Fig 2: CAPN5 co-fractionates with both soluble and synaptic vesicle proteins. (A) A schematic illustration of a photoreceptor cell maps the location of subcellular marker proteins. (B) Antibodies against CAPN5 were used to probe retinal protein fractions alongside protein markers for specific retinal compartments. CAPN5 was detected in the ROS, but most of it remained in the ROR fraction. After the ROR was fractionated to enrich for soluble (ROR-S1), membrane (ROR-P1), and synaptic (Syn-S2) proteins, CAPN5 was found in the soluble and synaptic vesicle pools (along with Actin and GAPDH, or RAB3 and VAMP2, respectively). NKA, sodium potassium ATPase; HCN1, hyperpolarization-activated and cyclic nucleotide-gated channel, family member 1.

Fig 3: CAPN5 Western blot shows specificity of multiple CAPN5 antibodies. (A) Schematic diagram of human CAPN5 protein domain structure. CAPN5 is composed of 640 amino acids that comprise four domains (A). Domain-I spans amino acid residues 1 to 25. Domain-II is the protease core and spans amino acid residues 26 to 343; it consists of two subdomains that together contain the catalytic triad and calcium binding sites. Domain II also contains the three different ADNIV-causing mutations.3 Domain-III spans amino acid residues 344 to 496 and contains a calcium-binding site leading to speculation that it is a regulatory region. Domain-IV (C2) also is believed to be regulatory; it spans amino acid residues 518 to 619, and contains three calcium-binding sites. (B) A diagram showing the epitopes of the seven different commercially available anti-CAPN5 antibodies tested, as well as the companies they were obtained from. Antibody Ab-1 (ab28280) mainly targeted domain I; Ab-2 (sc-50500) and Ab-3 (sc271271) targeted all of domain I, and part of domain II (catalytic domain); Ab-4 (ab38943) targeted the entire domain II and part of domain III; Ab-5 (12373-1-AP) and Ab-6 (ab97534) targeted domains I, II, and part of III. All antibodies were rabbit polyclonal antibodies, except for Ab-3, which was a mouse monoclonal antibody. (C) Immunoblots of HEK-293 cells transfected with full-length CAPN5 (tagged with c-myc and flag tag) and probed separately with three unique anti-CAPN5 antibodies. Anti-GAPDH antibody was used as a protein loading control. Immunoglobulin G (IgG) was used as a negative control. The closed arrows indicate CAPN5 bands at approximately 75 kDa, while the open arrowheads indicate GAPDH bands at approximately 37 kDa. Immunoblot images were captured with an ECL imager. Antibody Ab-7 consistently gave the best bands with the least amount of background, indicating higher specificity for CAPN5. (D) Immunoblots of HEK-293, SY-SH5Y, and Y79 cells transfected with full length CAPN5 and probed with Ab-4. (E) Immunoblots of retinas belonging to human and mice were probed separately with CAPN5 specific Ab-7. Immunoblot of protein obtained from wildtype (N2) C. elegans. 10 µg of protein were loaded.

Fig 4: Customized antigen raises highly specific CAPN5 antibodies. (A) Alignment of CAPN5 and Tra-3 sequences. Regions of homology are shown in black. Regions corresponding to the customized CAPN5 antigen are underlined with colors corresponding to the different CAPN5 domains. (B) A structural model of CAPN5. The highlighted regions correspond to those recognized by our custom antibody. (C) A diagram showing the epitopes of our custom anti-CAPN5 antibody. (D) Immunoblots of HEK-293 cells transfected with full-length CAPN5 and probed with our custom anti-CAPN5 antibody. We used GAPDH as a protein loading control. Preimmune-serum obtained from the same animal was used as a negative control. The closed arrows indicate CAPN5 bands at approximately 75 kDa, while the open arrowheads indicate GAPDH bands at approximately 37 kDa. Immunoblot images were captured with an ECL imager.

Fig 5: CAPN5 EVS Variants. Public exome databases show common variants in CAPN5 by amino acid position and type. The CAPN5 gene is 59 kilobases, contains 13 exons, and generates a 2791 nucleotide transcript (encompassing all purported isoforms) that encodes a 640 amino acid protein. Shown here, this data set serves as a normative database for CAPN5 variants, and depicts the relationship of these variants to functional residues and domains within CAPN5. Variants are rare within the proteolytic core, where retinal disease alleles are located. The EVS sequenced over 6476 individuals, with high CAPN5 coverage, and found no frameshift variants, two premature stop-codon variants, 843 missense variants (of which 237 were splice-site mutations), and 681 synonymous variants. Of these variants, the majority was scored as either “unknown” or “benign” by PolyPhen. PolyPhen predicted that 1% and 0.83% of the population has “Possibly Damaging” and “Probably Damaging” alleles, respectively.