PACE FURIN CAN PROCESS THE VITAMIN-K-DEPENDENT PRO-FACTOR-IX PRECURSOR WITHIN THE SECRETORY PATHWAY

Factor IX is synthesized as a precursor polypeptide which requires pro teolytic cleavage of the propeptide for functional activity. Expressio n of factor IX at high levels in Chinese hamster ovary (CHO) cells res ults in the secretion of a mixture of profactor IX and mature factor I X. We have studied whether the processing of profactor IX may be media ted by the recently discovered subtilisin-like serine proteases PACE/f urin and/or PACE4. Co-transfection of a PACE expression vector with a profactor IX expression vector resulted in the secretion of fully proc essed factor IX. In contrast, co-transfection of a PACE4 expression ve ctor with a profactor IX expression vector did not increase processing of profactor IX to the mature form. A factor IX Arg-to-Thr mutation a t the P1 position (residue 39) destroyed the ability for PACE to proce ss profactor IX. Amino-terminal sequence analysis demonstrated that pr ocessing mediated by PACE occurred at the authentic site within profac tor IX. The specificity of profactor IX processing by PACE was also ev aluated by transfection of a vector encoding the serine protease inhib itor alpha1-antitrypsin. Expression of wild-type alpha1-antitrypsin, w hich does not inhibit PACE, did not influence processing of profactor IX mediated by co-expression of PACE. In contrast, the alpha1-antitryp sin Pittsburgh mutant, which inhibits PACE, inhibited profactor IX pro cessing activity mediated by transfected PACE as well as the endogenou s CHO cell propeptide processing enzyme. Pulse-chase labeling indicate d 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.