ABSTRACT
This study with recombinant reconstituted system mimicking the cellular conditions of the native cones documents that photoreceptor ROS-GC1 is modulated by gaseous CO2. Mechanistically, CO2 is sensed by carbonic anhydrase (CAII), generates bicarbonate that, in turn, directly targets the core catalytic domain of ROS-GC1, and activates it to increased synthesis of cyclic GMP. This, then, functions as a second messenger for the cone phototransduction. The study demonstrates that, in contrast to the Ca2+-modulated phototransduction, the CO2 pathway is Ca2+-independent, yet is linked with it and synergizes it. It, through R787C mutation in the third heptad of the signal helix domain of ROS-GC1, affects cone-rod dystrophy, CORD6. CORD6 is caused firstly by lowered basal and GCAP1-dependent ROS-GC1 activity and secondly, by a shift in Ca2+ sensitivity of the ROS-GC1/GCAP1 complex that remains active in darkness. Remarkably, the first but not the second defect disappears with bicarbonate thus explaining the basis for CORD6 pathological severity. Because cones, but not rods, express CAII, the excessive synthesis of cyclic GMP would be most acute in cones.
Subject(s)
Carbon Dioxide/metabolism , Carbonic Anhydrase II/metabolism , Cone-Rod Dystrophies/enzymology , Guanylate Cyclase/metabolism , Receptors, Cell Surface/metabolism , Retinal Cone Photoreceptor Cells/enzymology , Retinal Rod Photoreceptor Cells/enzymology , Animals , COS Cells , Carbonic Anhydrase II/genetics , Catalysis , Cattle , Chlorocebus aethiops , Cone-Rod Dystrophies/genetics , Cone-Rod Dystrophies/pathology , Cyclic GMP/genetics , Cyclic GMP/metabolism , Guanylate Cyclase/genetics , Guanylate Cyclase-Activating Proteins/genetics , Guanylate Cyclase-Activating Proteins/metabolism , Receptors, Cell Surface/genetics , Retinal Cone Photoreceptor Cells/pathology , Retinal Rod Photoreceptor Cells/pathologyABSTRACT
We constructed a strain of Escherichia coli overproducing 6His-tagged Eco29kI by placing the coding sequence under control of a strong bacteriophage T5 promoter. The yield of 6His-Eco29kI restriction endonuclease expression could be increased to about 20% of the total cellular protein, but inclusion bodies formed consisting of insoluble 6His-Eco29kI protein. We developed a fast and effective protocol for purification of the homogeneous enzyme from both soluble and insoluble fractions and established their identity by catalytic activity assay. The isolated enzymes were tested for recognition specificity and optimal reaction conditions as a function of NaCl and KCl concentrations, temperature, and pH compared with the native Eco29kI restriction endonuclease. The 6His-tagged enzyme retained the specificity of the native protein but had an altered optimum of its catalytic reaction.
