Cloning and functional expression of Dfurin2, a subtilisin-like proprotein processing enzyme of Drosophila melanogaster with multiple repeats of a cysteine motif.
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.
Roebroek
A J
AJ
Laboratory for Molecular Oncology, University of Leuven, Belgium.
Creemers
J W
JW
Pauli
I G
IG
Kurzik-Dumke
U
U
Rentrop
M
M
Gateff
E A
EA
Leunissen
J A
JA
Van de Ven
W J
WJ
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D003545:Cysteine / Q000235:genetics*; D004331:Drosophila melanogaster / Q000201:enzymology* / Q000235:genetics*; D015870:Gene Expression; D006801:Humans; D007447:Invertebrate Hormones / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D016133:Polymerase Chain Reaction; D011499:Protein Processing, Post-Translational; D012091:Repetitive Sequences, Nucleic Acid; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000235:genetics; D012689:Sequence Homology, Nucleic Acid; D013381:Subtilisins / Q000235:genetics* / Q000378:metabolism
D007447:Invertebrate Hormones; D009838:Oligodeoxyribonucleotides; C076273:dfurin2; D013381:Subtilisins; D003545:Cysteine