Characterization of a membrane-bound metalloendoprotease of rat C6 glioblastoma cells.
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 neurons 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 characterize 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.
Amberger
V R
VR
Brain Research Institute, University of Zurich, Switzerland.
Paganetti
P A
PA
Seulberger
H
H
Eldering
J A
JA
Schwab
M E
ME
D000595:Amino Acid Sequence; D000818:Animals; D001253:Astrocytes / Q000473:pathology; D002455:Cell Division / Q000187:drug effects; D005909:Glioblastoma / Q000737:chemistry* / Q000378:metabolism / Q000473:pathology; D008565:Membrane Proteins / Q000737:chemistry; D008666:Metalloendopeptidases / Q000032:analysis*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009185:Myelin Proteins / Q000494:pharmacology; D009363:Neoplasm Proteins / Q000032:analysis*; D015260:Neprilysin / Q000032:analysis; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D051381:Rats; D014407:Tumor Cells, Cultured
D008565:Membrane Proteins; D009185:Myelin Proteins; D009363:Neoplasm Proteins; D010446:Peptide Fragments; D008666:Metalloendopeptidases; D015260:Neprilysin