Figure 1.
Domain structure and SNP locations within preproPC1/3.
The upward arrows indicate the cleavage sites required for PC1/3 maturation. The downward arrows indicate locations of previously described (black) and novel (purple) SNP. The dashed line between the pro and catalytic domains represents a primary cleavage site (occurring in the ER) that is required for activation. The dashed line in the middle of the prodomain indicates the secondary cleavage site (likely cleaved in the trans-Golgi network). The P or Homo B domain following the catalytic domain is important for the stabilization of the catalytic domain, as well as determining various enzymatic properties. The C-terminal domain plays a role in efficient routing of PC1/3 to the secretory granules, and contributes to substrate specificity as well as to specific activity and stability.
Table 1.
Potentially consequential variant alleles in PCSK1.
Figure 2.
Direct Sanger sequencing of genomic DNA from a subject bearing the Arg80Gln variant.
Figure 3.
Western blotting of wild-type and variant PC1/3 proteins expressed in HEK cells.
HEK cells were transiently transfected with empty pcDNA3 (E); pcDNA3 encoding either wild-type PC1/3; or PC1/3 proteins bearing the mutations under study. Western blots of cell lysates and media from transfected HEK cells were probed with amino-terminally directed PC1/3 primary antiserum for detection of recombinant proteins. The data shown represent 1 of 3 independent experiments performed in triplicate. Total secreted immunoreactive band intensity values, obtained through densitometry analysis and used to calculate specific activity for each variant, are represented above the Western blot and shown as the mean ± S.D.
Figure 4.
Specific activities of wild-type and variant PC1/3 proteins, expressed in HEK cells.
Enzymatic activities of secreted recombinant PC1/3 proteins in conditioned medium of transfected HEK cells were compared by measuring maximum cleavage rates using the fluorogenic substrate pyr-ERTKR-amc during a 1 h kinetic assay. Three replicates per transfection condition were assayed in triplicate, and maximum rates were divided by band intensity of immunoreactive protein in the spent medium of the same wells from which activity data were derived. Specific activity values are shown as the mean ± S.D (n = 3). Data represent one of 3 independent experiments performed in triplicate.
Figure 5.
Western blotting of wild-type and novel R80Q (rs1799904) variant PC1/3s, expressed in Neuro-2A cells.
Panel A: Neuro-2a cells were transiently transfected with equal amounts of empty pcDNA3 (E), or pcDNA3 encoding wild-type PC1/3 or the novel variant R80Q (rs1799904) PC1/3. Western blots of media were probed using amino-terminally directed PC1/3 primary antiserum. The data shown represent one of 3 independent experiments performed in triplicate. Panel B: Specific activities of wild-type PC1/3 and the R80Q PC1/3 variant. Enzymatic activities of secreted recombinant PC1/3 proteins in conditioned medium were compared by measuring maximum cleavage rates using the fluorogenic substrate pyr-ERTKR-amc during a 1 h kinetic assay. Three replicates per transfection condition were assayed in triplicate, and maximum rates were divided by band intensity of immunoreactive protein in the spent medium of the same wells from which the activity data were derived. Specific activity values are shown as the mean ± S.D. Data represent one of 3 independent experiments, each performed in triplicate.