title: The Dfur2 gene of Drosophila melanogaster: genetic organization, expression during embryogenesis, and pro-protein processing activity of its translational product Dfurin2.
abstract: The gene structure and expression of the Dfur2 gene of Drosophila melanogaster, which encodes the subtilisin-like serine endoprotease Dfurin2, was studied. The Dfur2 gene is very compact in contrast to the related Dfur1 gene, which has an estimated size of over 100 kbp. The 6-kb Dfur2 mRNA is encoded by 16 exons dispersed over a genomic region of about 9 kbp. The exon/intron organization shows conservation of intron positions not only in comparison with Dfur1, but also with the related mammalian genes FUR, PC1/PC3, PC2, and PC4. This conservation supports the hypothesis that all genes belonging to the family of subtilisin-like pro-protein processing enzymes are evolutionary related by descent from a common ancestral gene. In primer extension experiments, Dfur2 transcription initiation sites were identified in the presumed Dfur2 promoter region. This region was found to contain general RNA polymerase II promoter elements like a potential TATA box, a potential CAP signal, and several potential CCAAT boxes. Also, several sequence motifs putatively corresponding to binding sites for Drosophila transcription factors like zeste, bicoid, and engrailed were found to be present. RNA in situ hybridization experiments on Drosophila embryos revealed presumably maternal Dfur2 expression until the syncytial blastoderm (stage 5 of embryogenesis), no expression during gastrulation (stage 9), transient expression in a subset of neurons in the central nervous system of stage 12-13 embryos, and, from stage 13 onwards, expression in the developing tracheal tree. In a vaccinia expression system, the endoprotease Dfurin2 not only cleaved wild-type precursor of von Willebrand factor (pro-vWF) with pro-region cleavage site R-S-K-R decreases, but also, although to a lesser extent, pro-vWF mutants in which the P2 (vWFK-2A) or P4 (vWFR-4A) basic residue with respect to the pro-region cleavage site had been mutated. This cleavage specificity resembles that of human furin. The cleavage of pro-vWF by Dfurin2 shows that the previously reported lack of cleavage of the precursor of the beta A-chain of activin-A by Dfurin2 in this vaccinia expression system is substrate determined.
authors.affiliation: Laboratory for Molecular Oncology, University of Leuven, Belgium.
authors.forename: A J
authors.initials: AJ
authors.lastname: Roebroek
authors.forename: T A
authors.initials: TA
authors.lastname: Ayoubi
authors.forename: J W
authors.initials: JW
authors.lastname: Creemers
authors.forename: I G
authors.initials: IG
authors.lastname: Pauli
authors.forename: W J
authors.initials: WJ
authors.lastname: Van de Ven
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D004331:Drosophila melanogaster / Q000196:embryology* / Q000201:enzymology / Q000235:genetics*; D004625:Embryo, Nonmammalian / Q000201:enzymology; D005091:Exons; D015870:Gene Expression; D018507:Gene Expression Regulation, Developmental; D017344:Genes, Insect; D007438:Introns; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D011498:Protein Precursors / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D013381:Subtilisins / Q000235:genetics* / Q000378:metabolism; D014841:von Willebrand Factor / Q000378:metabolism*
chemical_list: D011498:Protein Precursors; D014841:von Willebrand Factor; C067003:von Willebrand factor propolypeptide; C076273:dfurin2; D013381:Subtilisins