was responsible for all cell cultures. or P8 (Lys191) positions. Notably, the thrombin-resistant R221A mutant is still cleaved by these PCs, revealing that convertase cleavage can precede thrombin activation. This conclusion was supported by the fact that the APC-specific activity in the medium of COS-1 cells is exclusively dependent on prior cleavage by the convertases, because both R198A and R221A lack protein C activity. Primary cultures of hepatocytes derived from wild-type or hepatocyte-specific furin, PC5/6, or complete PACE4 knock-out mice suggested that the cleavage of overexpressed proprotein C is predominantly performed by furin intracellularly and by all three proprotein convertases at the cell surface. Indeed, plasma analyses of single-proprotein convertase-knock-out mice showed that loss of the convertase furin or PC5/6 in hepatocytes results in a 30% decrease in APC levels, with no significant contribution from PACE4. We conclude that prior convertase cleavage of protein C in hepatocytes is critical for its thrombin activation. to = 0, 1, 2, or 3 spacer amino acids (aa); boldface and underlined basic residues critical for the recognition by proprotein convertases) occurring in the constitutive secretory pathway: (3), their inactivation in mice leads to specific phenotypes revealing that, and assay for the protein C activity, we further demonstrated that conversion of protein C to its active APC form by thrombin requires a prior cleavage by convertases at KKRKILKR198. Site-directed mutagenesis showed that the P1 Arg198 is Amicarbazone critical, as well the presence of two other basic residues at P2, P6, or P8. Finally, mice lacking furin or PC5/6 in hepatocytes exhibit a 30% decrease in APC levels in plasma, whereas those completely lacking PACE4 do not show significant changes in circulating APC levels. Results Processing Amicarbazone of mouse protein C in COS-1 cells It has been shown previously that upon overexpression of human protein C with furin in mouse mammary gland, it undergoes cleavage at Arg199 (22). Such cleavage occurs at the C terminus of a typical basic amino acid PC-like recognition sequence KKRSHLKR199 located in a linker region between the light and heavy chain domains of the zymogen proprotein C. This PC-like site is highly conserved between humans and mice (Fig. 1and supplemental Fig. S1). Because we aimed to analyze the activation of protein C in mice, we next tested the ability of PCs to process mouse proprotein C. C-terminally V5-tagged mouse protein C was co-expressed in COS-1 cells with either furin, PC5/6A, PC7, or PACE4 (1). Western blot analysis of the media with a V5-monoclonal antibody (V5-mAb) revealed two forms corresponding to the 65-kDa full-length protein and the 48-kDa C-terminal (CT) catalytic domain of mouse protein C (Fig. 1Golgi and endosomes (see supplemental Fig. S3 in Ref. 27)) but inhibits very well cell surface PCs (27, 28). These data indicate that, in COS-1 cells, convertase cleavage of mouse protein C occurs almost exclusively at the cell surface, as was the case for the growth factor Amicarbazone BMP10 (27). To support this finding, we performed a similar experiment where we compared the inhibition of the furin processing of proprotein C by RVKR and D6R with that of a potent cell-impermeable protein inhibitor, 1-antitrypsin Portland variant (1-PDX) (29) (Fig. 2). The data show that whereas incubation of cells with RVKR completely (100%) abrogated the furin processing, incubations with D6R or 1-PDX incompletely inhibited such processing by 80 and 88%, respectively. These data support the notion that in COS-1 cells furin processing happens mostly in the cell surface. Open in a separate window Number 2. Cellular protein C processing is definitely abrogated by incubation of cells with 1-PDX. The represents the production levels of V5-tagged 1-PDX in COS-1 cells. In the each display the R221A mutation does not prevent Personal computer5/6A from control mouse protein C, indicating that Personal computer cleavage is definitely thrombin-independent. Protein C activation by thrombin requires prior cleavage by Personal computers Because it is definitely hard to discriminate between Personal computers and thrombin cleavage products by SDS-PAGE, as they only differ by 1.2 kDa, we used an activity test to evaluate Rabbit polyclonal to IQGAP3 the part of Personal computers in mouse protein C activation into APC. Protein C activity was measured in 24-h conditioned press from COS-1 cells expressing Personal computer5/6A with either WT mouse protein C,.Desjardins, and R. cleavage from the convertases, because both R198A and R221A lack protein C activity. Main ethnicities of hepatocytes derived from wild-type or hepatocyte-specific furin, Personal computer5/6, or total PACE4 knock-out mice suggested the cleavage of overexpressed proprotein C is definitely mainly performed by furin intracellularly and by all three proprotein convertases in the cell surface. Indeed, plasma analyses of single-proprotein convertase-knock-out mice showed that loss of the convertase furin or Personal computer5/6 in hepatocytes results in a 30% decrease in APC levels, with no significant contribution from PACE4. We conclude that prior convertase cleavage of protein C in hepatocytes is critical for its thrombin activation. to = 0, 1, 2, or 3 spacer amino acids (aa); boldface and underlined fundamental residues critical for the acknowledgement by proprotein convertases) happening in the constitutive secretory pathway: (3), their inactivation in mice prospects to specific phenotypes exposing that, and assay for the protein C activity, we further demonstrated that conversion of protein C to its active APC form by thrombin requires a previous cleavage by convertases at KKRKILKR198. Site-directed mutagenesis showed the P1 Arg198 is critical, as well the presence of two additional fundamental residues at P2, P6, or P8. Finally, mice lacking furin or Personal computer5/6 in hepatocytes show a 30% decrease in APC levels in plasma, whereas those completely lacking PACE4 do not display significant changes in circulating APC levels. Results Control of mouse protein C in COS-1 cells It has been demonstrated previously that upon overexpression of human being protein C with furin in mouse mammary gland, it undergoes cleavage at Arg199 (22). Such cleavage happens in the C terminus of a typical basic amino acid PC-like acknowledgement sequence KKRSHLKR199 located in a linker region between the light and weighty chain domains of the zymogen proprotein C. This PC-like site is definitely highly conserved between humans and mice (Fig. 1and supplemental Fig. S1). Because we targeted to analyze the activation of protein C in mice, we next tested the ability of Personal computers to process mouse proprotein C. C-terminally V5-tagged mouse protein C was co-expressed in COS-1 cells with either furin, Personal computer5/6A, Personal computer7, or PACE4 (1). Western blot analysis of the media having a V5-monoclonal antibody (V5-mAb) exposed two forms related to the 65-kDa full-length protein and the 48-kDa C-terminal (CT) catalytic domain of mouse protein C (Fig. 1Golgi and endosomes (observe supplemental Fig. S3 in Ref. 27)) but inhibits very well cell surface Personal computers (27, 28). These data show that, in COS-1 cells, convertase cleavage of mouse protein C occurs almost exclusively in the cell surface, as was the case for the growth element BMP10 (27). To support this getting, we performed a similar experiment where we compared the inhibition of the furin processing of proprotein C by RVKR and D6R with that of a potent cell-impermeable protein inhibitor, 1-antitrypsin Portland variant (1-PDX) (29) (Fig. 2). The data show that whereas incubation of cells with RVKR completely (100%) abrogated the furin processing, incubations with D6R or 1-PDX incompletely inhibited such processing by 80 and 88%, respectively. These data support the notion that in COS-1 cells furin processing occurs mostly in the cell surface. Open in a separate window Number 2. Cellular protein C processing is definitely abrogated by incubation of cells with 1-PDX. The represents the production levels of V5-tagged 1-PDX in COS-1 cells. In the each display the R221A mutation does not prevent Personal computer5/6A from control mouse protein C, indicating that Personal computer cleavage is definitely thrombin-independent. Protein C activation by thrombin requires prior cleavage by Personal computers Because it is definitely hard to discriminate between Personal computers and thrombin cleavage products by SDS-PAGE, as they only differ by 1.2 kDa, we used an activity test to evaluate the part of Personal computers in mouse protein C activation into APC. Protein C activity was measured in 24-h conditioned press from COS-1 cells expressing Personal computer5/6A with either WT mouse protein C, its PC-resistant mutant R198A at P1, or its likely thrombin-resistant mutant R221A (Fig. 4show that PAR-1 inhibits 97% of the endogenous control of mouse protein C in these cells, suggesting that furin is the major endogenous mouse protein C control enzyme in COS-1 cells, because Personal computer5/6 and PACE4 cleave PAR-1 and are not inhibited by it (18). Overexpression.