Inactivation of proprotein convertase, PACE4, by alpha1-antitrypsin Portland (alpha1-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 alpha1-PDX.
PACE4 (SPC4), a member of the subtilisin-like proprotein convertase (SPC) family of proteases that cleave at paired basic amino acids, exhibits a dynamic expression pattern during embryogenesis and colocalizes with bone morphogenetic proteins (BMPs). Recently Cui et al. reported that the ectopic expression of alpha1-antitrypsin variant Portland (alpha1-PDX), an engineered serpin that contains the minimal SPC consensus motif in its reactive loop, blocks the proteolytic activation of BMP4, leading to abnormal embryogenic development [Cui, Y. et al. (1998) EMBO J. 17, 4735-4743]. TGFbeta-related factors such as BMPs are synthesized as inactive precursors and activated by limited proteolysis at multibasic amino acids. Therefore, an alpha1-PDX-inhibitable protease is thought to participate in BMP activation. However, conflicting properties, including sensitivity to alpha1-PDX, have been reported for PACE4. In this study, we examined whether alpha1-PDX is responsible for the inhibition of PACE4 by measuring the protease/inhibitor complex directly. Here we show that alpha1-PDX has the ability to form an SDS-stable acyl-intermediate (180 kDa) with PACE4 in vivo and in vitro. Further, we characterized the PACE4 secreted into the culture medium from Cos-1 cells by a specific immunological assay. An alpha1-PDX-insensitive and decanoyl-RVKR-chloromethylketone-sensitive 60-kDa protease(s) is greatly activated in conditioned medium by PACE4 overexpression, suggesting that the activation of an unknown protease(s) other than PACE4 is the cause of the variation in the properties of PACE4. PACE4 is a Ca(2+)-dependent protease with an optimal Ca(2+) requirement of 2 mM, and shows its highest activity at weakly basic pH. PACE4 activity is completely inhibited by EDTA and EGTA, but not by leupeptin. These results show that PACE4 activity can be inhibited by alpha1-PDX as well as furin (SPC1) and suggest that the inhibition of PACE4-mediated activation of factors such as BMPs by alpha1-PDX causes abnormal embryogenic development.
Tsuji
A
A
Department of Biological Science and Technology, Faculty of Engineering, The University of Tokushima, Tokushima, 770-8506, Japan.
Hashimoto
E
E
Ikoma
T
T
Taniguchi
T
T
Mori
K
K
Nagahama
M
M
Matsuda
Y
Y
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D001483:Base Sequence; D055415:Bone Morphogenetic Protein 4; D019485:Bone Morphogenetic Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D019556:COS Cells; D002118:Calcium / Q000378:metabolism; D002846:Chromatography, Affinity / Q000379:methods; D017077:Culture Media, Conditioned; D017931:DNA Primers; D005314:Embryonic and Fetal Development; D006868:Hydrolysis; D043484:Proprotein Convertases; D011480:Protease Inhibitors / Q000494:pharmacology; D051381:Rats; D012697:Serine Endopeptidases / Q000187:drug effects* / Q000276:immunology / Q000378:metabolism; D012967:Sodium Dodecyl Sulfate; D013379:Substrate Specificity; D000515:alpha 1-Antitrypsin / Q000494:pharmacology*
D000911:Antibodies, Monoclonal; C525811:Bmp4 protein, rat; D055415:Bone Morphogenetic Protein 4; D019485:Bone Morphogenetic Proteins; D017077:Culture Media, Conditioned; D017931:DNA Primers; D011480:Protease Inhibitors; D000515:alpha 1-Antitrypsin; C113421:alpha 1-antitrypsin Portland; D012967:Sodium Dodecyl Sulfate; C513087:Pcsk6 protein, rat; D043484:Proprotein Convertases; D012697:Serine Endopeptidases; D002118:Calcium