Abstract
Production of a variety of regulatory eukaryotic proteins, such as growth factors and polypeptide hormones, often involves endoproteolytic processing of proproteins at cleavage sites consisting of paired basic residues. The first known mammalian proprotein processing enzyme with such specificity is the human fur gene product furin. Structurally and functionally, furin is related to the subtilisin-like serine endoprotease kexin (EC 3.4.21.61) of yeast Saccharomyces cerevisiae; unlike kexin, it contains a cysteine-rich region with an unknown function. Here, we describe cloning and sequencing of a 5.8-kbp cDNA of the Dfur2 gene, a fur-like gene of Drosophila melanogaster, which we found expressed during various stages of development. This Dfur2 cDNA has an open reading frame for a 1680-residue protein, called Dfurin2. Dfurin2 contains similar protein domains as mammalian furin, however, it has an extended amino-terminal region and its cysteine-rich region is much larger than that of mammalian furin. Because of this latter phenomenon, we were able to identify a particular cysteine motif that was repeated multiple times in Dfurin2 but present only twice in mammalian furin. Furthermore, we show that Dfur2 encodes an endoproteolytic enzyme with specificity for paired basic amino acid residues as, in cotransfection experiments, correct cleavage was demonstrated of the precursor of the von Willebrand factor but not of a cleavage mutant. Finally, Dfur2 was mapped to region 14C of the X chromosome of D. melanogaster.
Highlights
From the $Laboratoryfor Molecular Oncology, Center for Human Genetics, Universityof Leuven, Herestraat 49, B-3000 Leuuen, Belgium, the §Institutefor Genetics, Johannes Gutenberg University, Saarstrasse 256,500 Mainz I, Germany, and the Womputer-assisted Organic SynthesislComputer-assistedMolecular Modelling Center, Faculty of Science, Universityof Nijmegen, Toernooiueld, 6525 ED Nijmegen, The Netherlands
We describe cloning coding such proprotein processing enzymes has opened a way and sequencing of a 5.8-kbp cDNA of the Dfur2gene, to study the protein domains that are relevant for the subcela fur-like gene of Drosophila melanogaster, which we lular distributionof these enzymes and implicated in substrate found expressedduring various stageosf development
We examined expression of the Dfurin2 mRNA in different developmental stages of D. melanogaster and assayed the proprotein processular probe, the amount and quality of the RNA samples on the blot were checked by rehyhridization with dhPR21, a genomic ribosomal DNA sequence from Drosophila hydei [35]
Summary
TGA replaced by glycine (R-S-K-G763)w, ere not processed by furin. At present, the furin proteins of mammalian species such as human [12, 17], mouse [18], and rat [19] and the amphibian Xenopus laeuis [20] have been characterized, and their deduced amino acid sequences are very similar. The open readingframe for Dfurin in the mammaliansubtilisin-likeproprotein processing enzymes were identified instudiesin which the polymerase chain reaction technology was used. These novel enzymes are PC1 ( called PC3 and,referred to hereafter as PC1/ PC3) and PC2 [21,22,23,24,25,26]. The known mammalian furins and Dfurinlof D. melanogaster After hybridization with the composite Dfur cDNA pIP67 asmolecdescribed earlier by us [31], protein features of the deduced Dfurin protein, among which an extremely large cysteinerich region, could be predicted. Recently discovered subtilisin-like proprotein processing en- Polymerase Chain Reaction-To fill in a gap between the cDNA zymes.
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