The destabilization of human GCAP1 by a proline to leucine mutation might cause cone-rod dystrophy

Guanylate cyclase activating protein-1 (GCAP1) is required for activation o f retinal guanylate cyclase-1 (RetGC1), which is essential for recovery of photoreceptor cells to the dark state. in this paper, experimentally derive d observations are reported that help in explaining why a proline-->leucine mutation at position 50 of human GCAP1 results in cone-rod dystrophy in a family carrying this mutation. The primary amino acid sequence of wild-type GCAP1 was mutated using site-directed mutagenesis to give a leucine at pos ition 50, In addition, serine replaced a glutamic acid residue at position 6 to promote N-terminal myristoylation, yielding the construct GCAP1 E6S/P5 0L. The enzyme was over-expressed in Escherichia coli cells, isolated and p urified before being used in assays with RetGC1, characterized by circular dichroism (CD) spectroscopy, and investigated for protease resistance and t hermal stability. Assays of cyclic guanosine monophosphate (cGMP) synthesis from guanosine triphosphate by RetGC1 in the presence of E6S/P50L showed t hat E6S/P50L could activate RetGC1 and displayed similar calcium sensitivit y to wild-type GCAP1, In addition, E6S/P50L and wild-type GCAP1 possess sim ilar CD spectra, However, there was a marked increase in the susceptibility to protease degradation and also a reduction in the thermal stability of E 6S/P50L as observed by both the cGMP assay and CD spectroscopy. It is there fore suggested that although GCAP1 E6S/P50L has a similar activity and calc ium dependency profile to the wild-type GCAP1, its lower stability could re duce its cellular concentration, which would in turn alter [Ca2+] and resul t in death of cells.