Novel Secretory Heparin-binding Factors from Human Glioma Cells (glia-activating Factors) Involved in Glial Cell Growth. Purification and Biological Properties

Growth factors for rat primary glial cells were identified in conditioned medium of a human glioma-derived cell line. The factors, designated glia-activating factors (GAFs), were purified to homogeneity by a combination of heparin affinity chromatography, gel filtration, and high performance liquid chromatography on a heparin affinity column and a C4 reversed-phase column. GAFs could be resolved into three peaks by C4 column chromatography. The M(r) values of these three proteins were estimated to be 30,000, 29,000, and 25,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. These M(r) values were in good agreement with the value of 26,000 +/- 3,000 estimated from the elution volume upon gel filtration chromatography under nondenaturing conditions. These data suggested that each of the GAFs consists of a single polypeptide chain and has no subunit structures. These three purified GAFs had almost the same growth-stimulating effect on glial cells in vitro, and the half-maximal dose was around 10(-11) M. Concanavalin A staining and glycopeptide N-glycosidase treatment of GAFs indicated that an asparagine-linked oligosaccharide chain(s) was attached to these three kinds of GAFs. Microsequencing of each GAF revealed a single amino-terminal sequence with no significant homology to any known protein, and the amino-terminal sequence of the 30-kDa GAF included that of the 29-kDa GAF. GAFs also stimulated the cell growth of oligodendrocyte type 2 astrocyte progenitor cells, BALB/c3T3 fibroblasts, and PC-12 cells but not that of human umbilical vein endothelial cells.