title: Secretion and maturation study of endothiapepsin in Saccharomyces cerevisiae. A first step toward improving its substrate specificity.
abstract: The gene encoding endothiapepsin (EAP), an extracellular aspartic proteinase from the filamentous ascomycete Cryphonectria parasitica, was expressed into Saccharomyces cerevisiae. Efficient secretion of an active and correctly processed enzyme was achieved when expressing the entire cDNA encoding prepro-EAP under the control of the galactose-inducible GRAP1 yeast promoter. Since three independent, site-directed mutations of EAP, including the substitution of an aspartyl catalytic residue, resulted in the intracellular accumulation of zymogen forms, we assumed that the EAP propeptide was autocatalytically processed. As a prerequisite to further improve the specificity of EAP, we therefore attempted to bypass this self-processing step in three different ways: 1) introduction of a Kex2-like recognition site between the pro and the mature part, 2) deletion of the prosequence (pre-EAP), and 3) co-expression in trans of the pre-EAP with its preprosequence. No improvement in the secretion of mutant enzymes was obtained in any of these experiments. As an alternative, we finally replaced the EAP processing site by the chymosin cleavage sequence of kappa-casein. Such a modification remained efficient in directing the secretion of active EAP only when a putative alpha-helix structural motif was conserved at the C terminus of the pro region.
authors.affiliation: Sanofi Recherche, Centre de Labège, Labège Innopole, BP 137, France.
authors.forename: V
authors.initials: V
authors.lastname: Valverde
authors.forename: P
authors.initials: P
authors.lastname: Delmas
authors.forename: M
authors.initials: M
authors.lastname: Kaghad
authors.forename: G
authors.initials: G
authors.lastname: Loison
authors.forename: P
authors.initials: P
authors.lastname: Jara
mesh_terms: D000595:Amino Acid Sequence; D016282:Aspartic Acid Endopeptidases / Q000235:genetics / Q000502:physiology*; D001483:Base Sequence; D008969:Molecular Sequence Data; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013379:Substrate Specificity
chemical_list: D016282:Aspartic Acid Endopeptidases; C042466:Endothia aspartic proteinase