Fig 2: Effect of CPA1 Ala-variants on cellular secretion and enzyme activity. Secretion of proCPA1 protein from transfected HEK 293T cells was assessed by SDS-PAGE and Coomassie Blue staining. Protein levels of the p.N256A variant in cell lysates were analyzed by Western blotting. Alpha-tubulin was measured as a loading control. (A) Effect of CPA1 variants p.V251A, p.P253A, p.R255A, p.N256A, and p.P270A. For comparison, variant p.N256K was also included. (B) Densitometric analysis of proCPA1 secretion to the growth medium. (C) CPA1 enzyme activity in the conditioned medium after activation of proCPA1. WT, wild-type.

Fig 3: Effect of natural CPA1 variants on endoplasmic reticulum stress markers in transfected HEK 293T cells. (A) Splicing of XBP1 mRNA (sXBP1) assessed by RT-PCR and agarose gel electrophoresis. GAPDH mRNA was measured as a loading control. Representative gel from two experiments is shown. (B) Expression of HSPA5 (BiP) mRNA measured by RT-qPCR. (C) Relationship between proCPA1 secretion and HSPA5 (BiP) mRNA levels. WT, wild-type.

Fig 4: Effect of the p.G250A CPA1 variant on endoplasmic reticulum stress markers in transfected HEK 293T cells. For comparison, the effect of wild-type CPA1 (WT), the reference pathogenic mutation p.N256K, and empty expression vector were studied. (A) Expression of HSPA5 (BiP) mRNA measured by RT-qPCR. (B) Expression of DDIT3 (CHOP) mRNA measured by RT-qPCR. (C) Splicing of XBP1 mRNA (sXBP1) assessed by RT-PCR and agarose gel electrophoresis. GAPDH mRNA was measured as a loading control. Representative gel from three experiments is shown.

Fig 5: Effect of the p.G250A CPA1 variant on cellular secretion, enzyme activity, intracellular protein levels, solubility, and BiP protein levels in transfected HEK 293T cells. For comparison, wild-type CPA1 (WT), the reference pathogenic mutation p.N256K, and empty expression vector were studied. (A) Secretion of proCPA1 to the growth medium analyzed by SDS-PAGE and Coomassie Blue staining. CPA1 enzyme activity in the medium after activation of proCPA1 is indicated in milliOD/min units. (B) Protein levels of proCPA1, BiP, and a-tubulin in total cell lysates, and in the insoluble and soluble cellular fractions. Proteins were detected by Western blotting. (C) Densitometric evaluation of proCPA1 and BiP levels in total cell lysates, and in the insoluble and soluble cellular fractions.