Inactivation of proprotein convertase, PACE4, by alpha 1-antitrypsin Portland (alpha 1-PDX), a blocker of proteolytic activation of bone morphogenetic protein during embryogenesis: Evidence that PACE4 is able to form an SDS-stable acyl intermediate with alpha 1-PDX

PACE4 (SPC4), a member of the subtilisin-like proprotein convertase (SPC) f amily of proteases that cleave at paired basic amino acids, exhibits a dyna mic expression pattern during embryogenesis and colocalizes with bone morph ogenetic proteins (BMPs), Recently Cui et at. reported that the ectopic exp ression of alpha 1-antitrypsin variant Portland (alpha 1-PDX), an engineere d serpin that contains the minimal SPC consensus motif in its reactive loop , blocks the proteolytic activation of BMP4, leading to abnormal embryogeni c development [Cui, Y, et at (1998) EMBO J, 17, 4735-4743], TGF beta-relate d factors such as BMPs are synthesized as inactive precursors and activated by limited proteolysis at multibasic amino acids. Therefore, an alpha 1-PD X-inhibitable protease is thought to participate in BMP activation, However , conflicting properties, including sensitivity to alpha 1-PDX, have been r eported for PACE4, In this study, we examined whether alpha 1-PDX is respon sible for the inhibition of PACE4 by measuring the protease/inhibitor compl ex directly. Here we show that alpha 1-PDX has the ability to form an SDS-s table acyl-intermediate (180 kDa) with PACE4 in vivo and in vitro. Further, we characterized the PACE4 secreted into the culture medium from Cos-1 cel ls by a specific immunological assay. An alpha 1-PDX-insensitive and decano yl-RVKR-chloromethylketone-sensitive 60-kDa protease(s) is greatly activate d in conditioned medium by PACE4 overexpression, suggesting that the activa tion of an unknown protease(s) other than PACE4 is the cause of the variati on in the properties of PACE4, PACE4 is a Ca2+-dependent protease with an o ptimal Ca2+ requirement of 2 mM, and shows its highest activity at weakly b asic pH, PACE4 activity is completely inhibited by EDTA and EGTA, but not b y leupeptin, These results show that PACE4 activity can be inhibited by alp ha 1-PDX as well as furin (SPC1) and suggest that the inhibition of PACE4-m ediated activation of factors such as BMPs by (alpha 1-PDX causes abnormal embryogenic development.