Expression of the Myelin‐Associated Glycoprotein in Cultures of Immortalized Schwann Cells

Abstract

Although the myelin-associated glycoprotein (MAG) cannot be detected in primary cultures of rat Schwann cells in the absence of neurons, MAG expression was demonstrated in some lines of cultured Schwann cells that had been immortalized by repetitive passaging. Radioimmunoassay of one such Schwann cell line, S-16, showed a remarkably high MAG concentration of about 1 ng/microgram of total protein, a level that is comparable to the MAG concentration in adult sciatic nerve. The S-16 cells divide very rapidly, are rounder than normal Schwann cells, and elaborate many processes after reaching high density. The cells are galactocerebroside positive, but express little or no P0 glycoprotein or myelin basic protein. As in nerve, the MAG synthesized by the cultured cells is primarily the shorter isoform (S-MAG). Furthermore, the posttranslational processing resembles that occurring in vivo including a similar degree of glycosylation, sulfation of oligosaccharides, and phosphorylation of the polypeptide. The sensitivity of MAG to treatment of the intact cells with trypsin or neuraminidase, as well as surface labeling with [3H]borohydride reduction after periodate oxidation, demonstrated that most of the MAG expressed by the S-16 cells is located on the cell surface. This line of immortalized Schwann cells expressing a remarkably high level of MAG should be useful for investigating the cell biology and function of this glycoprotein.

SOX10 regulates an alternative promoter at the Charcot-Marie-Tooth disease locusMTMR2

“…PMP22, MPZ, and SOX10) (22)(23)(24). Briefly, we cloned each genomic segment upstream of a minimal promoter directing the expression of a firefly luciferase reporter gene (25).…”

“…Immortalized rat Schwann cells (S16) (22), mouse spinal motor neurons (MN-1) (26), and HeLa cells were grown under standard conditions in DMEM with 10% fetal bovine serum, 2mM L-glutamine, 50 U/ml penicillin, and 50 g/ ml streptomycin. For luciferase assays, $1Â10 4 S16 or MN-1 cells were plated in each well of a 96-well plate.…”

Ceramide displaces cholesterol from lipid rafts and decreases the association of the cholesterol binding protein caveolin-1

“…The basic premise of SILAC is that two populations of cells are grown in medium containing either unlabeled amino acids or an amino acid containing deuterium or 13 C substitutions. After a suffi-cient incubation period to completely incorporate the labeled amino acid into the cellular proteome, one population of the cells serves as a control and the other is subjected to treatment.…”

Evidence for Regulation of Myelin Protein Synthesis by Contact between Adjacent Schwann Cell Plasma Membranes

“…Immortalized S16 Schwann cells [3,4] were grown on 150-mm dishes (Falcon) in DMEM supplemented with 10 % fetal bovine serum and 50 Ìg/ml of gentamycin at a density of more than 3.4 ! 10 4 cells/cm 2 .…”

“…They include interactions with the axon and basal lamina, rate of cell division, growth factors, cAMP levels and transcription factors [1,2]. Immortalized Schwann cell lines (S16 and S42) expressing myelin-associated glycoprotein (MAG), PO glycoprotein and galactocerebroside have been generated in our laboratory [3] and in many ways biochemically resemble Schwann cells at an early stage of differentiation toward myelin formation [4]. Previous examination of the cells under different conditions of growth showed that high levels of MAG, PO and their messenger RNAs correlated with decreased proliferation brought about by serum deprivation and with increased cell density [5].…”