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Characterization of a Membrane-bound Metalloendoprotease of Rat C6 Glioblastoma Cells¹

Brain Research Institute, University of Zurich, August-Forel-Str. 1, 8029 Zurich [V. R. A., P. A. P., H. S., M. E. S.], and Institute for Biochemistry and Molecular Biology, University of Berne, 3001 Bern [J. A. E.], Switzerland

C6 rat glioblastoma cells are able to attach to and to spread on culture dishes which are coated with purified central nervous system myelin, in contrast to normal astrocytes, fibroblasts or neuronas which adhere poorly and are unable to spread on this substrate. The metalloprotease blockers o-phenanthroline and a newly developed oligopeptide could specifically inhibit C6 cell spreading on central nervous system myelin, suggesting a crucial role for a metalloprotease. Here we characteriz this metalloproteolytic activity of C6 cells using a peptide degradation assay with the iodinated tetrapeptide carbobenzoxy-Phe-Ala-Phe-¹²⁵I-Tyr-amide as a substrate. Purified, salt-washed C6 plasma membranes cleaved the peptide between alanine and phenylalanine, an effect which is strongly inhibited by o-phenanthroline, but not by thiol-blocking agents or aspartic and serine protease inhibitors. The metalloendoprotease is highly sensitive to phosphoramidon but insensitive to thiorphan. The enzyme is tightly bound to the plasma membrane but not G protein-phosphatidylinositol linked. It can be solubilized in part by the detergents 3-(3-cholamidopropyldimethylamino)-1-propanesulfonate or Triton X-114. Gel filtration chromatography using the Triton X-114-solubilized proteins or the proteins removed by a short trypsin treatment revealed a molecular weight range for the C6 enzyme of 60,000–100,000. Polymerase chain reaction with primers corresponding to endopeptidase 24.11 or to the highly conserved motif of the "astacin family" showed that both enzymes were not detectable in the C6 glioblastoma cells.

¹ This study was supported by the Swiss National Science Foundation (Grant 31-29981.90), the Cancer Foundation of the Kanton of Zurich, the Slack-Gyr-Foundation (Zurich), and Regeneron Pharmaceuticals, Inc., Tarrytown, NY.

² To whom requests for reprints should be addressed.

³ Present address: Sandoz Forschungsinstitut AG Bern, 3001 Bern, Switzerland.

⁴ Present address: BASF, Abteilung ZHB, 6700 Ludwigshafen, Germany.

Received 1/31/94. Accepted 5/31/94.

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