Abstract

Endothelin-converting enzyme-1 (ECE-1) cleaves big endothelins, as well as bradykinin and beta-amyloid peptide. Several isoforms of ECE-1 (a-d) have been identified to date; they differ only in their NH(2) terminus but share the catalytic domain located in the COOH-terminal end. Using quantitative PCR, we found ECE-1d to be the most abundant type in several endothelial cells (EC) types. In addition to full-length ECE-1 forms we have identified novel, alternatively spliced mRNAs of ECE-1 b-d. These splice variants (SVs) lack exon 3', which codes for the transmembrane region and is present in full-length forms. SVs mRNA were highly expressed in EC derived from macro and microvascular beds but much less so in other, non-endothelial cells expressing ECE-1, which suggests that the splicing mechanism is cell-specific. Analyses of ECE-1d and its SV form in stably transfected HEK-293 cells revealed that both proteins were recognized by anti COOH-terminal ECE-1 antibodies, but anti NH(2)-terminal antibodies only bound ECE-1d. The novel protein, designated ECE-1 sv, has an apparent molecular mass of 75 kDa; by using site-directed mutagenesis its start site was identified in a region common to all ECE-1 forms suggesting that ECE-1 b-d SV mRNAs are translated into the same protein. In agreement with the findings demonstrating common COOH terminus for ECE-1sv and ECE-1d, both exhibited a similar catalytic activity. However, immunofluorescence staining and differential centrifugation revealed a distinct intracellular localization for these two proteins. The presence of ECE-1sv in different cellular compartments than full-length forms of the enzyme may suggest a distinct physiological role for these proteins.

Highlights

  • Endothelin-converting enzyme-1 (ECE-1)2 is a type II membrane protease that belongs to the neprilysin (NEP) family of zinc metallopeptidases [1, 2]

  • The mRNA levels of forms b and c were expressed in all three cell types, but the relative expression level of 1c was higher in BAEC than in the two luteal endothelial cells (EC) types

  • The mRNA of isoforms ECE-1b and -c were rather scarce, with levels 6 –10-fold lower than that of ECE-1d. These were not the only ECE-1 forms found in EC, we have identified novel splice variants of ECE-1b, -c, and -d mRNA, which lacked exon 3Ј that codes for the TM domain of the enzyme

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Summary

EXPERIMENTAL PROCEDURES

Materials—Dulbecco’s minimum essential medium (DMEM) low glucose, DMEM with Ham’s F-12 1:1 (v/v) nutrient mixture, SuperScriptII RNase H-reverse transcriptase, calf serum, and ultra pure electrophoresis agarose gel were obtained from Invitrogen. Type I collagen from Cohesion Technologies (Palo Alto, CA). Penicillin, streptomycin, and fetal calf serum were from Biological Industries

A Novel Alternatively Spliced Variant of ECE-1
RESULTS
DISCUSSION
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