Fig 1: Syncytin-2 is cleaved by PCSK7.(A) Principle of the PCSK capture assay. (I) A 96-well plate is coated with antibodies against the AU1 tag or the respective isotype control. (II) Lysates of HEK293T cells overexpressing individual, AU1-tagged PCSKs are added, and (III) proteases are captured and washed. (IV) Captured proteases are incubated with AMC reporter substrates harboring the cleavage sites of Syncytin-1, Syncytin-2, or a furin consensus cleavage site, and fluorescence signal is integrated with a plate reader. (B) PCSK-mediated cleavage of Syncytins. The ability of furin, PCSK5, PCSK6, and PCSK7 to cleave Syncytin-1 or Syncytin 2 was determined as described in (A). Mean values of three independent experiments ± SD are shown. A paired t test was performed (*P < 0.05). RFU, relative fluorescence units. (C) Western blot analysis of captured proteases. Capture efficiency of PCSKs was monitored by Western blotting. One exemplary Western blot of the experiments shown in (B) is shown. (D) Furin- and PCSK7-mediated cleavage of mutated Syncytin substrates. AMC reporter substrates harboring the cleavage sites (P1 to P9) of Syncytin-1 or Syncytin-2 are shown on the top left. The chimeras that were generated to map determinants of PCSK7 cleavage are shown below. Amino acids found in Syncytin-1 and Syncytin-2 are shown in purple and orange, respectively. The AMC reporter substrates shown on the left were either incubated with proteases captured from transfected HEK293T cells as described in Fig. 3A (middle) or commercially available proteases produced in myeloid cells (right). Substrate cleavage was monitored over time, and the area under the curve was calculated. Mean values of three to six independent experiments ± SD are shown. A one-way ANOVA with multiple comparisons (Dunnett’s test) was performed (*P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001).
Fig 2: PCSK7 is resistant to GBP-mediated restriction.(A) Interaction of furin or PCSK7 with GBPs. HEK293T cells were cotransfected with expression plasmids for the indicated PCSKs and GBPs or the respective vector controls. One day posttransfection, cells were harvested, and furin or PCSK7 were pulled down via their C-terminal AU1 tag. Western blotting was performed to detect PCSKs and GBPs in whole cell lysates (left) and the pull down (right). The results of one of three independent experiments are shown. IP, immunoprecipitation; #, protein size marker. (B and C) Furin- and PCSK7-mediated Syncytin cleavage in the presence of GBPs. Furin and PCSK7 were captured from cell lysates of transfected HEK293T cells essentially as described in Fig. 3A. However, cells were additionally cotransfected with expression plasmids for GBP2 or GBP5 (WT or isoprenlyation mutant) or the respective vector control. The ability of captured furin/PCSK7 to process a consensus furin cleavage site (B) or the Syncytin-1 and Syncytin-2 cleavage sites (C) were analyzed using the AMC reporter assay described in Fig. 3A. Mean values of three independent experiments ± SD are shown on top. A one-way ANOVA with multiple comparisons (Dunnett’s test) was performed (*P < 0.05, **P < 0.01, and ***P < 0.001). Capture efficiency of PCSKs was monitored by Western blotting and is shown at the bottom.
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