In vitro maturation of Drosophila melanogaster Spätzle protein with refolded Easter reveals a novel cleavage site within the prodomain
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Christian Ursel
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.
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Superscript residue numbers for Easter correspond to the chymotrypsin(ogen) numbering system for trypsin-like serine proteases (Shotton and Hartley, 1970)
The authors are grateful to Pia Rosenburg for technical assistance and Hauke Lilie and Andrea Sinz for valuable discussions. This work was supported by Deutsche Forschungsgemeinschaft (DFG) Grant SFB610 ‘Protein-Zustände mit Zellbiologischer und Medizinischer Relevanz’ to MTS.
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©2013 by Walter de Gruyter Berlin Boston
Articles in the same Issue
- Masthead
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- Guest Editorial
- Highlight: Protein states with cell biological and medicinal relevance
- HIGHLIGHT: PROTEIN STATES WITH CELL BIOLOGICAL AND MEDICAL RELEVANCE
- Towards improved receptor targeting: anterograde transport, internalization and postendocytic trafficking of neuropeptide Y receptors
- Progress in demystification of adhesion G protein-coupled receptors
- The unresolved puzzle why alanine extensions cause disease
- Molecular function of the prolyl cis/trans isomerase and metallochaperone SlyD
- Structure and allosteric regulation of eukaryotic 6-phosphofructokinases
- Polyionic and cysteine-containing fusion peptides as versatile protein tags
- p0071/PKP4, a multifunctional protein coordinating cell adhesion with cytoskeletal organization
- Lysine-specific histone demethylase LSD1 and the dynamic control of chromatin
- Methylation of the nuclear poly(A)-binding protein by type I protein arginine methyltransferases – how and why
- Oxidative in vitro folding of a cysteine deficient variant of the G protein-coupled neuropeptide Y receptor type 2 improves stability at high concentration
- Identification of prolyl oligopeptidase as a cyclosporine-sensitive protease by screening of mouse liver extracts
- In vitro maturation of Drosophila melanogaster Spätzle protein with refolded Easter reveals a novel cleavage site within the prodomain
- Subcellular localization and RNP formation of IGF2BPs (IGF2 mRNA-binding proteins) is modulated by distinct RNA-binding domains
- High level expression of the Drosophila Toll receptor ectodomain and crystallization of its complex with the morphogen Spätzle
Articles in the same Issue
- Masthead
- Masthead
- Guest Editorial
- Highlight: Protein states with cell biological and medicinal relevance
- HIGHLIGHT: PROTEIN STATES WITH CELL BIOLOGICAL AND MEDICAL RELEVANCE
- Towards improved receptor targeting: anterograde transport, internalization and postendocytic trafficking of neuropeptide Y receptors
- Progress in demystification of adhesion G protein-coupled receptors
- The unresolved puzzle why alanine extensions cause disease
- Molecular function of the prolyl cis/trans isomerase and metallochaperone SlyD
- Structure and allosteric regulation of eukaryotic 6-phosphofructokinases
- Polyionic and cysteine-containing fusion peptides as versatile protein tags
- p0071/PKP4, a multifunctional protein coordinating cell adhesion with cytoskeletal organization
- Lysine-specific histone demethylase LSD1 and the dynamic control of chromatin
- Methylation of the nuclear poly(A)-binding protein by type I protein arginine methyltransferases – how and why
- Oxidative in vitro folding of a cysteine deficient variant of the G protein-coupled neuropeptide Y receptor type 2 improves stability at high concentration
- Identification of prolyl oligopeptidase as a cyclosporine-sensitive protease by screening of mouse liver extracts
- In vitro maturation of Drosophila melanogaster Spätzle protein with refolded Easter reveals a novel cleavage site within the prodomain
- Subcellular localization and RNP formation of IGF2BPs (IGF2 mRNA-binding proteins) is modulated by distinct RNA-binding domains
- High level expression of the Drosophila Toll receptor ectodomain and crystallization of its complex with the morphogen Spätzle
