Human Molecular Genetics, 2001, Vol. 10, No. 1 47-54
© 2001 Oxford University Press
The destabilization of human GCAP1 by a proline to leucine mutation might cause cone-rod dystrophy
1School of Biological Sciences, Queen Mary, University of London, Mile End Road, London E1 4NS, UK, 2Department of Molecular Genetics, Institute of Ophthalmology, 11–43 Bath Street, London EC1V 9EL, UK and 3Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560 012, India
Guanylate cyclase activating protein-1 (GCAP1) is required for activation of retinal guanylate cyclase-1 (RetGC1), which is essential for recovery of photoreceptor cells to the dark state. In this paper, experimentally derived 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 position 50. In addition, serine replaced a glutamic acid residue at position 6 to promote N-terminal myristoylation, yielding the construct GCAP1 E6S/P50L. The enzyme was over-expressed in Escherichia coli cells, isolated and purified before being used in assays with RetGC1, characterized by circular dichroism (CD) spectroscopy, and investigated for protease resistance and thermal stability. Assays of cyclic guanosine monophosphate (cGMP) synthesis from guanosine triphosphate by RetGC1 in the presence of E6S/P50L showed that E6S/P50L could activate RetGC1 and displayed similar calcium sensitivity to wild-type GCAP1. In addition, E6S/P50L and wild-type GCAP1 possess similar CD spectra. However, there was a marked increase in the susceptibility to protease degradation and also a reduction in the thermal stability of E6S/P50L as observed by both the cGMP assay and CD spectroscopy. It is therefore suggested that although GCAP1 E6S/P50L has a similar activity and calcium dependency profile to the wild-type GCAP1, its lower stability could reduce its cellular concentration, which would in turn alter [Ca2+] and result in death of cells.
+ To whom correspondence should be addressed. Tel: +44 207 882 7718; Fax: +44 208 983 0973; Email: m.j.warren@qmw.ac.uk
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  I. Sokal, W. J. Dupps, M. A. Grassi, J. Brown Jr, L. M. Affatigato, N. Roychowdhury, L. Yang, S. Filipek, K. Palczewski, E. M. Stone, et al. A Novel GCAP1 Missense Mutation (L151F) in a Large Family with Autosomal Dominant Cone-Rod Dystrophy (adCORD) Invest. Ophthalmol. Vis. Sci., April 1, 2005; 46(4): 1124 - 1132. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. M. Nishiguchi, I. Sokal, L. Yang, N. Roychowdhury, K. Palczewski, E. L. Berson, T. P. Dryja, and W. Baehr A Novel Mutation (I143NT) in Guanylate Cyclase-Activating Protein 1 (GCAP1) Associated with Autosomal Dominant Cone Degeneration Invest. Ophthalmol. Vis. Sci., November 1, 2004; 45(11): 3863 - 3870. [Abstract] [Full Text] [PDF]
|
|