Sunu Budhi Raharjo

Molecular Identification and Characterization of Novel Membrane-bound Metalloprotease, the Soluble Secreted Form of Which Hydrolyzes a Variety of Vasoactive Peptides

One class of zinc metalloproteases, represented by neutral endopeptidase 24.11 and endothelin-converting enzyme, has been shown to be involved in proteolytic activation or inactivation of many regulatory peptides. Here, we report molecular cloning and characterization of a novel member of this type II membrane-bound metalloprotease family, termed soluble secreted endopeptidase (SEP). Alternative splicing results in the generation of another transcript, SEPΔ, which lacks a 69-base pair nucleotide segment following the transmembrane helix. Both SEP and SEPΔmRNA are detected in all mouse tissues examined. Transfection of an SEP cDNA expression construct resulted in the expression of the membrane-bound form of SEP in the early secretory pathway as well as the soluble secreted form of the enzyme in the culture medium. In contrast, transfection of the SEPΔ cDNA only results in the expression of the membrane-bound form. In vitroenzymological analysis of the recombinant soluble form of SEP demonstrated that it hydrolyzes a variety of vasoactive peptides, including endothelin-1, atrial natriuretic peptide, and angiotensin I. This activity of SEP was inhibited by phosphoramidon and the neutral endopeptidase 24.11 specific inhibitor thiorphan, but it was only partially inhibited by the endothelin-converting enzyme specific inhibitor FR901533. These findings suggest that SEP is a novel metalloprotease that possesses a broad substrate specificity and that it may be involved in the metabolism of biologically active peptides intracellulary as well as extracellularly.

Dual ECE/NEP Inhibition on Cardiac and Neurohumoral Function During the Transition From Hypertrophy to Heart Failure in Rats

CGS 26303 is a vasopeptidase inhibitor that simultaneously inhibits endothelin-converting enzyme (ECE) and neutral endopeptidase (NEP). We compared the effects of chronic treatment with CGS 26303 to the selective inhibition of angiotensin-converting enzyme (ACE) and NEP during the transition from left ventricular hypertrophy (LVH) to congestive heart failure (CHF) in hypertensive rats. LV geometry and function were assessed in Dahl salt-sensitive rats placed on a high-salt diet from age 6 weeks (hypertensive rats) and in control rats fed a low-salt diet. The hypertensive rats were randomized into groups that received no treatment or were treated with an ACE inhibitor (temocapril), an ECE/NEP inhibitor (CGS 26303), or a NEP inhibitor (CGS 24592) from the LVH stage (11 weeks) to the CHF stage (17 weeks). All treatments decreased the systolic blood pressure equally and significantly improved LV fractional shortening. Both temocapril and CGS 26303 ameliorated LV perivascular fibrosis, reduced mRNA levels of types I and III collagen, and decreased the heart weight/body weight ratio. CHF rats had increased plasma ET-1 levels compared with control rats. Only CGS 26303 reduced ET-1 to normal levels. ET-1 levels were found to correlate with heart/body weight, right ventricle/body weight and perivascular fibrosis ratios. During the transition to CHF, CGS 26303 produces effects that are comparable to temocapril and superior to CGS 24592. The beneficial effects of CGS 26303 are likely caused in part by the greater reduction of plasma ET-1. Dual ECE/NEP inhibitor may provide a new strategy for the treatment of human heart failure.

Alternative Splicing Regulates the Endoplasmic Reticulum Localization or Secretion of Soluble Secreted Endopeptidase

A subfamily of zinc metalloproteases, represented by Neutral endopeptidase (EC 3.4.24.11) and endothelin-converting enzyme, is involved in the metabolism of a variety of biologically active peptides. Recently, we cloned and characterized a novel member of this metalloprotease family termed soluble secreted endopeptidase (SEP), which hydrolyzes many vasoactive peptides. Here we report that alternative splicing of the mouseSEP gene generates two polypeptides, SEPΔ and SEP. After synthesis, both isoforms are inserted into the endoplasmic reticulum (ER) as type II membrane proteins. SEPΔ then becomes an ER resident, whereas SEP, which differs by only the presence of 23 residues at the beginning of its luminal domain, is proteolytically cleaved by membrane secretase(s) in the ER and transported into the extracellular compartment. An analysis of the chimeric proteins between SEPΔ and bovine endothelin-converting enzyme-1b (bECE-1b) demonstrated that the retention of SEPΔ in the ER is mediated by the luminal domain. In addition, the dissection of the chimeric bECE-1b/SEP insertion showed that its insertion domain is obviously responsible for its secretion. A series of mutagenesis in this region revealed that the minimal requirement for cleavage was found to be a WDERTVV motif. Our results suggest that the unique subcellular localization and secretion of SEP proteins provide a novel model of protein trafficking within the secretory pathway.

The effects of phosphoramidon on the expression of human endothelin-converting enzyme-1 (ECE-1) isoforms.

&NA; Endothelin‐1 (ET‐1) is generated from big ET‐1 by endothelin converting enzyme‐1 (ECE‐1). This process is inhibited by phosphoramidon through binding to the catalytic domain of ECE‐1. There are four isoforms of human ECE‐1 (ECE‐1a, ECE‐1b, ECE‐1c and ECE‐1d) which possess a conserved catalytic domain. Interestingly, a recent study has shown that in ECE‐1b‐transfected CHO cells phosphoramidon increases the expression and activity of ECE‐1b. It is not known, however, whether phosphoramidon has similar effects on the expression of other ECE‐1 isoforms. To address this point, we have established recombinant CHO cell lines that permanently express either human ECE‐1a, ECE‐1b or ECE‐1c. Incubation of CHO/ECE‐1a, ‐1b, and ‐1c with phosphoramidon (100 &mgr;M) for 16 hours markedly elevated the intracellular expression of ECE‐1a and ECE‐1b, but not ECE‐1c protein, as indicated by Western blotting and immunocytochemistry. These increases appear to be due to inhibition of intracellular degradation of the protein because metabolic labeling followed by immunoprecipitation showed ECE‐1a and ECE‐1b proteins had prolonged half‐lives in the phosphoramidon‐treated cells. This is further supported by the finding that ECE‐1 mRNA levels were unchanged following phosphoramidon treatment. Taken together, our results demonstrate that phosphoramidon differentially affects the expression of three human ECE‐1 isoforms.