FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2007435025> ?p ?o ?g. }

• W2007435025 endingPage "19293" @default.
• W2007435025 startingPage "19283" @default.
• W2007435025 abstract "The poor axonal regeneration that follows lesions of the central nervous system (CNS) is crucially influenced by the local CNS tissue environment through which neurites have to grow. In addition to an inhibitory role of the glial scar, inhibitory substrate effects of CNS myelin and oligodendrocytes have been demonstrated. Several proteins including NI-35/250, myelin-associated glycoprotein, tenascin-R, and NG-2 have been described to have neurite outgrowth inhibitory or repulsive properties in vitro. Antibodies raised against NI-35/250 (monoclonal antibody IN-1) were shown to partially neutralize the growth inhibitory effect of CNS myelin and oligodendrocytes, and to result in long distance fiber regeneration in the lesioned adult mammalian CNS in vivo. We report here the purification of a myelin protein to apparent homogeneity from bovine spinal cord which exerts a potent neurite outgrowth inhibitory effect on PC12 cells and chick dorsal root ganglion cells, induces collapse of growth cones of chick dorsal root ganglion cells, and also inhibits the spreading of 3T3 fibroblasts. These activities could be neutralized by the monoclonal antibody IN-1. The purification procedure includes detergent solubilization, anion exchange chromatography, gel filtration, and elution from high resolution SDS-polyacrylamide gel electrophoresis. The active protein has a molecular mass of 220 kDa and an isoelectric point between 5.9 and 6.2. Its inhibitory activity is sensitive to protease treatment and resists harsh treatments like 9m urea or short heating. Glycosylation is, if present at all, not detectable. Microsequencing resulted in six peptides and strongly suggests that this proteins is novel. The poor axonal regeneration that follows lesions of the central nervous system (CNS) is crucially influenced by the local CNS tissue environment through which neurites have to grow. In addition to an inhibitory role of the glial scar, inhibitory substrate effects of CNS myelin and oligodendrocytes have been demonstrated. Several proteins including NI-35/250, myelin-associated glycoprotein, tenascin-R, and NG-2 have been described to have neurite outgrowth inhibitory or repulsive properties in vitro. Antibodies raised against NI-35/250 (monoclonal antibody IN-1) were shown to partially neutralize the growth inhibitory effect of CNS myelin and oligodendrocytes, and to result in long distance fiber regeneration in the lesioned adult mammalian CNS in vivo. We report here the purification of a myelin protein to apparent homogeneity from bovine spinal cord which exerts a potent neurite outgrowth inhibitory effect on PC12 cells and chick dorsal root ganglion cells, induces collapse of growth cones of chick dorsal root ganglion cells, and also inhibits the spreading of 3T3 fibroblasts. These activities could be neutralized by the monoclonal antibody IN-1. The purification procedure includes detergent solubilization, anion exchange chromatography, gel filtration, and elution from high resolution SDS-polyacrylamide gel electrophoresis. The active protein has a molecular mass of 220 kDa and an isoelectric point between 5.9 and 6.2. Its inhibitory activity is sensitive to protease treatment and resists harsh treatments like 9m urea or short heating. Glycosylation is, if present at all, not detectable. Microsequencing resulted in six peptides and strongly suggests that this proteins is novel. Neurite growth in the mammalian CNS 1The abbreviations used are: CNS, central nervous system; PAGE, polyacrylamide gel electrophoresis; DMEM, Dulbecco's modified Eagle's medium; NGF, nerve growth factor; FCS, fetal calf serum; PBS, phosphate-buffered saline; mAb, monoclonal antibody; E, embryonic day; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; MAG, myelin-associated glycoprotein. 1The abbreviations used are: CNS, central nervous system; PAGE, polyacrylamide gel electrophoresis; DMEM, Dulbecco's modified Eagle's medium; NGF, nerve growth factor; FCS, fetal calf serum; PBS, phosphate-buffered saline; mAb, monoclonal antibody; E, embryonic day; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; MAG, myelin-associated glycoprotein. ceases at the end of the developmental period. Although CNS neurons maintain some ability to rearrange their axonal and dendritic arbors in the adult brain, regeneration of severed CNS axons over long distances is absent. Transplantations of peripheral nerve explants into various parts of the brain and spinal cord revealed that the lack of regeneration is not primarily due to intrinsic properties of CNS neurons but is instead dependent on the microenvironment encountered by the regenerating fibers (1Richardson P.M. McGuinness U.M. Aguayo A.J. Nature. 1980; 284: 264-265Crossref PubMed Scopus (774) Google Scholar, 2David S. Aguayo A.J. Science. 1981; 214: 931-933Crossref PubMed Scopus (1447) Google Scholar); CNS axons were able to grow over long distances in the peripheral nerve segments, but ceased to grow as they entered the CNS tissue again (2David S. Aguayo A.J. Science. 1981; 214: 931-933Crossref PubMed Scopus (1447) Google Scholar). Several lines of evidence suggest that the presence of inhibitory factors rather than the lack of growth promoting molecules is responsible to the non-conducive properties of CNS tissue in adult vertebrates (for review, see Ref. 3Schwab M.E. Bartholdi D. Physiol. Rev. 1996; 76: 319-370Crossref PubMed Scopus (987) Google Scholar). In vitro experiments demonstrated that adult optic nerve explants were not invaded by neurites, although high amounts of neurotrophic factors were provided (4Schwab M.E. Thoenen H. J. Neurosci. 1985; 5: 2415-2423Crossref PubMed Google Scholar). Similarly, cryostat sections of adult CNS tissue were shown to be non-permissive substrates for neurite outgrowth, especially the densely myelinated areas (5Carbonetto S. Evans D. Cochard P. J. Neurosci. 1987; 7: 610-620Crossref PubMed Google Scholar, 6Crutcher K.A. Exp. Neurol. 1989; 104: 39-54Crossref PubMed Scopus (86) Google Scholar, 7Crutcher K.A. Privitera M. Ann. Neurol. 1989; 26: 580-583Crossref PubMed Scopus (11) Google Scholar, 8Savio T. Schwab M.E. J. Neurosci. 1989; 9: 1126-1133Crossref PubMed Google Scholar, 9Tuttle R. Matthew W.D. J. Neurosci. Methods. 1991; 39: 193-202Crossref PubMed Scopus (22) Google Scholar). Differentiated oligodendrocytes in culture and CNS myelin exerted a strong inhibitory effect on adhesion and outgrowth of primary neurons, neuroblastoma cells, and also for spreading of 3T3 fibroblasts (10Schwab M.E. Caroni P. J. Neurosci. 1988; 8: 2381-2393Crossref PubMed Google Scholar, 11McKerracher L. David S. Jackson D.L. Kottis V. Dunn R.J. Braun P.E. Neuron. 1994; 13: 805-811Abstract Full Text PDF PubMed Scopus (995) Google Scholar, 12Ng W.P. Cartel N. Roder J. Roach A. Lozano A. Brain Res. 1996; 720: 17-24Crossref PubMed Scopus (27) Google Scholar). Growth cones of rat dorsal root ganglion (DRG) neurons interacting with differentiated oligodendrocytes were arrested and collapsed (13Fawcett J.W. Rokos J. Bakst I. J. Cell Sci. 1989; 92: 93-100PubMed Google Scholar, 14Bandtlow C.E. Zachleder T. Schwab M.E. J. Neurosci. 1990; 10: 3837-3848Crossref PubMed Google Scholar). In vivo experiments demonstrated that regeneration of lesioned axons over long distances could be observed in myelin-free spinal cord or optic nerve, which has been obtained by killing the dividing oligodendrocyte precursors by repeated x-irradiation of newborn rats (15Savio T. Schwab M.E. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 4130-4133Crossref PubMed Scopus (173) Google Scholar, 16Weibel D. Cadelli D. Schwab M.E. Brain Res. 1994; 642: 259-266Crossref PubMed Scopus (117) Google Scholar) or by the suppression of the onset of myelination by immunocytolysis in chicken (17Keirstead H.S. Hasan S.J. Muir G.D. Steeves J.D. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 11664-11668Crossref PubMed Scopus (146) Google Scholar, 18Hasan S.J. Nelson B.H. Valenzuela J.I. Keirstead H.S. Shull S.E. Ethell D.W. Steeves J.D. Rest. Neurol. Neurosci. 1991; 2: 137-154PubMed Google Scholar). Moreover, high levels of GAP-43, a protein related to axonal growth (19Meiri K.F. Willard M. Johnson M.I. J. Neurosci. 1988; 8: 2571-2581Crossref PubMed Google Scholar, 20Kapfhammer J.P. Schwab M.E. Eur. J. Neurosci. 1994; 6: 403-411Crossref PubMed Scopus (59) Google Scholar, 21Kapfhammer J.P. Schwab M.E. J. Comp. Neurol. 1994; 340: 194-206Crossref PubMed Scopus (183) Google Scholar), and a greatly increased structural plasticity as reflected by collateral sprouting of sensory fibers in response to dorsal root lesions could be observed in these myelin deficient zones (22Schwegler G. Schwab M.E. Kapfhammer J.P. J. Neurosci. 1995; 15: 2756-2767Crossref PubMed Google Scholar). All these results are consistent with a strong growth-restricting function of adult CNS myelin. When CNS myelin was separated by ether/ethanol extraction into a lipid and a protein fraction, the neurite growth inhibitory activity was associated with the protein fraction (23Caroni P. Schwab M.E. J. Cell Biol. 1988; 106: 1281-1288Crossref PubMed Scopus (761) Google Scholar). Proteins eluted from gel slices containing molecules with an apparent molecular mass of 35 and 250 kDa (SDS-PAGE) showed a very potent inhibitory activity (23Caroni P. Schwab M.E. J. Cell Biol. 1988; 106: 1281-1288Crossref PubMed Scopus (761) Google Scholar, 24Bandtlow C.E. Schmidt M.F. Hassinger T.D. Schwab M.E. Kater S.B. Science. 1993; 259: 80-83Crossref PubMed Google Scholar). A monoclonal antibody (mAb IN-1), which has been raised against rat NI-250 (25Caroni P. Schwab M.E. Neuron. 1988; 1: 85-96Abstract Full Text PDF PubMed Scopus (757) Google Scholar), neutralizes the neurite growth inhibitory property of differentiated oligodendrocytes and NI-35 and NI-250 (14Bandtlow C.E. Zachleder T. Schwab M.E. J. Neurosci. 1990; 10: 3837-3848Crossref PubMed Google Scholar, 24Bandtlow C.E. Schmidt M.F. Hassinger T.D. Schwab M.E. Kater S.B. Science. 1993; 259: 80-83Crossref PubMed Google Scholar, 25Caroni P. Schwab M.E. Neuron. 1988; 1: 85-96Abstract Full Text PDF PubMed Scopus (757) Google Scholar, 26Bastmeyer M. Beckmann M. Schwab M.E. Stuermer C.A. J. Neurosci. 1991; 11: 626-640Crossref PubMed Google Scholar, 27Lang D.M. Rubin B.P. Schwab M.E. Stuermer C.A.O. J. Neurosci. 1995; 15: 99-109Crossref PubMed Google Scholar, 28Varga Z.M. Schwab M.E. Nicholls J.G. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10959-10963Crossref PubMed Scopus (43) Google Scholar, 29Rubin B.P. Spillmann A.A. Bandtlow C.E. Keller F. Schwab M.E. Eur. J. Neurosci. 1995; 7: 2524-2529Crossref PubMed Scopus (26) Google Scholar, 30Spillmann A.A. Amberger V.R. Schwab M.E. Eur. J. Neurosci. 1997; 9: 549-555Crossref PubMed Scopus (46) Google Scholar). Immunoprecipitation of CNS myelin proteins by the mAb IN-1 removed more than 50% of inhibitory substrate properties (25Caroni P. Schwab M.E. Neuron. 1988; 1: 85-96Abstract Full Text PDF PubMed Scopus (757) Google Scholar, 31Bandtlow C. Schiweck W. Tai H.H. Schwab M.E. Skerra A. Eur. J. of Bioch. 1996; 241: 468-475Crossref PubMed Scopus (28) Google Scholar). Immunohistochemistry revealed that mAb IN-1 stains white matter and myelin in the whole brain and spinal cord from adult rats (32Rubin B.P. Dusart I. Schwab M.E. J. Neurocytol. 1994; 23: 209-217Crossref PubMed Scopus (53) Google Scholar).In vivo, the application of mAb IN-1 to lesioned nerve fiber tracts in adult rats resulted in long distance growth of regenerating fibers in the CNS (spinal cord, optic nerve, septo-hippocampal tract) (16Weibel D. Cadelli D. Schwab M.E. Brain Res. 1994; 642: 259-266Crossref PubMed Scopus (117) Google Scholar, 33Schnell L. Schwab M.E. Nature. 1990; 343: 269-272Crossref PubMed Scopus (1076) Google Scholar, 34Cadelli D. Schwab M.E. Eur. J. Neurosci. 1991; 3: 825-832Crossref PubMed Scopus (74) Google Scholar, 35Schnell L. Schneider R. Kolbeck R. Barde Y.A. Schwab M.E. Nature. 1994; 367: 170-173Crossref PubMed Scopus (813) Google Scholar, 36Bartsch U. Bandtlow C.E. Schnell L. Bartsch S. Spillmann A.A. Rubin B.P. Hillenbrand R. Montag D. Schwab M.E. Schachner M. Neuron. 1995; 15: 1376-1381Abstract Full Text PDF Scopus (218) Google Scholar) and in a recovery of specific reflex and locomotor functions after spinal cord injury (37Bregman B.S. Kunkel-Bagden E. Schnell L. Dai H.N. Gao D. Schwab M.E. Nature. 1995; 378: 498-501Crossref PubMed Scopus (654) Google Scholar). In addition, an increase of collateral growth from intact fibers could be observed in spinal cord and brainstem after IN-1 application following unilateral pyramidal tract lesion (38Thallmair M. Metz G.A.S. Z'Graggen W.J. Raineteau O. Kaitje G.L. Schwab M.E. Nat. Neurosci. 1998; 1: 124-131Crossref PubMed Scopus (339) Google Scholar, 39Z'Graggen W.J. Metz G.A.S. Kaitje G.L. Thallmair M. Schwab M.E. J. Neurosci. 1998; 18: 4744-4757Crossref PubMed Google Scholar). Here we describe the purification and biochemical characterization of a high molecular mass protein of bovine spinal cord myelin which exerts potent inhibition of neurite outgrowth of NGF-primed PC12 cells and chick DRG, inhibits spreading of 3T3 fibroblast, and induces collapse of chick DRG growth cones. The mAb IN-1 is able to fully neutralize this inhibitory activity, indicating that the purified protein is an IN-1 antigen. Bovine spinal cords were obtained from the local slaughterhouse. The columns, Q-Sepharose and Superdex 200, were obtained from Amersham Pharmacia Biotech (Uppsala, Sweden). CHAPS and all other analytical grade chemicals, unless otherwise mentioned, were purchased from Sigma. Mouse NIH 3T3 fibroblasts (from the American Type Culture Collection, Rockville, MD) were cultured and assayed for cell spreading in Dulbecco's modified Eagle's medium (DMEM) (Life Technologies, Inc.) containing 10% fetal calf serum (FCS) (Biological Industries, Kibbutz Beth Haemek, Israel), 100 units/ml Penicillin, and 100 μg/ml streptomycin PS (Life Technologies, Inc.) as described below. The cells were passaged 1 day before the assay. A pheochromocytoma cell line (PC12 subclone, obtained from M. V. Chao, New York), which grows neurites rapidly in the presence of NGF, but independently of laminin (40Benedetti M. Levi A. Chao M.V. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7859-7863Crossref PubMed Scopus (271) Google Scholar), was passaged by mechanical detachment in Hank's balanced salt solution and cultivated in RPMI 1640 medium (Life Technologies, Inc.), which contained 10% horse serum (Sera-lab, Sussex, United Kingdom), 5% FCS, 100 units/ml penicillin, and 0.5 mg/ml streptomycin. Prior to the bioassays, PC12 cells were primed for 2 days with NGF (100 ng/ml, 2.5 s, Harlan Bioproducts, Indianapolis, IN) in 2% horse serum, 1% FCS. The following monoclonal antibodies were used in this study: IN-1 (IgM, used as hybridoma supernatant, 1:1 for functional assays; Ref. 25Caroni P. Schwab M.E. Neuron. 1988; 1: 85-96Abstract Full Text PDF PubMed Scopus (757) Google Scholar), O1 (IgM, used as hybridoma supernatant, recognizes galactocerebroside on the membrane of oligodendrocytes, 1:1 for functional assays, gift of Prof. M. Schachner; Ref. 41Sommer I. Schachner M. Dev. Biol. 1981; 83: 311-327Crossref PubMed Scopus (953) Google Scholar), monoclonal anti-human tenascin-C (1:100 for Western blots, clone HT64, gift of Dr. R. Odermatt, University Hospital Zürich), and monoclonal anti-human spectrin (α + β, 1:400 for Western blots, clone SB-SP1, Sigma). Spreading of 3T3 cells was determined as described previously (23Caroni P. Schwab M.E. J. Cell Biol. 1988; 106: 1281-1288Crossref PubMed Scopus (761) Google Scholar, 25Caroni P. Schwab M.E. Neuron. 1988; 1: 85-96Abstract Full Text PDF PubMed Scopus (757) Google Scholar). Briefly, fractions (100 μl) were coated overnight on four-well plates (well area: 1 cm2, Greiner, Nürtingen, Germany) at 4 °C in a humid chamber. Unbound material was removed by three washes with Ca2+/Mg2+-free Hank's solution. 3T3 NIH fibroblasts were detached from approximately 80% confluent cultures by brief 0.1% (w/v) trypsin treatment in prewarmed phosphate-buffered saline (PBS) with 0.025% (w/v) EDTA, pH 8.0. Trypsinization was stopped by addition of a 10-fold excess of serum-containing DMEM; cells were collected and resuspended in DMEM plus 10% FCS. 50 μl (8000 cells/cm2) were added to each well, and cells were scored after 1 h in culture (37 °C, 5% CO2), a time at which more than 70% of the cells had spread to large, typical fibroblasts on plastic control substrates. The percentage of cells that remained round, i.e. did not spread on the test substrate, was determined by counting in five randomly chosen areas of the dish. To determine the protein concentration that had a half-maximal inhibitory effect (EC50 values), different protein concentrations were coated and the percentage of cells that remained round was calculated for each concentration. The EC50 value corresponded to the concentration where 65% of the cells showed inhibition (half-maximal inhibitory effect over a background of ±30% round cells on control substrates). To assess neutralization of inhibitory activity, substrate-coated wells were incubated with 100 μl of Hank's buffer, IN-1, or O1 hybridoma supernatant for 20 min at 37 °C. The wells were then washed briefly with Hank's and cells were applied in the presence of Hank's, IN-1, or O1 hybridoma supernatant diluted 1: 1 with medium. All assays were done in duplicate, and about 150–200 cells were counted per condition tested. The test was performed as described previously (29Rubin B.P. Spillmann A.A. Bandtlow C.E. Keller F. Schwab M.E. Eur. J. Neurosci. 1995; 7: 2524-2529Crossref PubMed Scopus (26) Google Scholar). Briefly, NGF-primed PC12 cells were detached mechanically, trypsinized for 5 min at 37 °C with 0.05% (w/v) trypsin in Ca2+/Mg2+-free Hank's solution, and plated at a density of 3000–5000 cells/cm2 in culture medium with 100 ng/ml NGF. Assays were stopped after 24 h in culture by adding 4% (m/v) formalin buffered with NaCl/Pi(137 mm NaCl, 2.7 mm KCl, 1.5 mmKH2PO4, 8 mm NaHPO4, pH 7.4) and quantified in duplicates. In six randomly chosen fields per well, we determined the percentage of PC12 cells with neurites shorter than two diameters of the cell body. Treatment with the mAb IN-1 was performed in the same way as for the 3T3 fibroblasts (see above). About 150–200 cells were evaluated per condition tested. EC50values were determined as for the 3T3 assay (see above). 22-mm glass coverslips were coated with 0.1 mg/ml collagen (from rat tail; Ref.42), 1 μg/ml laminin (Life Technologies, Inc.) for 6 h at 37 °C and air-dried. 10 μl of one of the following substrates was coated: 10 μg/ml gel-eluted bNI-220, 10 μg/ml semaIII/collapsin 1 (COS supernatant), or an equivalent amount of gel-eluted control protein (120 kDa, bC120) for 2 h at 37 °C. Coverslips were then blocked with F-12/DMEM culture medium (Dulbecco's MEM NUT MIX F-12 (Ham), Life Technologies, Inc.), containing 10% FCS, 100 units/ml penicillin, and 0.5 mg/ml streptomycin or incubated with hybridoma supernatant (mAb O1 or mAb IN-1) containing 6% FCS for 1 h at 37 °C. Four embryonic day 11–13 chick DRG explants were grown on these coverslips with 200 μl of F-12/DMEM culture medium containing 50 ng/ml NGF. Results are from three independent experiments for SemaIII, laminin, and bC120, and five independent experiments for bNI-220. Serial dilutions of purified protein were assayed for growth cone collapse on explanted chick embryonic day 11–13 (E11–E13) DRG similar as described previously (43Luo Y. Raible D. Raper J.A. Cell. 1993; 75: 217-227Abstract Full Text PDF PubMed Scopus (998) Google Scholar). Briefly, explants were dissected from chick embryos and incubated at 37 °C overnight on a plastic dish coated with 1 μg of laminin (area 1 cm2) in 60 μl of L15 medium (L15 medium = 60 mg/liter imidazole, 15 mg/liter aspartic acid, 15 mg/liter glutamic acid, 15 mg/liter cystine, 5 mg/liter β-alanine, 2 mg/liter vitamin B12, 10 mg/liter inositol, 10 mg/liter choline-Cl, 0.5 mg/literdl-thioeticacid lipoic acid, 0.02 mg/liter biotin, 5 mg/liter p-aminobenzoic acid, 25 mg/liter fumaric acid, 0.4 mg/liter coenzyme A; pH 7.35) containing 50 ng/ml NGF and 0.3% methocel. The following day, 20 μl of liposomes containing different amounts of gel-eluted protein (24Bandtlow C.E. Schmidt M.F. Hassinger T.D. Schwab M.E. Kater S.B. Science. 1993; 259: 80-83Crossref PubMed Google Scholar) were applied to the explanted culture. Following 1 h of incubation at 37 °C, the explants were fixed in 4% paraformaldehyde sucrose/PBS solution. Growth cones were scored as being either spread or collapsed. The percentage of collapsed growth cones was then plotted against the concentration of the purified proteins added to the cultured explant. At least 50 growth cones were counted per explants, and for one condition, experiments were performed in duplicate. Data are the mean of three independent experiments. All purification steps were carried out at 4 °C. Inhibitory substrate activity of the obtained fractions was routinely determined by the 3T3 spreading or by the PC12 neurite outgrowth assay. Bovine spinal cord tissue (30 min post mortem frozen to −80 °C) was carefully cleaned by stripping off the meninges and cut into small pieces. Myelin was prepared by the method of Colmanet al. (44Colman D.R. Kraibich G. Frey A.B. Sabatini D.D. J. Cell Biol. 1982; 95: 598-608Crossref PubMed Scopus (340) Google Scholar). The obtained myelin was then extracted in extraction buffer (60 mm CHAPS, 100 mm Tris-Cl, pH 8.0, 10 mm EDTA buffer, pH 8.0, 2.5 mmiodacetamide, 1 mm phenylmethylsulfonyl fluoride, 0.1 μg/ml aprotinin, 1 μg/ml leupeptin, 1 μg/ml pepstatin A). To obtain spinal cord extract, the tissue was homogenized directly in CHAPS extraction buffer in a ratio of (1: 1; w: v). The homogenate was centrifuged twice at 100,000 × g (Kontron type: K50.13, fixed angle) for 1 h at 4 °C. The clear supernatant (extract) was immediately applied to a Q-Sepharose column (2.6 × 11.5 cm), equilibrated in buffer A (20 mm Tris-Cl, pH 8.0, 0.5% (w/v) CHAPS). Bound proteins were eluted with a five-bed volume linear gradient from 0 to 1 m NaCl in buffer A (100-ml gradient in 50 min). Active fractions containing bNI-220 eluted around 0.4 m NaCl and were pooled (q-pool 1) for subsequent application on a Superdex 200 (2.6 × 60 cm) column, equilibrated in buffer B (150 mm NaCl, 20 mm Tris-Cl, pH 8.0, 0.5% (w/v) CHAPS). Active fractions after gel filtration (s-pool 1) were separated by 6% SDS-PAGE (10 × 24 × 0.01 cm gel) under reducing conditions and low constant power (2 watts/gel) to a total of 2500 Vh. Bands and gel regions were identified after Coomassie Blue staining (0.1% w/v R250 in 50% methanol and 10% acetic acid), cut out, and extracted in 800 μl of gel elution buffer (0.5% (w/v) CHAPS, 20 mm Tris-Cl, pH 8.0, 10 mm EDTA, pH 8.0, 2.5 mm iodacetamide, 1 mmphenylmethylsulfonyl fluoride, 0.1 μg/ml aprotinin, 1 μg/ml leupeptin, 1 μg/ml pepstatin A) for at least 48 h at 4 °C (30Spillmann A.A. Amberger V.R. Schwab M.E. Eur. J. Neurosci. 1997; 9: 549-555Crossref PubMed Scopus (46) Google Scholar). The IN-1 neutralizable active gel-eluted material of several gels was re-run on a 10% SDS-polyacrylamide gel under reducing conditions, and stained with 0.1% (w/v) Coomassie Blue R250 in 50% methanol and 10% acetic acid. The 220-kDa band was cut out, and endoproteinase Lys-C digestion (1:50 molar ratio) was performed directly in the gel. The sample was acidified and applied to a reverse phase high performance liquid chromatography column, peptides were separated with a linear gradient (0–100%) of 0.04% trifluoroacetic acid and 80% acetonitrile, and fractions containing single peptide species were subjected to automated Edman degradation. High resolution SDS-PAGE was carried out using 6% (w/v) SDS-polyacrylamide gels (10 × 24 × 0.01 cm) under reducing conditions (100 mmdithiothreitol) according to the method of Laemmli (45Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (205531) Google Scholar). Transfer onto Immobilon-P membranes (Millipore) was performed in 20 mmTris base, 192 mm glycine, pH 8.3, 0.037% (w/v) SDS, 20% methanol (81Towbin H. Staehelin T. Gordon J. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 4350-4354Crossref PubMed Scopus (44644) Google Scholar) with a semidry transfer apparatus (Bio-Rad, Trans Blot SD). Transfer time was 2 h at 0.8 mA/cm2. Blocking reagent (1 h at room temperature) was 3% gelatin in PBS (phosphate-buffered saline, pH 7.2, 8 g NaCl, 0.2 g of KH2PO4, 2.8 g of Na2HPO4·12H2O, and 0.2 g of KCl, dissolved in 1 liter of water) and the washing solution contained 20 mm Tris-Cl, pH 7.5, 150 mm NaCl, and 0.4% Tween (3 × 10 min at room temperature). Incubation time for the first antibody (for dilution with 1% gelatin in PBS, see “Antibodies”) was usually overnight at 4 °C. Horseradish peroxidase-conjugated anti-mouse IgG secondary antibody (1:2000) was incubated for 1 h at room temperature. Finally, the ECL chemiluminescence system was used for detection (Amersham Pharmacia Biotech). Gel-eluted proteins from high resolution SDS-PAGE were precipitated by addition of a 9-fold excess of acetone (1 day at −20 °C), dried in a stream of nitrogen and solubilized in 10 μl of buffer that contained 2% w/v CHAPS, 0.1% w/v Triton X-100, 9 m urea, and 2% v/v ampholines (one-third volume pH 3–10, two-thirds volume pH 4–8; Millipore) (46Persson H. Overholm T. Electrophoresis. 1990; 11: 642-648Crossref PubMed Scopus (9) Google Scholar). Undissolved material was pelleted for 5 min at 13000 rpm in a microcentrifuge. The supernatant was applied to isoelectric focusing gels (3.5% acrylamide, 9 m urea, 2% w/v Triton X-100, 0.3 w/v CHAPS, and 6% v/v ampholines (1/3 vol. pH 3–10, 2/3 vol. pH 4–8; Millipore) in 5 cm 0.1 mm capillaries, (46Persson H. Overholm T. Electrophoresis. 1990; 11: 642-648Crossref PubMed Scopus (9) Google Scholar). The isoelectrically focused proteins were separated in the second dimension by 6% (w/v) SDS-PAGE (6 × 8 × 0.0075 cm) under reducing conditions (47Boxberg Y.V. Anal Biochem. 1988; 169: 372-375Crossref PubMed Scopus (24) Google Scholar). Gel-eluted fractions after high resolution SDS-PAGE as described above were precipitated by addition of a 9-fold excess of acetone (1 day at −20 °C), dried in a stream of nitrogen, and solubilized in 100 μl of buffer, which contained various concentrations of different detergents. Undissolved material was pelleted for 5 min at 13,000 rpm in a microcentrifuge. The supernatant was then tested in the PC12 neurite outgrowth assay. Urea was added to the gel eluted fractions to the desired final concentration. After incubation for 2 h at 37 °C, the solution was tested in the PC12 neurite outgrowth assay. Trypsin was added to gel-eluted fractions resulting in a final concentration of 0.1% (w/v), incubated for 1 min at 37 °C and then put on ice. As a control, either heat-inactivated trypsin (5 min, 100 °C) or trypsin inhibitor 0.2% (w/v) together with trypsin 0.1% (w/v) were used and processed in the same way. After centrifugation for 5 min at 13,000 rpm in a microcentrifuge, supernatants were analyzed in the PC12 neurite outgrowth assay and by SDS-PAGE. Gel-eluted fractions were incubated for 0, 5, or 30 min at 100 °C and then put on ice. After centrifugation for 5 min at 13,000 rpm in a microcentrifuge, supernatants were analyzed in the PC12 neurite outgrowth assay. To verify the presence of sugars in a possible glycoconjugate, the digoxigenin glycan detection kit from Boehringer Mannheim was used. The principle is as follows. Hydroxyl groups of sugars of glycoconjugates are oxidized to aldehyde groups by mild periodate treatment. Digoxigenin is then covalently attached to these aldehydes via a hydrazide group. Digoxigenin is detected in an enzyme immunoassay using a digoxigenin specific antibody conjugated with alkaline phosphatase. N-Glycosidase F treatment was performed according to the protocol of the manufacturer (Boehringer Mannheim). Briefly, samples were boiled for 5 min at 100 °C and cooled on ice. Then, 2 units ofN-glycosidase F (Boehringer) was added and the mixture was incubated for 14 h at 37 °C. Samples were incubated for 1 h at 37 °C in 0.1 m NaOH. 50 μl of gel-eluted fractions (∼0.4 μg of protein) were incubated with 20 milliunits of affinity-purified chondroitinase ABC (Sigma) in 40 mmTris-Cl, pH 8.0, for 3 h at 37 °C (48Watanabe E. Aono S. Matsui F. Yamada Y. Naruse I. Oohira A. Eur. J. Neurosci. 1995; 7: 547-554Crossref PubMed Scopus (37) Google Scholar) and then put on ice. After centrifugation for 5 min at 13,000 rpm in a microcentrifuge, supernatants were analyzed either by SDS-PAGE or in the PC12 neurite outgrowth assay. All data are expressed as means ± standard error of the mean (S.E.). Statistical analysis was performed according to the one-tailed, paired Student's ttest. Protein determination was carried out by the method of Bradford (49Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (211983) Google Scholar) or was estimated after silver-stained SDS-PAGE (50Merril C.R. Goldman D. Sedman S.A. Ebert M.H. Science. 1981; 211: 1437-1438Crossref PubMed Scopus (2085) Google Scholar). For the purification of the IN-1 antigen present in bovine CNS myelin, two culture assays in combination with the neutralizing mAb IN-1 were used. The first bioassay tests the effect of substrates on the spreading behavior of 3T3 fibroblasts, which has been shown previously to be strongly impaired by oligodendrocytes and CNS myelin (10Schwab M.E. Caroni P. J. Neurosci. 1988; 8: 2381-2393Crossref PubMed Google Scholar). The second bioassay analyzes substrate effects on the neurite outgrowth response of PC12 cells (29Rubin B.P. Spillmann A.A. Bandtlow C.E. Keller F. Schwab M.E. Eur. J. Neurosci. 1995; 7: 25" @default.
• W2007435025 created "2016-06-24" @default.
• W2007435025 creator A5000585501 @default.
• W2007435025 creator A5015489575 @default.
• W2007435025 creator A5041718533 @default.
• W2007435025 creator A5053830487 @default.
• W2007435025 creator A5057817479 @default.
• W2007435025 date "1998-07-01" @default.
• W2007435025 modified "2023-09-28" @default.
• W2007435025 title "Identification and Characterization of a Bovine Neurite Growth Inhibitor (bNI-220)" @default.
• W2007435025 cites W1480202189 @default.
• W2007435025 cites W1565947331 @default.
• W2007435025 cites W1599255496 @default.
• W2007435025 cites W1619042305 @default.
• W2007435025 cites W1668379041 @default.
• W2007435025 cites W1676877312 @default.
• W2007435025 cites W1876601469 @default.
• W2007435025 cites W1964834956 @default.
• W2007435025 cites W1966778303 @default.
• W2007435025 cites W1969642885 @default.
• W2007435025 cites W1971304382 @default.
• W2007435025 cites W1971697475 @default.
• W2007435025 cites W1973316814 @default.
• W2007435025 cites W1978704453 @default.
• W2007435025 cites W1985431192 @default.
• W2007435025 cites W1985783459 @default.
• W2007435025 cites W1990346699 @default.
• W2007435025 cites W1992601290 @default.
• W2007435025 cites W1993713261 @default.
• W2007435025 cites W1999035185 @default.
• W2007435025 cites W2002422337 @default.
• W2007435025 cites W2009191172 @default.
• W2007435025 cites W2012443200 @default.
• W2007435025 cites W2013035816 @default.
• W2007435025 cites W2014321075 @default.
• W2007435025 cites W2025778924 @default.
• W2007435025 cites W2025851321 @default.
• W2007435025 cites W2027474757 @default.
• W2007435025 cites W2029768920 @default.
• W2007435025 cites W2032304318 @default.
• W2007435025 cites W2036567890 @default.
• W2007435025 cites W2036750380 @default.
• W2007435025 cites W2040310971 @default.
• W2007435025 cites W2041700814 @default.
• W2007435025 cites W2047363293 @default.
• W2007435025 cites W2047762215 @default.
• W2007435025 cites W2050415048 @default.
• W2007435025 cites W2052003685 @default.
• W2007435025 cites W2057858723 @default.
• W2007435025 cites W2064093191 @default.
• W2007435025 cites W2065298253 @default.
• W2007435025 cites W2066337898 @default.
• W2007435025 cites W2072401558 @default.
• W2007435025 cites W2074830774 @default.
• W2007435025 cites W2076744147 @default.
• W2007435025 cites W2078316783 @default.
• W2007435025 cites W2078956426 @default.
• W2007435025 cites W2081870622 @default.
• W2007435025 cites W2083369535 @default.
• W2007435025 cites W2085893808 @default.
• W2007435025 cites W2091855895 @default.
• W2007435025 cites W2093425928 @default.
• W2007435025 cites W2100837269 @default.
• W2007435025 cites W2101108802 @default.
• W2007435025 cites W2103511872 @default.
• W2007435025 cites W2106973256 @default.
• W2007435025 cites W2107445843 @default.
• W2007435025 cites W2110773974 @default.
• W2007435025 cites W2116181102 @default.
• W2007435025 cites W2116858049 @default.
• W2007435025 cites W2119267047 @default.
• W2007435025 cites W2122301782 @default.
• W2007435025 cites W2124248841 @default.
• W2007435025 cites W2127810698 @default.
• W2007435025 cites W2147149841 @default.
• W2007435025 cites W2147786624 @default.
• W2007435025 cites W2148411419 @default.
• W2007435025 cites W2151630250 @default.
• W2007435025 cites W2152980841 @default.
• W2007435025 cites W2161154195 @default.
• W2007435025 cites W2166086027 @default.
• W2007435025 cites W2171946109 @default.
• W2007435025 cites W2345949243 @default.
• W2007435025 cites W2470301900 @default.
• W2007435025 cites W2470318754 @default.
• W2007435025 cites W4293247451 @default.
• W2007435025 doi "https://doi.org/10.1074/jbc.273.30.19283" @default.
• W2007435025 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/9668118" @default.
• W2007435025 hasPublicationYear "1998" @default.
• W2007435025 type Work @default.
• W2007435025 sameAs 2007435025 @default.
• W2007435025 citedByCount "154" @default.
• W2007435025 countsByYear W20074350252012 @default.
• W2007435025 countsByYear W20074350252013 @default.
• W2007435025 countsByYear W20074350252014 @default.
• W2007435025 countsByYear W20074350252015 @default.
• W2007435025 countsByYear W20074350252016 @default.
• W2007435025 countsByYear W20074350252017 @default.

• next