In vivo expression of mutant preproendothelins: hierarchy of processing events but no strict requirement of Trp-Val at the processing site.
Endothelin-1 (ET-1), a 21-residue vasoconstrictor peptide, originates in human cells from a 212-amino acid precursor (preproET-1). Big ET-1, an intermediate form of 38 amino acids, is generated by cleavage at basic-pair residues of proET-1, while a specific "ET-converting enzyme" was proposed to process the unusual Trp-Val site at positions 21 and 22 of big ET-1. We have previously shown that expression of synthetic RNA encoding human preproET-1 in Xenopus oocytes results in secretion of putative ET-1 and big ET-1. Here, to further dissect the processing pathway of preproET-1, we designed and expressed in oocytes a set of preproET-1 mutants. Four mutants affecting the Trp-Val site always originated putative ET-1(s) at levels comparable to the wild type, suggesting that there is only a conformational requirement for cleavage at this site. An Arg-->Ile mutation at the basic-pair site after the C terminus of big ET-1 fully inhibited the formation of both big ET-1 and ET-1, indicating that processing at this site is an early event and that big ET-1 is an obligate intermediate for the synthesis of ET-1 in vivo. Also, a truncated mutant bearing a stop codon after the C terminus of the big ET-1 sequence was totally stable and further processed into mature big ET-1 and ET-1, indicating that the second part of the precursor is not necessary for maturation.
Fabbrini
M S
MS
Istituto Biosintesi Vegetali, Consiglio Nazionale delle Ricerche, Milan, Italy.
Vitale
A
A
Pedrazzini
E
E
Nitti
G
G
Zamai
M
M
Tamburin
M
M
Caiolfa
V R
VR
Patrono
C
C
Benatti
L
L
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D003062:Codon / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D019332:Endothelin-1; D016232:Endothelins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D005260:Female; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D009865:Oocytes / Q000378:metabolism; D011498:Protein Precursors / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D011499:Protein Processing, Post-Translational; D011863:Radioimmunoassay; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D014158:Transcription, Genetic; D014981:Xenopus
D003062:Codon; D019332:Endothelin-1; D016232:Endothelins; D009838:Oligodeoxyribonucleotides; D011498:Protein Precursors; D011994:Recombinant Proteins