title: In vitro maturation of Drosophila melanogaster Spätzle protein with refolded Easter reveals a novel cleavage site within the prodomain.
abstract: Dorsoventral patterning during Drosophila melanogaster embryogenesis is mediated by a well-defined gradient of the mature NGF-like ligand Spätzle. Easter, the ultimate protease of a ventrally-restricted serine protease cascade, plays a key role in the regulation of the morphogenic gradient, catalyzing the activation cleavage of proSpätzle. As a result of alternative splicing, proSpätzle exists in multiple isoforms, almost all of which differ only in their prodomain. Although this domain is unstructured in isolation, it has a stabilizing influence on the mature cystine knot domain and is involved in the binding to the Toll receptor. Here, we report the expression and refolding of Easter, and show that the renatured enzyme performs the activation cleavage of two Spätzle isoforms. We determine the affinity of the prodomain for the cystine knot domain, and show that Easter performs a previously unknown secondary cleavage in each prodomain.
authors.affiliation: Institut für Biochemie und Biotechnologie, Martin-Luther- Universität Halle-Wittenberg, Kurt-Mothes-Strasse 3, D-06120 Halle Saale, Germany
authors.forename: Christian
authors.initials: C
authors.lastname: Ursel
authors.forename: Uwe
authors.initials: U
authors.lastname: Fandrich
authors.forename: Anita
authors.initials: A
authors.lastname: Hoffmann
authors.forename: Torsten
authors.initials: T
authors.lastname: Sieg
authors.forename: Christian
authors.initials: C
authors.lastname: Ihling
authors.forename: Milton T
authors.initials: MT
authors.lastname: Stubbs
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D029721:Drosophila Proteins / Q000737:chemistry / Q000378:metabolism*; D004331:Drosophila melanogaster / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D020033:Protein Isoforms / Q000737:chemistry / Q000378:metabolism; D058849:Protein Refolding; D017434:Protein Structure, Tertiary; D012697:Serine Endopeptidases / Q000737:chemistry / Q000378:metabolism*
chemical_list: D029721:Drosophila Proteins; D020033:Protein Isoforms; C086179:spz protein, Drosophila; D012697:Serine Endopeptidases; C078099:ea protein, Drosophila