sulfated chondroitin sulfate/dermatan sulfate (CS/DS) chains from various marine organisms exhibit growth factor binding activities and neurite outgrowth-promoting activities in embryonic mouse hippocampal neurons in vitro. In this study we demonstrated that CS/DS hybrid chains purified from embryonic pig brain displayed marked neuritogenic activity and growth factor binding activities toward fibroblast growth factor 2 (FGF2), FGF10, FGF18, pleiotrophin, and midkine, all of which exhibit neuroregulatory activities in the brain. In contrast, the CS/DS preparation from adult pig brain showed considerably less activity to bind these growth factors and no neuritogenic activity. Structural analysis indicated that the average size of the CS/DS chains was similar (40 kDa) between these two preparations, but the disaccharide compositions differed considerably, with a significant proportion of l-iduronic acid (IdoUA)-containing disaccharides (8∼9%) in the CS/DS chains from embryos but not in those from adults (<1%). Interestingly, both neurite outgrowth-promoting activity and growth factor binding activities of the CS/DS chains from embryos were abolished by digestion not only with chondroitinase ABC but also with chondroitinase B, suggesting that the IdoUA-containing motifs are essential for these activities. These findings imply that the temporal expression of CS/DS hybrid structures containing both GlcUA and IdoUA and binding activities toward various growth factors play important roles in neurogenesis in the early stages of the development of the brain. Chondroitin sulfate proteoglycans (CS-PGs) 1The abbreviations used are: CS, chondroitin sulfate; PG, proteoglycan; GAG, glycosaminoglycan; DS, dermatan sulfate; HA, hyaluronic acid; E-CS/DS, embryonic pig brain-derived CS/DS; A-CS/DS, adult pig brain-derived CS/DS; GlcUA, d-glucuronic acid; IdoUA, l-iduronic acid; HPLC, high performance liquid chromatography; 2AB, 2-aminobenzamide; MALDI-TOF MS, matrix-assisted laser desorption ionization time-of-flight mass spectrometry; FGF, fibroblast growth factor; MK, midkine; PTN, pleiotrophin; HB-EGF, heparin binding epidermal growth factor-like growth factor; PBS, phosphate-buffered saline; ΔHexUA, 4-deoxy-l-threo-hex-4-enepyranosyluronic acid; ΔDi-4S, ΔHexUAα1–3GalNAc(4-O-sulfate); rh, recombinant human; mIU, milli-international units; PTP, protein-tyrosine phosphatase; P-ORN, poly-dl-ornithine. are complex macromolecules consisting of a protein core and at least one covalently linked CS glycosaminoglycan (GAG) chain and are ubiquitous components in the extracellular matrices of connective tissues and at the surfaces of many cell types (for reviews, see Refs. 1Bandtlow C.E. Zimmermann D.R. Physiol. Rev. 2000; 80: 1267-1290Crossref PubMed Scopus (552) Google Scholar, 2Sugahara K. Kitagawa H. Curr. Opin. Struct. Biol. 2000; 10: 518-527Crossref PubMed Scopus (359) Google Scholar, 3Sugahara K. Yamada S. Trends Glycosci. Glycotechnol. 2000; 12: 321-349Crossref Scopus (96) Google Scholar). In the mammalian brain CS-PGs are common extracellular matrix components with a highly regulated spatiotemporal expression (4Margolis R.K. Margolis R.U. Experientia (Basel). 1993; 49: 429-446Crossref PubMed Scopus (243) Google Scholar, 5Herndon M. Lander A.D. Neuron. 1990; 4: 949-961Abstract Full Text PDF PubMed Scopus (231) Google Scholar, 6Fernaud-Espinosa I. Nieto-Sampedro M. Bovolenta P. J. Neurobiol. 1996; 30: 410-424Crossref PubMed Scopus (42) Google Scholar, 7Clement M.A. Nadanaka S. Masayama K. Mandl C. Sugahara K. Faissner A. J. Biol. Chem. 1998; 273: 28444-28453Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar, 8Maeda 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 (99) Google Scholar). Although the CS chains attached to CS-PGs have attracted little attention until recently compared with heparan sulfate (9Perrimon N. Bernfield M. Nature. 2000; 404: 725-728Crossref PubMed Scopus (667) Google Scholar), recent advances in the structural biology of CS chains have suggested important biological functions in the development of the brain (10Sugahara K. Mikami T. Uyama T. Mizuguchi S. Nomura K. Kitagawa H. Curr. Opin. Struct. Biol. 2003; 13: 612-620Crossref PubMed Scopus (607) Google Scholar). Several studies have demonstrated that the composition of CS chains changes with aging and normal brain maturation and that some CS epitopes are only found in specific sections of the avian and mammalian brain (7Clement M.A. Nadanaka S. Masayama K. Mandl C. Sugahara K. Faissner A. J. Biol. Chem. 1998; 273: 28444-28453Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar, 8Maeda 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 (99) Google Scholar, 11Kitagawa H. Tsutsumi K. Tone Y. Sugahara K. J. Biol. Chem. 1997; 272: 31377-31381Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar). The developmentally regulated expression and tissue-specific distribution of CS variants suggest that CS chains differing in the degree and profile of sulfation exhibit distinct functions during the development of the brain. The functions of CS-PGs and CS chains in the central nervous system can be categorized as the regulation of cell adhesion and migration, neurite formation, polarization of neurons, synaptic plasticity, survival of neurons, etc. (for reviews, see Ref. 1Bandtlow C.E. Zimmermann D.R. Physiol. Rev. 2000; 80: 1267-1290Crossref PubMed Scopus (552) Google Scholar and 4Margolis R.K. Margolis R.U. Experientia (Basel). 1993; 49: 429-446Crossref PubMed Scopus (243) Google Scholar). Concerning the neurite formation, studies have shown that CS-PGs and CS chains exhibit primarily inhibitory effects on neurite outgrowth on defined growth-promoting substrata (4Margolis R.K. Margolis R.U. Experientia (Basel). 1993; 49: 429-446Crossref PubMed Scopus (243) Google Scholar) and axon guidance in vivo (12Ichijo H. Connect. Tissue Res. 2003; 35: 11-17Google Scholar, 13Morgenstern D.A. Asher R.A. Fawcett J.W. Prog. Brain Res. 2002; 137: 313-332Crossref PubMed Scopus (387) Google Scholar). The inhibitory function of CS chains was supported by recent studies demonstrating that degradation of CS chains by in vivo injection of chondroitinase ABC permitted axonal regeneration after spinal cord injury (14Bradbury 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 (1951) Google Scholar) and reactivation of ocular dominance plasticity in the adult visual cortex (15Pizzorusso T. Medini P. Berardi N. Chierzi S. Fawcett J.W. Maffei L. Science. 2002; 298: 1248-1251Crossref PubMed Scopus (1296) Google Scholar). By contrast, Faissner et al. (16Faissner A. Clement A. Lochter A. Streit A. Mandl C. Melitta S. J. Cell Biol. 1994; 126: 783-799Crossref PubMed Scopus (347) Google Scholar) report that DSD-1-PG, a CS-PG derived from neonatal mouse brain, displayed significant neurite outgrowth-promoting activity in vitro toward hippocampal neurons from embryonic day 18 (E18) rats, and this activity was neutralized by the monoclonal antibody 473 HD, which recognizes a structure embedded in the CS chains (referred to as the DSD-1 epitope). Clement et al. (7Clement M.A. Nadanaka S. Masayama K. Mandl C. Sugahara K. Faissner A. J. Biol. Chem. 1998; 273: 28444-28453Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar) show that the CS side chains of DSD-1-PG contain a small but significant proportion of an over-sulfated D disaccharide unit (GlcUA[2-O-sulfate]β1–3GalNAc[6-O-sulfate]). Over-sulfated CS-D from shark cartilage contains a high proportion (∼20%) of the D unit and exhibits neurite outgrowth-promoting activity, whereas CS-A and CS-C do not (17Nadanaka S. Clement A.M. Masayama K. Faissner A. Sugahara K. J. Biol. Chem. 1998; 273: 3296-3307Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). Clement et al. (18Clement A.M. Sugahara K. Faissner A. Neurosci. Lett. 1999; 269: 125-128Crossref PubMed Scopus (120) Google Scholar) further demonstrate that another over-sulfated CS variant CS-E from squid cartilage, which contains a high proportion (more than 60%) of the E unit, GlcUAβ1-3GalNAc(4,6-O-disulfate), promotes neurite outgrowth in a DSD-1 epitope-independent fashion, giving a long prominent neurite with an axon-like morphology. Recently, Hikino et al. (19Hikino 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 (112) Google Scholar) have shown that the over-sulfated dermatan sulfate (DS) chains purified from various marine organisms also exhibit such activities in a sulfation pattern-dependent manner and that the alleged CS side chains of DSD-1-PG are actually of the DS-type, which implies the possible involvement of DS chains in the neuritogenesis during brain development. Lafont et al. (20Lafont F. Rouget M. Triller A. Prochiantz A. Rousselet A.D. Development. 1992; 114: 17-29Crossref PubMed Google Scholar) previously reported that soluble DS chains prepared from bovine mucosa increased dendrite growth in mesencephalic neurons. These findings suggest that not only over-sulfated CS but also over-sulfated DS chains are neuritogenic. Thus, CS/DS chains cannot simply be classified as either supportive or inhibitory of neurite outgrowth; the sulfation pattern and specific sugar sequence may define these properties (3Sugahara K. Yamada S. Trends Glycosci. Glycotechnol. 2000; 12: 321-349Crossref Scopus (96) Google Scholar, 10Sugahara K. Mikami T. Uyama T. Mizuguchi S. Nomura K. Kitagawa H. Curr. Opin. Struct. Biol. 2003; 13: 612-620Crossref PubMed Scopus (607) Google Scholar, 21Snow D.M. Smith J.D. Cunningham A.T. McFarlin J. Goshorn E.C. Exp. Neurol. 2003; 182: 310-321Crossref PubMed Scopus (47) Google Scholar). Compared with CS-PGs, DS-PGs are minor components of the adult mammalian brain, and so far only a few DS-PG species, such as decorin and biglycan, have been partially characterized (22Kappler J. Stichel C.C. Gleichmann M. Gillen C. Junghans U. Kress H. Müller H.W. Brain Res. 1998; 18: 328-332Crossref Scopus (21) Google Scholar). Thus, DS-type GAG chains appear to exist in the brain, but their distribution, developmental changes, and functions remain unclear. The CS/DS variants, from marine organisms used in our previous studies, include CS-D, CS-E, and hagfish notochord-derived CS-H (the major H disaccharide unit is IdoUAα1–3GalNAc[4,6-O-disulfate]), all of which contain unusually high proportions of over-sulfated disaccharide units. However, they are only minor components in the brain (7Clement M.A. Nadanaka S. Masayama K. Mandl C. Sugahara K. Faissner A. J. Biol. Chem. 1998; 273: 28444-28453Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar, 8Maeda 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 (99) Google Scholar, 11Kitagawa H. Tsutsumi K. Tone Y. Sugahara K. J. Biol. Chem. 1997; 272: 31377-31381Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar, 23Ueoka 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, 24Zou K. Muramatsu H. Ikematsu S. Sakuma S. Salama R.H. Shinomura K. Kimata K. Muramatsu T. Eur. J. Biochem. 2000; 267: 4046-4053Crossref PubMed Scopus (72) Google Scholar). For example, CS-PGs from E18 rat brain contained 1.7% D unit and 1.2% E unit (23Ueoka 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). To evaluate the contribution of the CS/DS chains to the neurite extension, it is essential to characterize CS/DS chains purified from mammalian brains. The developmentally regulated disaccharide composition of brain CS/DS chains suggests that at different developmental stages the chains may have distinct biological functions in neurite formation and growth factor signaling (8Maeda 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 (99) Google Scholar, 11Kitagawa H. Tsutsumi K. Tone Y. Sugahara K. J. Biol. Chem. 1997; 272: 31377-31381Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar, 25Zou P. Zou K. Muramatsu H. Ichihara-Tanaka K. Habuchi O. Ohtake S. Ikematsu S. Sakuma S. Muramatsu T. Glycobiology. 2003; 13: 35-42Crossref PubMed Scopus (75) Google Scholar). In this study, we purified CS/DS chains from embryonic and adult pig brains and investigated their effects on neurite outgrowth and growth factor binding. We also characterized the structures responsible for these activities. Preliminary results were reported in abstract form (26Bao X. Nishimura S. Yamada S. Sugahara K. Seikagaku. 2003; 75: 908Google Scholar). Animals—Embryonic pigs (body weight: 315 g) and adult pig brains were purchased from a local pig-raising company. ddY mice were used for the preparation of E16 hippocampal neurons. Materials—The following enzymes were purchased from Seikagaku Corp. (Tokyo, Japan): chondroitinase ABC (EC 4.2.2.4), chondro-4-sulfatase (EC 3.1.6.9) and chondro-6-sulfatase (EC 3.1.6.10) from Proteus vulgaris; chondroitinase AC-I (EC 4.2.2.5), chondroitinase B (EC 4.2.2), heparitinase (EC 4.2.2.8), and heparinase (EC 4.2.2.7) from Flavobacterium heparinum; hyaluronidase (EC 4.2.2.1) from Streptomyces hyalurolyticus. Recombinant human (rh)-midkine (MK) expressed in Escherichia coli and rh-fibroblast growth factor 1 (FGF1) (acidic FGF) expressed in E. coli were purchased from PeproTech EC LTD (London, UK). rh-pleiotrophin (PTN) expressed in E. coli was from RELIA Tech GmbH (Braunschweig, Germany), and rh-heparin binding epidermal growth factor-like growth factor (HB-EGF) expressed in Sf 21 insect cells was from R&D systems (Minneapolis). rh-FGF2 (basic FGF) expressed in E. coli was from Genzyme Techne (Minneapolis, MN), and rh-FGF10 expressed in E. coli was provided by Takashi Katsumata (Sumitomo Pharmaceutical Research Center, Osaka, Japan). Recombinant mouse FGF18 was prepared as reported (27Hu M.C. Qiu W.R. Wang Y.P. Hill D. Ring B.D. Scully S. Bolon B. DeRose M. Luethy R. Simonet W.S. Arakawa T. Danilenko D.M. Mol. Cell. Biol. 1998; 18: 6063-6074Crossref PubMed Scopus (116) Google Scholar). Actinase E was obtained from Kaken Pharmaceutical Co. (Tokyo, Japan). Sep-Pak Plus Accell™ anion-exchange cartridges were obtained from Waters Corp. (Milford, MA), and a Superdex 75 HR column (10 × 300 mm), a Superdex Peptide HR column (10 × 300 mm), a Superdex 200 HR column (10 × 300 mm), and prepacked disposable PD-10 columns were from Amersham Biosciences. Size-defined dextrans (average Mr 65,500, 37,500, and 18,000), bovine intestinal mucosa HS (average Mr 7,500), and low molecular weight heparin from porcine intestinal mucosa (average Mr 6,000) were purchased from Sigma. All other chemicals and reagents were of the highest quality available. Preparation and Purification of CS/DS Chain Fractions—Embryonic (9.7 g) and adult (108.0 g) pig brains were homogenized with 2.5 volumes of ice-cold phosphate-buffered saline (PBS) containing 20 mm EDTA (11Kitagawa H. Tsutsumi K. Tone Y. Sugahara K. J. Biol. Chem. 1997; 272: 31377-31381Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar). The homogenates were centrifuged at 15,000 × g for 25 min at 4 °C, and to the resultant supernatant 4 volumes of ethanol was added, which precipitated PBS-soluble CS/DS-PGs. The PBS-insoluble fraction was further homogenized with 1.5 volumes of acetone, and the acetone-insoluble materials were collected by centrifugation. After drying, both PBS-soluble and -insoluble CS/DS-PG-containing fractions from embryo and adult pig brains were extensively digested with actinase E at 60 °Cin0.1 m borate-sodium buffer, pH 8.0, containing 10 mm CaCl2. After incubation and subsequent treatment with 5% trichloroacetic acid, the GAG-containing fractions were recovered by ethanol precipitation. Each GAG fraction was desalted by gel filtration on a PD-10 column and then fractionated by anion-exchange chromatography on an Accell QMA Plus cartridge using stepwise elution with 0.3 m phosphate buffers, pH 6.0, containing 0.15, 1.0, and 1.5 m NaCl. All subfractions were desalted by gel filtration on a PD-10 column. The total amount of GAGs in each subfraction was evaluated by the carbazole reaction (28Bitter T. Muir H.M. Anal. Biochem. 1962; 4: 330-334Crossref PubMed Scopus (5311) Google Scholar), and the CS/DS disaccharide composition was analyzed by chondroitinase ABC digestion followed by HPLC (29Sugahara K. Okumura Y. Yamashina I. Biochem. Biophys. Res. Commun. 1989; 162: 189-197Crossref PubMed Scopus (80) Google Scholar). The 1.0 m NaCl-eluted fractions from both PBS-soluble and -insoluble fractions, which contained more than 90% of the recovered CS/DS chains of the embryo or adult pig brain-derived sample, was used for further purification. To remove hyaluronic acid (HA) PBS-soluble and -insoluble fractions obtained from both embryonic and adult pig brains were subjected to digestion with Streptomyces hyaluronidase at 60 °C in 20 mm acetate buffer, pH 5.0 (30Ohya T. Kaneko Y. Biochim. Biophys. Acta. 1970; 198: 607-609Crossref PubMed Scopus (460) Google Scholar). After 4 h of incubation, the digests were subjected to gel filtration chromatography on a column of Superdex 75 (10 × 300 mm) that was eluted with 0.2 m NH4HCO3 at a flow rate of 0.4 ml/min. Absorption at 210 nm was used to monitor GAG chains. The flow-through fractions, which contained GAG chains, were collected, pooled, and evaporated to dryness. The hyaluronidase-resistant GAGs were subsequently digested with a mixture of heparitinase and heparinase in 20 mm sodium acetate buffer, pH 7.0, containing 2 mm calcium acetate for 4 h (31Sugahara K. Yamada S. Yoshida K. de Waard P. Vliegenthart J.F.G. J. Biol. Chem. 1992; 267: 1528-1533Abstract Full Text PDF PubMed Google Scholar). The polysaccharides resistant to heparinase and heparitinase were recovered by gel filtration as described above. After the removal of HA and heparan sulfate, the CS/DS-containing materials were passed through a C-18 cartridge to remove trace amounts of free peptides. The CS/DS fractions thus prepared from PBS-soluble and -insoluble fractions of embryonic pig brains were designated Es-CS/DS and Ei-CS/DS, whereas those of adult pig brains were As-CS/DS and Ai-CS/DS, respectively. The purity of all these preparations was checked by gel filtration after chondroitinase ABC digestion and by amino acid analysis after acid hydrolysis with 3 m HCl in the gas phase at 100 °C for 16 h. Determination of Molecular Size—An aliquot (10.0 μg) of Es-, Ei-, As-, or Ai-CS/DS was chromatographed on a gel filtration column of Superdex 200 (10 × 300 mm) that had been calibrated using a series of size-defined commercial polysaccharides (32Yamada S. Okada Y. Ueno M. Iwata S. Deepa S.S. Nishimura S. Fujita M. van Die I. Hirabayashi Y. Sugahara K. J. Biol. Chem. 2002; 277: 31877-31886Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). The column was eluted with 0.2 m NH4HCO3 at a flow rate of 0.3 ml/min. Fractions were collected at 3-min intervals, lyophilized, and digested with 10 mIU of chondroitinase ABC as described below. The resultant digests were analyzed by anion-exchange HPLC as described previously. Analysis of Disaccharide Composition—An aliquot (4.0 μg) of Es-, Ei-, As-, or Ai-CS/DS was incubated with 40 mIU of chondroitinase ABC in a 50mm Tris-HCl buffer, pH 8.0, containing 60 mm sodium acetate in a total volume of 40 μlat37 °C for 4 h (33Saito H. Yamagata T. Suzuki S. J. Biol. Chem. 1968; 243: 1536-1542Abstract Full Text PDF PubMed Google Scholar). One-fourth of the digest was subsequently incubated with 40 mIU of chondro-4-sulfatase or chondro-6-sulfatase in a total volume of 50 μl at 37 °C for 1 h (34Sugahara K. Shigeno K. Masuda M. Fujii N. Kurosaka A. Takeda K. Carbohydr. Res. 1994; 255: 145-163Crossref PubMed Scopus (82) Google Scholar). Each digest corresponding to one-fourth of the starting materials was analyzed by anion-exchange HPLC on an amino-bound silica PA-03 column (4.6 × 250 mm, YMC Co., Kyoto, Japan) as reported (29Sugahara K. Okumura Y. Yamashina I. Biochem. Biophys. Res. Commun. 1989; 162: 189-197Crossref PubMed Scopus (80) Google Scholar). Identification and quantification of the resulting disaccharides were achieved by comparison with CS-derived authentic unsaturated disaccharides: ΔHexUAα1–3GalNAc, ΔHexUAα1–3GalNAc(6-O-sulfate), ΔHexUAα1–3GalNAc(4-O-sulfate) (ΔDi-4S), ΔHexUA(2-O-sulfate)α1–3GalNAc(6-O-sulfate), ΔHexUAα1–3GalNAc(4,6-O-disulfate), and ΔHexUA(2-O-sulfate)α1–3GalNAc(4,6-O-disulfate). Analysis of the Glucuronate: Iduronate Ratio—An aliquot (2 μg) of Es-, Ei-, As-, or Ai-CS/DS was exhaustively digested with chondroitinases ABC (20 mIU), AC-I (20 mIU), or B (10 mIU) in the appropriate buffer, 50 mm Tris-HCl buffer, pH 7.3 (for AC-I) (19Hikino 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 (112) Google Scholar) or 100 mm Tris-HCl buffer, pH 8.0 (for B) (35Sugahara K. Ohkita Y. Shibata Y. Yoshida K. Ikegami A. J. Biol. Chem. 1995; 270: 7204-7721Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar) (for ABC, see above). Each digestion was initiated with half the amount of the enzyme described above and run at 37 °C for 12 h, after which the rest of the enzyme was added, and incubation was continued for another 2 h. Half of each digest was analyzed by anion-exchange HPLC for identification and quantification of the resultant unsaturated disaccharides as described above. Analysis of the Iduronate Distribution—An aliquot (0.4 μg) of Es- or Ei-CS/DS was digested with chondroitinase AC-I (6 mIU) and another with chondroitinase B (6 mIU) as described above. The reaction mixtures were lyophilized and derivatized with a fluorophore 2-aminobenzamide (2AB), then the excess 2AB reagent was removed by paper chromatography (36Kinoshita A. Sugahara K. Anal. Biochem. 1999; 269: 367-378Crossref PubMed Scopus (194) Google Scholar). The 2AB derivatives were subjected to gel filtration on a column (10 × 300 mm) of Superdex Peptide using 0.025 m NH4HCO3 containing 7% 1-propanol as an effluent at a flow rate of 0.4 ml/min. Eluates were monitored by fluorescence with excitation and emission wavelengths of 330 and 420 nm, respectively. Identification and quantification of 2AB-labeled oligosaccharides were achieved by comparison with size-defined 2AB-labeled standard unsaturated CS disaccharides and sulfated oligosaccharides 2S. S. Deepa and K. Sugahara, unpublished results. prepared by 2AB derivatization of CS-E oligosaccharides generated by testicular hyaluronidase digestion (37Kinoshita A. Yamada S. Haslam S.M. Morris H.R. Dell A. Sugahara K. J. Biol. Chem. 1997; 272: 19656-19665Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). The percentage of galactosaminidic bonds cleaved by each specific enzyme was calculated from the distribution of fluorescence intensity across peaks relative to the total amount of the starting material. Delayed Extraction Matrix-assisted Laser Desorption Ionization Time-of-flight Mass Spectrometry (MALDI-TOF MS)—The chondroitinase AC-I-resistant 2AB-labeled oligosaccharides, which were obtained as described above, were mixed with a matrix of 2,5-dihydroxybenzoic acid and used for MALDI-TOF MS analyses in a positive ion mode as described previously (38Sugiyama E. Mizuno N. Uemura K. Taketomi T. Glycobiology. 1997; 7: 719-724Crossref PubMed Scopus (59) Google Scholar). The MS spectra were recorded on a Voyager DE-RP/Pro (PerSeptive Biosystems, Framingham, MA) in the linear mode. Preparation of Substrates for Cell Culture—An aliquot (2 μg) of Es-CS/DS or As-CS/DS was digested with 5 mIU of chondroitinases ABC, AC-I, or B in a total volume of 20 μl of the appropriate buffer at 37 °C (or 30 °C for chondroitinase B) for 120 min, and the digests were used to prepare substrates for cell cultures. Aliquots (2 μg) of the parent CS/DS fractions or their chondroitin lyase-treated preparations were individually coated onto plastic coverslips (10 × 10 mm) that had been precoated with poly-dl-ornithine (P-ORN) (Sigma, Tokyo, Japan) dissolved in 0.1 m borate buffer, pH 8.2, at a concentration of 1.5 μg/ml. Heat-inactivated chondroitin lyases with their buffers were used as controls. Cell Culture and Neurite Outgrowth Promotion Assays—Cultures of mouse hippocampal neurons were established from E16 animals as described (7Clement M.A. Nadanaka S. Masayama K. Mandl C. Sugahara K. Faissner A. J. Biol. Chem. 1998; 273: 28444-28453Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar, 17Nadanaka S. Clement A.M. Masayama K. Faissner A. Sugahara K. J. Biol. Chem. 1998; 273: 3296-3307Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar) with some modifications (19Hikino 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 (112) Google Scholar). Briefly, hippocampi were dissected from mouse E16 embryonic brains and dissociated into a single cell suspension. The cells were seeded on coverslips coated with defined GAG substrates at a density of 10,000 cell/cm2 and cultivated in Eagle's modified essential medium containing N2 supplements (Invitrogen), 0.1 mm pyruvate, 0.1% (w/v) ovalbumin, 0.029% (w/v) l-glutamine, 0.2% (w/v) sodium hydrogen carbonate, and 5 mm HEPES. The cultures were incubated in a humidified atmosphere with 5% CO2 at 37 °C. After 24 h of incubation the cells were fixed with 4% (w/v) paraformaldehyde for 30 min at room temperature and then immunostained with anti-microtubule-associated protein 2 (Leico Technologies Inc., St. Louis, MO) and anti-neurofilament (Sigma) at 1:100 and 1:250 dilutions, respectively, in PBS containing 3% (w/v) bovine serum albumin. The antibodies were then detected using a Vectastain ABC kit (Vector Laboratories Inc., Burlingame, CA) with 3,3′-diaminobenzidine as a chromogen. At least three independent experiments in duplicate were carried out for each culture condition. The stained hippocampal neurons were analyzed with a morphometric station equipped with an optical microscope (BH-2, Olympus, Tokyo, Japan), a digital camera (HC-300Z/OL, Olympus), and software (Mac Scope Mitani Corp., Tokyo, Japan). Clearly isolated cells with at least one neurite longer than the diameter of the cell body were chosen at random. The length of the neurites and the number of primary neurites (longer than the cell body diameter) were determined by drawing and counting, respectively. The data are expressed as the mean ± S.E., and the significance of difference between the means was evaluated using Mann-Whitney's U test. Growth Factor Binding Assays Using a BIAcore System—Binding reactions were carried out at 25 °C on a BIAcore™ J Biosensor (BIAcore AB, Uppsala, Sweden) using streptavidin-derivatized sensor chips. Es-CS/DS and As-CS/DS were biotinylated as described (39Deepa S.S. Umehara Y. Higashiyama S. Itoh N. Sugahara K. J. Biol. Chem. 2002; 277: 43707-43716Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar), and the excess biotinylation reagent was removed using an ultrafree centrifugal filter tube (molecular mass cut-off, 10 kDa; Millipore, Bedford, MA). The biotinylated Es-CS/DS and As-CS/DS chains were then immobilized on the surface of the streptavidin-derivatized sensor chip. The amount of Es-CS/DS or As-CS/DS, immobilized by repeated injections, was controlled at 320 ± 8 resonance units, which corresponds to 0.37 ng of sugar chain. Growth factors (200 ng each) including FGF1, FGF2, FGF10, FGF18, PTN, MK, and HB-EGF in the running buffer, pH 7.4 (HBS-EP), containing 10 mm HEPES, 0.15 m NaCl, 3 mm EDTA, and 0.005% (w/v) Tween 20 were individ
Год издания: 2004
Авторы: Xingfeng Bao, Shuji Nishimura, Tadahisa Mikami, Shuhei Yamada, Nobuyuki Itoh, Kazuyuki Sugahara
Издательство: Elsevier BV
Источник: Journal of Biological Chemistry
Ключевые слова: Proteoglycans and glycosaminoglycans research, Seaweed-derived Bioactive Compounds, Glycosylation and Glycoproteins Research
Другие ссылки: Journal of Biological Chemistry (PDF)
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PubMed (HTML)
Journal of Biological Chemistry (HTML)
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Открытый доступ: hybrid
Том: 279
Выпуск: 11
Страницы: 9765–9776