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J. Biol. Chem., Vol. 268, Issue 12, 8458-8465, 04, 1993
LC Wasley, A Rehemtulla, JA Bristol and RJ Kaufman
Factor IX is synthesized as a precursor polypeptide which requires
proteolytic cleavage of the propeptide for functional activity. Expression
of factor IX at high levels in Chinese hamster ovary (CHO) cells results in
the secretion of a mixture of profactor IX and mature factor IX. We have
studied whether the processing of profactor IX may be mediated by the
recently discovered subtilisin-like serine proteases PACE/furin and/or
PACE4. Co-transfection of a PACE expression vector with a profactor IX
expression vector resulted in the secretion of fully processed factor IX.
In contrast, co-transfection of a PACE4 expression vector with a profactor
IX expression vector did not increase processing of profactor IX to the
mature form. A factor IX Arg- to-Thr mutation at the P1 position (residue
39) destroyed the ability for PACE to process profactor IX. Amino-terminal
sequence analysis demonstrated that processing mediated by PACE occurred at
the authentic site within profactor IX. The specificity of profactor IX
processing by PACE was also evaluated by transfection of a vector encoding
the serine protease inhibitor alpha 1-antitrypsin. Expression of wild-type
alpha 1- antitrypsin, which does not inhibit PACE, did not influence
processing of profactor IX mediated by co-expression of PACE. In contrast,
the alpha 1-antitrypsin Pittsburgh mutant, which inhibits PACE, inhibited
profactor IX processing activity mediated by transfected PACE as well as
the endogenous CHO cell propeptide processing enzyme. Pulse-chase labeling
indicated that PACE processed profactor IX late within the secretory
pathway, although a secreted soluble mutant PACE was also capable of
processing profactor IX in the conditioned medium. The results implicate
PACE as a candidate for the enzyme that processes profactor IX in vivo.
PACE/furin can process the vitamin K-dependent pro-factor IX precursor within the secretory pathway
Genetics Institue, Cambridge, Massachusetts 02140-2387.
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