phosphoramidon and Glioma

Deposition of beta-amyloid (Abeta) peptides in the brain is an early and invariant feature of all forms of Alzheimer's disease. As with any secreted protein, the extracellular concentration of Abeta is determined not only by its production but also by its catabolism. A major focus of Alzheimer's research has been the elucidation of the mechanisms responsible for the generation of Abeta. Much less, however, is known about the mechanisms responsible for Abeta removal in the brain. In this report, we describe the identification of endothelin-converting enzyme-1 (ECE-1) as a novel Abeta-degrading enzyme. We show that treatment of endogenous ECE-expressing cell lines with the metalloprotease inhibitor phosphoramidon causes a 2-3-fold elevation in extracellular Abeta concentration that appears to be due to inhibition of intracellular Abeta degradation. Furthermore, we show that overexpression of ECE-1 in Chinese hamster ovary cells, which lack endogenous ECE activity, reduces extracellular Abeta concentration by up to 90% and that this effect is completely reversed by treatment of the cells with phosphoramidon. Finally, we show that recombinant soluble ECE-1 is capable of hydrolyzing synthetic Abeta40 and Abeta42 in vitro at multiple sites.

Topics: Alzheimer Disease; Amino Acid Sequence; Amyloid beta-Peptides; Animals; Aspartic Acid Endopeptidases; Cell Line; CHO Cells; Chromatography, High Pressure Liquid; Cloning, Molecular; Cricetinae; Endothelin-Converting Enzymes; Enzyme-Linked Immunosorbent Assay; Glioma; Glycopeptides; Humans; Hydrolysis; Metalloendopeptidases; Molecular Sequence Data; Tumor Cells, Cultured

Endothelin-converting enzyme is a phosphoramidon-sensitive metalloprotease that cleaves big endothelin to the potent vasoconstrictor peptide, endothelin. The converting enzyme is expressed in endothelial cells in a variety of tissues and in some secretory cells. In the present study, phosphoramidon-sensitive endothelin-converting enzyme activity has been demonstrated by radioimmunoassay in the neuroblastoma cell line, SH-SY5Y, and in Bu17 and C6 glioma lines. The identity of the activity was confirmed by immunoblotting, revealing a polypeptide of approximately 120 kDa in each of these lines, in D384 glioma cells, and in primary astrocytes. Immunofluorescence revealed the cell-surface location of endothelin-converting enzyme in the neuronal and glial cell lines and in primary astrocytes. Pretreatment of SH-SY5Y and Bu17 cells with phosphoramidon resulted in an apparent concentration of the enzyme protein in an intracellular compartment. Immunoperoxidase-staining of rat brain sections located this metalloprotease to the pyramidal cells of the hippocampus. Endothelin-converting enzyme-1 was revealed by in situ hybridisation in the neuronal and glial cell lines.

Topics: Animals; Antibodies, Monoclonal; Aspartic Acid Endopeptidases; Astrocytes; CD13 Antigens; Cell Membrane; Cells, Cultured; Cloning, Molecular; DNA, Complementary; Endothelin-Converting Enzymes; Fluorescent Antibody Technique; Gene Expression Regulation, Enzymologic; Glial Fibrillary Acidic Protein; Glioma; Glycopeptides; Golgi Apparatus; Hippocampus; Immunoenzyme Techniques; In Situ Hybridization; Metalloendopeptidases; Neprilysin; Neuroblastoma; Neuroglia; Protease Inhibitors; Rats; RNA, Messenger; Tumor Cells, Cultured