Human preproendothelin-1 is converted into active endothelin-1 by baculovirus-infected insect cells.
To investigate biochemical and biological parameters involved in preproendothelin-1 (preproET-1) maturation we infected Spodoptera frugiperda (Sf21) cells with a suitable engineered baculovirus vector carrying the cDNA encoding the entire human 212 amino acids precursor. Culture supernatants were tested by RIA using an anti-ET-1 serum, ET-1-like immunoreactive material (IRM) was detected in the infected Sf21 cells but not in control, wild-type or mock-infected cells. Fractionation of the culture supernatant by RP-HPLC coupled to an ET-1 specific RIA yielded two main peaks corresponding to the retention times of human bigET-1 and ET-1. Furthermore, culture supernatant of preproET-1 expressing Sf21 cells elicited a characteristic dose-response vasoconstrictive activity on rabbit vena cava, consistent with the amount of ET-1 as estimated by RP-HPLC coupled to RIA. These results suggest that insect cells possess the enzymatic activities necessary for human preproET-1 full maturation even though no such peptide has ever been found in insect cells.
Benatti
L
L
Biotechnology Department, Farmitalia Carlo Erba Research Laboratories, Nerviano, Italy.
Cozzi
L
L
Zamai
M
M
Tamburin
M
M
Vaghi
F
F
Caiolfa
V R
VR
Fabbrini
M S
MS
Sarmientos
P
P
D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D001483:Base Sequence; D001681:Biological Assay; D002460:Cell Line; D019332:Endothelin-1; D016232:Endothelins / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D005822:Genetic Vectors; D006801:Humans; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D009036:Moths; D009119:Muscle Contraction / Q000187:drug effects; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000502:physiology; D009838:Oligodeoxyribonucleotides; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism*; D011817:Rabbits; D014162:Transfection; D014661:Vasoconstriction / Q000187:drug effects; D014684:Venae Cavae / Q000187:drug effects / Q000502:physiology
D019332:Endothelin-1; D016232:Endothelins; D009838:Oligodeoxyribonucleotides; D011498:Protein Precursors