Heterologous in vivo processing of human preproendothelin 1 into bioactive peptides.

Abstract

Endothelin (ET) is an extremely potent vasoconstrictor peptide of 21 amino acids, originally found in the supernatant of cultured vascular endothelial cells. To gain insights into its biosynthetic pathway, we expressed a synthetic RNA coding for the 212-amino acid precursor of human ET-1 (preproET-1) in Xenopus oocytes. Cell homogenates and oocyte incubation medium were tested by RIA using an anti-ET-1 serum. ET-1-like immunoreactivity was detected in oocytes njected with preproET-1 synthetic RNA but not in control oocytes and was much higher in medium than in cell homogenates. When preproET-1 was expressed in oocytes treated with monensin, a dramatic decrease in secretion of immunoreactive material was observed, indicating that secretion is mediated by the Golgi complex. ET-1-like immunoreactive material present in oocyte incubation medium was fractionated by reverse-phase HPLC into two main peaks, corresponding to the retention times of human big ET-1 and ET-1. Incubation medium of oocytes expressing the synthetic preproET-1 RNA elicited a characteristic vasoconstrictor response on rabbit vena cava, consistent with the biological activity that would be predicted from the amount of ET-1-like immunoreactivity measured.These results suggest that common pathways of ET maturation exist in widely different cells and that Xenopus oocytes may represent a useful tool in studying the cell biology of ET-1 synthesis.Endothelin (ET), a 21-residue peptide, is one of the most potent vasoconstrictors known (1). In addition to its cardiovascular effects, recent studies indicate that ET could also act as a neurotransmitter or neurosecretory hormone (2). The regulation of ET biosynthesis in mammalian tissues is largely unknown. Interestingly, the peptide sequence shows a high degree of homology to the N terminus of a-scorpion neurotoxins (2) and particularly to the snake sarafotoxins that affect the heart (3), suggesting that similar mechanisms of synthesis and processing of the precursors could have survived in organisms as distant as mammals and reptiles. Sequencing of a human preproET-1 cDNA clone revealed that ET originates from a 212-amino acid precursor (4). The first 17 amino acids showed the sequence characteristics of a signal peptide for the translocation into the endoplasmic reticulum. Human ET spans the region from Cys53 to Trp73 and a 38-amino acid intermediate, termed big ET-1, has a 17-amino acid extension after Trp73. A possible pathway of ET biosynthesis has been proposed (1). In this pathway, the precursor is cleaved at a dibasic pair of residues and further processed by a specific ET converting enzyme that cleaves between Trp2l and Val22 of the last intermediate big

In vivo expression of mutant preproendothelins: hierarchy of processing events but no strict requirement of Trp-Val at the processing site.

“…This activity is intracellular, since synthetic big ET-1 added to the medium of Xenopus oocytes is fully stable (5). In the present study we have also provided evidence that before this enzyme can act to form ET-1, big ET-1 must be cleaved from proET-1.…”

“…Many efforts have been made to search for a specific endothelinconverting enzyme (ECE) responsible for the cleavage of the unusual Trp-Val site of big ET-1 as a key element in the regulation of ET-1 biosynthesis (3,4). However, expression of human preproET-1 in Xenopus oocytes (5) or insect cells (6) results in constitutive secretion of peptides with immunological and biological characteristics of ET-1 and big ET-1.…”

“…PCR was performed according to the manufacturer's instructions (Perkin-Elmer/Cetus). All mutagenized sequences were controlled by sequencing them before they were cloned into the vector psp64T (9) by the same strategy used to obtain "wild-type" CNR1 (5). In vitro transcription with SP6 RNA polymerase was essentially as described (10).…”

Two preproendothelin 1 mRNAs transcribed by alternative promoters.

“…In the last three years, many attempts have been focused to understand the mechanisms controlling the processing of its inactive precursor. Recently, we reported that human preproET-1 follows the constitutive secretory pathway and undergoes posttranslational polypeptide maturation when expressed in Xenopus oocytes (21 ). We therefore suggested that regulation of ET-1 secretion occurs mainly at transcriptional level.…”

“…The existence of an endothelin-converting enzyme cleaving bigET-1 into mature 2 1-amino acid peptide ET-I has been postulated ( 1 ). Recently we demonstrated that human preproET-1 is fully processed by microinjected Xenopus oocytes (21 ) and recombinant baculovirus-infected insect cells (22), suggesting that common pathways of preproET-1 maturation exist among different cells. Moreover, ultrastructural analysis demonstrates that endothelial cells do not possess substantial numbers of secretory granules (23).…”