Apoptosis-inducing signal sequence mutation in carbonic anhydrase IV identified in patients with the RP17 form of retinitis pigmentosa

Abstract

Genetic and physical mapping of the RP17 locus on 17q identified a 3.6-megabase candidate region that includes the gene encoding carbonic anhydrase IV (CA4), a glycosylphosphatidylinositol-anchored protein that is highly expressed in the choriocapillaris of the human eye. By sequencing candidate genes in this region, we identified a mutation that causes replacement of an arginine with a tryptophan (R14W) in the signal sequence of the CA4 gene at position -5 relative to the signal sequence cleavage site. This mutation was found to cosegregate with the disease phenotype in two large families and was not found in 36 unaffected family members or 100 controls. Expression of the mutant cDNA in COS-7 cells produced several findings, suggesting a mechanism by which the mutation can explain the autosomal dominant disease. In transfected COS-7 cells, the R14W mutation (i) reduced the steady-state level of carbonic anhydrase IV activity expressed by 28% due to a combination of decreased synthesis and accelerated turnover; (ii) led to up-regulation of immunoglobulin-binding protein, double-stranded RNA-regulated protein kinase-like ER kinase, and CCAAT/enhancer-binding protein homologous protein, markers of the unfolded protein response and endoplasmic reticulum stress; and (iii) induced apoptosis, as evidenced by annexin V binding and terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling staining, in most cells expressing the mutant, but not the WT, protein. We suggest that a high level of expression of the mutant allele in the endothelial cells of the choriocapillaris leads to apoptosis, leading in turn to ischemia in the overlying retina and producing autosomal dominant retinitis pigmentosa.

Fig. 1.

Effect of the R14W mutation on the steady-state level of CA IV protein. Aliquots of lysates from COS-7 cells transfected with WT or R14W mutant (MT) CA4 cDNAs containing 2 or 5 μg of cell protein were analyzed by SDS/PAGE under nonreducing conditions. The forms of CA IV were characterized by Western blot by using CA IV specific antibodies (Upper). The 37-kDa unfolded precursor (P) migrates slower than the mature 35-kDa protein (M). The polypeptide band intensities were analyzed by an image analyzer and the relative intensity for the bands plotted (Lower).

Fig. 2.

Effect of the R14W mutation on the stability of CA IV protein in the absence and presence of MG 132. COS-7 cells transfected with WT or R14W mutant (MT) CA4 cDNAs were pulse-labeled for 30 min and chased for 15-60 min in the absence (-) and presence (+) of MG 132 (10 μg/ml). CA IV immunoprecipitates were analyzed by SDS/PAGE under nonreducing conditions followed by fluorography. Precursor (P) and mature (M) polypeptides are marked (Upper). The percent residual protein remaining was calculated from the incorporation of radioactive amino acids in the total polypeptides (precursor + mature) (Lower). The mutant CA IV was degraded faster than the WT and was protected by the proteasomal inhibitor, MG 132.

Fig. 3.

Effect of R14W mutation on induction of the ER proteins, BiP, and PERK, in transfected COS-7 cells. (A) One of many fields of COS-7 cells transfected with WT or R14W mutant (R14W) CA4 cDNA that were fixed, permeabilized, and treated with a mixture of anti-CA IV and anti-BiP antibodies, followed by secondary antibodies conjugated with FITC or rhodamine, respectively. The slides were visualized for DAPI (blue; for cell nucleus), CA IV (red), and BiP (green). (B) Data summarizing counts from 500 WT and 350 R14W cells total in many fields, showing the percent of total cells that were positive for CA IV and BiP expression. (C) COS-7 cells transfected with WT or R14W mutant (R14W) CA4 cDNA were fixed, permeabilized, and treated with a mixture of anti-CA IV and anti-PERK antibodies, followed by secondary antibodies conjugated with FITC or rhodamine, respectively. The cells were visualized for DAPI (blue), CA IV (red), and PERK (green). (D) Data summarizing analysis of many fields containing 580 WT and 377 R14W cells total, showing percent of total cells positive for CA IV and PERK expression.

Fig. 4.

Effect of R14W mutation on induction of CHOP in transfected COS-7 cells. Western blot analysis of control COS-7 cells expressing vector-only (lane 1, negative control) or vector-only exposed to 10 μg/ml (lane 2, positive control) or 20 μg/ml (lane 3, positive control) tunicamycin for 3 h, which is known to cause ER stress and up-regulation of CHOP. Lanes 4 and 5 are lysates from COS-7 cells transfected with WT and R14W mutant CA4 cDNAs, respectively. The arrow indicates CHOP (28 kDa).

Fig. 5.

Effect of R14W mutation on induction of apoptosis in transfected COS-7 cells. (A) One of many fields of COS-7 cells transfected with WT or R14W (R14W) mutant CA4 cDNAs, which were fixed and incubated with anti-CA IV followed by secondary antibody conjugated with rhodamine or FITC-annexin V. The slides were visualized for DAPI (blue), CA IV (red), and annexin V (green). (B) Data summarizing counts from many fields of 121 WT and 330 R14W cells total, showing the percent of total cells stained for CA IV and annexin V. (C) One of many fields of COS-7 cells transfected with WT or R14W (R14W) mutant CA4 cDNAs that were fixed, permeabilized, and incubated with anti-CA IV antibody followed by secondary antibody conjugated with rhodamine. The slides were visualized for DAPI (blue), CA IV expression (red), and TUNEL reaction (green). (D) Data summarizing counts from many fields of 54 WT and 63 R14W cells total, showing the percent of total cells positive for CA IV expression and TUNEL reaction.