IDENTIFICATION AND CHARACTERIZATION OF A BOVINE NEURITE GROWTH INHIBITOR (BNI-220)

The poor axonal regeneration that follows lesions of the central nervo us system (CNS) is crucially influenced by the local CNS tissue enviro nment through which neurites have to grow. In addition to an inhibitor y role of the glial scar, inhibitory substrate effects of CNS myelin a nd 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 prope rties in vitro. Antibodies raised against NI-35/250 (monoclonal antibo dy IN-1) were shown to partially neutralize the growth inhibitory effe ct of CNS myelin and oligodendrocytes, and to result in long distance! fiber regeneration in the lesioned adult mammalian CNS in vivo, We re port here the purification of a myelin protein to apparent homogeneity from bovine spinal; cord which exerts a potent neurite outgrowth inhi bitory effect on PC12 cells and chick dorsal root ganglion cells, indu ces 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 proc edure 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 kD a and an isoelectric point between 5.9 and 6.2, Its inhibitory activit y is sensitive to protease treatment and resists harsh treatments like 9 M urea or short heating, Glycosylation is, if present at all, not d etectable. Microsequencing resulted ill six peptides and strongly sugg ests that this proteins is novel.