Characterization and partial purification of a plasma protein that induces rat cerebral type 2 astroglia from bipotential glial progenitors, or, Ode to a glial cell's differentiation

Studies from many laboratories have demonstrated that fetal bovine serum (FBS) contains a factor that induces cultured bipotential glial precursors known as O-2A progenitors to become type 2 astroglia rather than oligodendroglia. The goal of this research project was to characterize and purify this factor, which I will refer to as the astroglia inducing molecule (AIM). Using an indirect enzyme immunoassay for the astroglial marker glial fibrillary acidic protein I have determined that AIM is present in human and bovine sera, and that because of its high specific activity, fetal bovine serum qualifies as the best serum source of AIM. Evidence is presented that AIM is a protein and that it may be a plasma glycoprotein. Applying an ammonium sulfate precipitate of FBS to heparin, then lentil lectin agarose, followed by gel filtration chromatography produced an AIM preparation that was enriched 240 fold. Gel filtration chromatography on an ammonium sulfate precipitate of serum revealed activity at approximately 50 kilodaltons (kDa). However, with enrichment, activity was seen at several molecular masses, all of which were approximate multiples of 50 kDa. Since AIM activity was shifted to much higher molecular mass proteins after exposure to low pH's, and re-chromatographing a high molecular mass AIM in the presence of the chaotropic agent guanidine hydrochloride shifted AIM to an average molecular mass between 12 and 18 kDa, it is likely that AIM forms higher molecular mass aggregates during the purification procedure. On a preparative isoelectric focusing gel AIM activity most frequently migrated between the pH's of 3 and 4; however, proteins with isoelectric points at 6 and greater than 9 also had activity in several experiments. While the data support the existence of multiple AIMs, I postulate that this heterogeneity may be due to the association of a 12-18 kDa AIM with a carrier protein. Finally, double-label immunofluorescence for the lineage markers GD{dollar}sb3{dollar} and A2B5 confirmed that AIM preparations induce type 2 astroglia from bipotential glial progenitors, and that AIM appears to have much less of an effect on the type 1 astroglial lineage. Since none of the known growth factors that have been tested to date mimic its effects, AIM may be a novel differentiation factor.