Neurostatin was originally described as an inhibitor of astroblast and astrocytoma division present in rat brain extracts and immunologically related to the sugar moiety of epidermal growth factor receptor and to blood group antigens. It was purified recently from mammalian brain extracts and characterized as a glycosphingolipid, but its precise structure remained unknown. Neurostatin has now been purified to apparent homogeneity from ganglioside extracts of rat, bovine, and porcine brain. It is cytostatic for astroblasts, C6 glioma cells, and various human astrocytomas grades III and IV, with IC 50 values ranging from 250 to 450 nM, but does not affect the division of primary or transformed fibroblasts up to concentrations Ͼ4 M. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry of purified pig neurostatin showed a molecular ion of 1,905 Da and ions of 1,863 and 1,934 Da, compatible with a disialoganglioside. Mono-and bidimensional NMR spectra, together with biochemical studies, suggest that neurostatin may be the 9-O-monoacetyl ester of GD1b. Key Words: Astrocytosis-Antimitotic-Growth inhibition-Tumoral growth. J. Neurochem. 74, 2547Neurochem. 74, -2556Neurochem. 74, (2000.The number of astrocytes in mammalian brain remains stationary throughout adulthood (Sturrock, 1974;Korr, 1986), probably thanks to the concomitant presence of specific mitogens and mitogen inhibitors (NietoSampedro et al., 1985). Definite evidence for the existence in rat brain of specific astroblast mitogen inhibitors was presented by Nieto-Sampedro (1988) and confirmed by Nieto-Sampedro and Broderick (1989). The inhibitor, which had epitopes in common with both the carbohydrate moiety of the epidermal growth factor (EGF) receptor and with human blood groups (Nieto-Sampedro, 1988;Nieto-Sampedro and Broderick, 1989), was recently purified from rat and bovine brain extracts (AbadRodríguez et al., 1998). It was a very-low-abundance ganglioside that in reference to its source and biological activity was called neurostatin. Neurostatin and its synthetic oligosaccharide analogues inhibited the division of astrocytes, glioma, and neuroblastoma cells in culture (Nieto-Sampedro, 1988;Santos-Benito et al., 1992) and promoted the destruction in vivo of an experimental rat brain glioma (Nieto-Sampedro et al., 1996).Determination of the precise structure of neurostatin required the purification of comparatively large amounts of the molecule. The fractionation of larger amounts of brain tissue was facilitated by preparation of brain ganglioside extracts (Tettamanti et al., 1973). Neurostatin was then purified from the extracts using a combination of conventional and high-performance ion-exchange chromatographies.The purification to homogeneity reported here permitted the determination of the inhibitor's structure. Combined bidimensional NMR, matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS), and biochemical studies permitted us to conclude that neurostatin is O-acetylated G...
