Title: Modulation of furin-mediated proprotein processing activity by site-directed mutagenesis
Authors: Creemers, John
Siezen, R J
Roebroek, Anton
Ayoubi, T A
Huylebroeck, Danny
Van de Ven, Willem # ×
Issue Date: Nov-1993
Series Title: Journal of Biological Chemistry vol:268 issue:29 pages:21826-21834
Abstract: The proprotein processing activity of mutants of the subtilisin-like enzyme furin was studied in transfected mammalian cells. Our studies indicate that the three residues of the catalytic triad of furin, Asp46, His87, and Ser261, are critical not only for substrate processing but also for maturation of furin. Furthermore, evidence is provided that maturation of furin occurs through an intramolecular autocatalytic process. Substitution of the asparagine residue (Asn188) of the oxyanion hole by an alanine residue appears to block substrate processing but not furin maturation. Analysis of carboxyl-terminal deletion mutants revealed that the segment encompassing residues Glu449 to Glu469 of the "middle" domain, which is more than 100 residues downstream of the predicted catalytic domain, contains residues that seem to be critical for processing activity but that the more carboxyl-terminal cysteine-rich region, the transmembrane region, and the cytosolic tail are dispensable. Finally, we made mutants in the substrate binding region of human furin and studied their ability to process von Willebrand factor (pro-vWF) substrates, including wild-type pro-vWF as well as pro-vWF mutants in which the P1 (vWFR-1G), P2 (vWFK-2A), or P4 (vWFR-4A) basic residue with respect to the pro region cleavage site had been mutated. It is demonstrated that particular negatively charged residues in or near the substrate binding region of furin are critical for cleavage activity and specificity of the enzyme for multiple basic residues in the substrate. Furthermore, substrate binding region mutants of furin were obtained, which cleaved either the pro-vWFK-2A or pro-vWFR-4A mutant of pro-vWF more efficiently than wild-type pro-vWF.
ISSN: 0021-9258
Publication status: published
KU Leuven publication type: IT
Appears in Collections:Molecular Biology (Celgen) (-)
Molecular Genetics Section (-)
Department of Human Genetics - miscellaneous
× corresponding author
# (joint) last author

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