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
Summary
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
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