Purification and characterization of glia maturation factor beta: a growth regulator for neurons and glia.

Abstract

A protein has been isolated from bovine brains by using a modification of the procedure used to purify glia maturation factor. The method consists of ammonium sulfate precipitation, chromatography with DEAE-Sephacel, Sephadex G-75, and hydroxylapatite columns, passage through a heparin-Sepharose column, and rinally fractionation by reverse-phase HPLC with a C4 column. The isolated protein reacts strongly with the mouse monoclonal antibody G2-09 and has a molecular weight of =17,000 and an isoelectric point of pH 4.9. The N terminus is blocked, but tryptic digestion releases 28 peptides, 8 of which have been sequenced. The total known residues add up to more than two-thirds of the entire 140-residue protein, estimated from amino acid composition, and show no sequence homology with any known protein.Reversible thermal renaturation greatly enhances its biological activity. The purified protein stimulates differentiation of normal neurons as well as glial cells. It inhibits the proliferation of the N-18 neuroblastoma line and the C6 glioma line while promoting their phenotypic expression. We designate this protein glia maturation factor f.The trophic function of the nervous system on peripheral organs was detected in an in vivo system as early as 1823 (1). The in vitro counterpart of this experiment was conducted in 1939 (2-4), showing the mitogenic effect of brain homogenates on cultured fibroblasts. However, it was not until 1972t that an autoregulatory role of brain-derived growth factors on brain cells was first demonstrated in this laboratory (5,6). This factor was named glia maturation factor (GMF), based on the bioassay system in which the activity was first observed. Since then this laboratory has engaged in an intensive search for the molecule responsible for this important function. The effort culminated in the isolation of a brain protein with a unique amino acid sequence documented below. MATERIALS AND METHODSPreliminary Purification of GMF-j3. The published procedure (7, 8) through the Sephadex G-75 step was followed with slight modifications. Briefly, four beef brains [1.0 kg (total wet weight)] were homogenized and centrifuged to obtain the crude extract. The ammonium sulfate precipitate between 45% and 70% saturation was dissolved in 100 ml of water and dialyzed for two 6-hr periods against 10 liters of water. The sample was adjusted to contain 0.02 M Tris HCl (pH 7.45) and applied to a DEAE-Sephacel column (2.5 x 37 cm). After eluting with 1.25 liters of a linear gradient of 0-0.3 M NaCl in the same buffer at 50 ml/hr, the fractions that showed mitogenic and morphologic activities on astrocytes were pooled (500 ml) and concentrated to 50 ml by Amicon PM10 filtration. The sample was applied to a Sephadex G-75 column (5 x 90 cm) and eluted with 0.15 M NaCl containing the above buffer at 40 ml/hr. The fractions active on astrocytes were pooled (500 ml) and used for the final steps of purification (see Results).Bioassay Production of Antibodies and Immunoassay. The mouse monoclonal antibody G2-0...

Glia maturation factor overexpression in neuroblastoma cells activates glycogen synthase kinase-3β and caspase-3

“…Glia maturation factor (GMF), a 17 kDa highly conserved protein, with 99% homology between humans and rodents, was isolated, sequenced and cloned in our laboratory (Kaplan et al, 1991;Lim et al, 1989;Zaheer et al, 1993). The GMF gene is localized to the long arm of human chromosome 14 (Lander et al, 2001).…”

“…The Glia Maturation Factor (GMF), a highly conserved brain-specific protein, was isolated, sequenced and cloned in our laboratory (Kaplan et al, 1991;Lim et al, 1989;Lim et al, 1990;Zaheer et al, 1993;Zaheer et al, 1995a;Zaheer et al, 1995b). The functional property of GMF was largely unknown.…”

Diminished degradation of myelin basic protein by anti-sulfatide antibody and interferon-γ in myelin from glia maturation factor-deficient mice

“…This event apparently thought to be the result of a combined autoimmune response to some of the myelin components (Glynn and Linington 1989;Bernard et al 1997;Steinman and Zamvil 2005;Gold et al 2006). Research efforts in recent years on glia maturation factor (GMF), a highly conserved brain-specific protein, isolated, sequenced and cloned in our laboratory (Lim et al 1989;Lim et al 1990;Kaplan et al 1991;Zaheer et al 1993), have demonstrated an immunomodulatory function for GMF. Recently, it has been established that overexpression of GMF in astrocytes leads to immune activation of microglia through secretion of granulocyte-macrophage-colony stimulating factor (GM-CSF) (Zaheer et al J. Neurochem.…”

Antiproliferative function of glia maturation factor beta.

“…normal (Similar curves were obtained using immortalized Schwann ( Figure 4B). The morphologic effect of GMF-,B on C6 cells, consisting of cell elongation, has been published (Lim et al, 1989) (Lim et al, 1976;Bosch et al, 1984). Schwannoma and glioma cells responded to the same preparation by an initial surge in cell division when the cells were sparse, but exhibited contact inhibition when the culture became confluent (Lim et al, 1981(Lim et al, , 1986 Figure 6.…”

“…Because of the presence of other growth factors in the brain, the question arose as to whether the mitogenicity was indeed an intrinsic function of GMF or a result of contamination. Our purification effort led to the isolation of a 17-kDa acidic protein designated GMF-,B (Lim et al, 1989). The protein has now been sequenced (Lim et al, 1990b), cloned, and expressed in E. cofi (Kaplan et al, 1990).…”