Purification and characterization of furin, a Kex2-like processing endoprotease, produced in Chinese hamster ovary cells.
Furin, a mammalian homolog of the yeast Kex2 protease, is associated with Golgi membranes and is involved in cleavage of precursor proteins at sites marked by the Arg-X-Lys/Arg-Arg (RXK/RR) motif. We have recently shown that a furin mutant lacking the transmembrane domain can be secreted from cDNA-transfected cells with proteolytic activity for the fluorogenic peptide t-butoxycarbonyl-Arg-Val-Arg-Arg-4-methylcoumarin-7- amide. In this study, we purified and characterized the recombinant furin from the conditioned medium of these cells. Furin was purified as a mixture of 83- and 81-kDa forms and a 96-kDa form. The differences in molecular mass were not due to differences in molecular mass were not due to differences in glycosylation. Moreover, all forms had the same NH2-terminal sequence beginning at the residue after the Arg-Ala-Lys-Arg sequence. These data suggest that the three different forms may be produced by differential COOH-terminal processing of a furin molecule and that mature furin may be autocatalytically produced. Both enzyme preparations showed a pH optimum at 7.0, required Ca2+ for the activity, and showed essentially the same inhibitor profile. These properties resembled those of the Kex2 protease. Both preparations efficiently cleaved fluorogenic peptides with an RXK/RR sequence and moderately cleaved a peptide with an RXXR sequence, but did not cleave dibasic peptides. The sequence requirements determined in vitro were compatible with those determined by expression studies in cultured cells. These data unequivocally demonstrate that furin is an endogenous cellular protease responsible for cleavage of precursor proteins mainly at RXK/RR sites.
Hatsuzawa
K
K
Institute of Applied Biochemistry, University of Tsukuba, Ibaraki, Japan.
Nagahama
M
M
Takahashi
S
S
Takada
K
K
Murakami
K
K
Nakayama
K
K
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D015153:Blotting, Western; D016466:CHO Cells; D002852:Chromatography, Ion Exchange; D006224:Cricetinae; D004591:Electrophoresis, Polyacrylamide Gel; D045683:Furin; D006801:Humans; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010957:Plasmids; D043484:Proprotein Convertases; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D012689:Sequence Homology, Nucleic Acid; D012697:Serine Endopeptidases / Q000378:metabolism*; D013379:Substrate Specificity; D013381:Subtilisins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D014162:Transfection
D029701:Saccharomyces cerevisiae Proteins; D011505:Protein-Tyrosine Kinases; D043484:Proprotein Convertases; D012697:Serine Endopeptidases; D013381:Subtilisins; C058751:KEX2 protein, S cerevisiae; D045683:Furin