PMC4182778_01
Alignment of N309K missense variant to members of the PC1/3 gene family.. The location of the missense mutation within a conserved region in the catalytic domain of the PC1/3 gene family.
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S8 family domain) is conserved in all vertebrates (Fig 3). Sanger sequencing of other affected and unaffected family members confirmed that this mutation segregated in the family, with patients II-2, II-4 and II-5 being homozygous and the parents (I-1 and I-2) and patient II-3 heterozygous (Fig 4).
Sanger sequencing validation of the N309K variant.. Sanger sequencing of the results for the proband, parent and unaffected control at nucleotide position 5: 95746646.
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S8 family domain) is conserved in all vertebrates (Fig 3). Sanger sequencing of other affected and unaffected family members confirmed that this mutation segregated in the family, with patients II-2, II-4 and II-5 being homozygous and the parents (I-1 and I-2) and patient II-3 heterozygous (Fig 4).
Lack of activity of N309K PC1/3 despite robust secretion.. PC/3-encoding vectors were transfected into HEK (A panels, left side) or Neuro2A cells (B panels, right side) and the conditioned media subjected to either enzymatic assay (A1, B1) using the standard fluorogenic assay; or to Western blotting using PC1/3 antiserum (A2, B2). Cell extracts were also subjected to Western blotting (A3, B3). Panel C shows the maturation of 35S-methionine-labeled wild-type and N309K precursor proteins during a 20-minute pulse followed by a 2 h chase in Neuro2A cells. WT, wild-type PC1/3. Panel D shows a Western blot of Neuro2A cell lysate, previously transfected with either N309K and G593R PC1/3 cDNAs and incubated with alpha-FLAG M1 or alpha-FLAG M2 antibodies to discriminate the 94 kDa PC1/3 prodomain ER-retained form.
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HEK293 and Neuro2A cells (Fig 5). However, the secreted enzyme showed no catalytic activity (panels A1-B1) and was not processed into the 66 kDa form independently of cell context (panels A2-B3), indicating no in trans activity. Fig 5, Panel C shows that the N309K mutant underwent only slight prodomain removal (prodomain  = 94 kDa form; mature form  = 87 kDa) within the 20 minute pulse period, but showed complete prodomain removal within the 2 h chase period in media samples.