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Membrane peptidase expression in mammalian cell lines

Membrane peptidase expression in mammalian cell lines
Brendan James Walkden


Department of Biochemistry and Molecular Biology, University of Leeds, Leeds LS2 9JT, UNITED KINGDOM.


Endothelin-1 (ET-1) is a potent vasoconstrictive peptide which is produced by the cleavage of the inactive precursor, big ET-1 at the Trp21-Val22 bond by endothelin converting enzyme (ECE). ECE has been found to be expressed by a variety of cell types and tissues.

Using the human endothelial cell line, EA.hy926, ECE has been demonstrated, by analogy to the related protein, E-24.11 to be an integral transmembrane protein which is not anchored by a G-PI moiety. The relationship between E-24.11 and ECE was further investigated by screening of antibodies raised to E-24.11 for cross-reactivity with the ECE protein. Immobilised GK4A9, a monoclonal antibody to E-24.11 was found to immunoprecipitate >80 % of the total E-24.11 and ECE activity from Triton X-100 solubilised membrane preparations of EA. hy926 cells. The immunoprecipitation was reproduced using a transfected cell line which expressed high levels of ECE activity but was shown not to express E-24.11. Immunoprecipitation of ECE from transfected cells was confirmed by enzyme assay and immunoblotting. The immunoreactivity of GK4A9 was shown to be specific for ECE and E-24.11 suggesting a shared epitope between the two proteins. These data were consistent with the hypothesis that E-24.11 and ECE originated from the same protein family, sharing significant structural and sequence homology.

The cell specific expression of ECE was demonstrated by radioimmunoassay in the neuroblastoma cell line, SH-SY5Y, and the Bu17, C6 and D384 glioma lines. The identity of the activity was confirmed by immunoblotting which revealed a polypeptide of approximately 120 kDa in each of these cell lines.

Pre-treatment of EA. hy926, Bu17 and SH-SYSY cells with the zinc metalloprotease inhibitor phosphoramidon (PR) resulted in an accumulation of the ECE protein. This increase was reproduced by treatment of cells with thiorphan and suggests the possible involvement of a novel PR and thiorphan-sensitive metalloprotease in the turnover of ECE.

Other possible regulatory mechanisms involved in ECE production were investigated. The protein tyrosine phosphatase inhibitor vanadate was found to cause a 2-3-fold upregulation of ECE in EA. hy926 and Bu17 cells and appeared to induce ECE expression in rat pheochromocytoma PC12 cells which had been stimulated to differentiate to a neuronal phenotype in response to vanadate treatment.

The identification of two distinct human isoforms of ECE-1, α and ß (Schmidt et al.,1994; Shimada et al., 1995) which were found to differ only in N-terminal amino acid sequence allowed the generation of antibodies specific for each isoform. Peptides corresponding to immunogenic regions of the N-terminal sequence in each isoform were synthesised attached to a carrier protein and used to raise anti-peptide antibodies. The immunoreactivity of the antiserum was monitored using an E.L.I.S.A. with the peptide as the antigen. The most immunoreactive antiserum was affinity purified and the antibody characterised by detection of each ECE-1 isoform using transfected cell lines expressing high levels of each isoform. One antibody (ECE1a I was successfully raised to ECE-1 α and found to be specific for this isoform.