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Dysfunction of outer segment guanylate cyclase caused by retinal disease related mutations.

Zägel P, Koch KW - Front Mol Neurosci (2014)

Bottom Line: The mutation H1019P caused the cyclase to become more labile.The different biochemical consequences of these mutations seem to reflect the different clinical symptoms.In contrast, a strong reduction in cGMP synthesis due to an inactive or structurally unstable ROS-GC1 would trigger more severe forms of retinal diseases.

View Article: PubMed Central - PubMed

Affiliation: Biochemistry Group, Department of Neurosciences, Carl von Ossietzky University Oldenburg Oldenburg, Germany.

ABSTRACT
Membrane bound guanylate cyclases are expressed in rod and cone cells of the vertebrate retina and mutations in several domains of rod outer segment guanylate cyclase 1 (ROS-GC1 encoded by the gene GUCY2D) correlate with different forms of retinal degenerations. In the present work we investigated the biochemical consequences of three point mutations, one is located in position P575L in the juxtamembrane domain close to the kinase homology domain and two are located in the cyclase catalytic domain at H1019P and P1069R. These mutations correlate with various retinal diseases like autosomal dominant progressive cone degeneration, e.g., Leber Congenital Amaurosis and a juvenile form of retinitis pigmentosa. Wildtype and mutant forms of ROS-GC1 were heterologously expressed in HEK cells, their cellular distribution was investigated and activity profiles in the presence and absence of guanylate cyclase-activating proteins were measured. The mutant P575L was active under all tested conditions, but it displayed a twofold shift in the Ca(2) (+)-sensitivity, whereas the mutant P1069R remained inactive despite normal expression levels. The mutation H1019P caused the cyclase to become more labile. The different biochemical consequences of these mutations seem to reflect the different clinical symptoms. The mutation P575L induces a dysregulation of the Ca(2) (+)-sensitive cyclase activation profile causing a slow progression of the disease by the distortion of the Ca(2) (+)-cGMP homeostasis. In contrast, a strong reduction in cGMP synthesis due to an inactive or structurally unstable ROS-GC1 would trigger more severe forms of retinal diseases.

No MeSH data available.


Related in: MedlinePlus

Cellular localization of WT and ROS-GC1 mutants in HEK293 cells. (A) Cells were stably transfected with human ROS-GC1 constructs and were probed with different antibodies; the corresponding ROS-GC1 construct used for transfection is indicated in the corner of the upper left panel. Anti-Na+/K+-ATPase was used as plasma membrane marker (upper left panel, red staining); anti-calnexin antibodies as marker for the endoplasmic reticulum (top right panel, red staining); anti-ROS-GC1 antibody (middle panels, blue staining); overlay of ROS-GC1 staining and membrane specific localization (lower panels). Secondary antibodies were indicated in the Methods section. Visualization was done in an Olympus fluorescence microscope. Cells were fixed either with paraformaldehyde (localization of plasma membrane) or with paraformaldehyde-methanol (endoplasmic reticulum). Scale bar = 10 μm. (B) Western blot of HEK293 cells expressing human ROS-GC1, WT and the mutants P575L, H1019P, and P1069R. Blots were probed with a primary antibody against ROS-GC1 (GC1#3, dilution 1:2000) and a secondary anti-rabbit antibody from Dianova (dilution 1:4000) as described (Zägel et al., 2013).
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Figure 2: Cellular localization of WT and ROS-GC1 mutants in HEK293 cells. (A) Cells were stably transfected with human ROS-GC1 constructs and were probed with different antibodies; the corresponding ROS-GC1 construct used for transfection is indicated in the corner of the upper left panel. Anti-Na+/K+-ATPase was used as plasma membrane marker (upper left panel, red staining); anti-calnexin antibodies as marker for the endoplasmic reticulum (top right panel, red staining); anti-ROS-GC1 antibody (middle panels, blue staining); overlay of ROS-GC1 staining and membrane specific localization (lower panels). Secondary antibodies were indicated in the Methods section. Visualization was done in an Olympus fluorescence microscope. Cells were fixed either with paraformaldehyde (localization of plasma membrane) or with paraformaldehyde-methanol (endoplasmic reticulum). Scale bar = 10 μm. (B) Western blot of HEK293 cells expressing human ROS-GC1, WT and the mutants P575L, H1019P, and P1069R. Blots were probed with a primary antibody against ROS-GC1 (GC1#3, dilution 1:2000) and a secondary anti-rabbit antibody from Dianova (dilution 1:4000) as described (Zägel et al., 2013).

Mentions: Previous work has shown that ROS-GC1 can be expressed heterologously in HEK293 cells in sufficient quantity to allow subsequent biochemical studies (Hwang et al., 2003; Koch and Helten, 2008; Zägel et al., 2013). In HEK293 cells the enzyme was found to be present in cell membranes and mainly co-localized with the endoplasmic reticulum. Probing the cells with an anti-ROS-GC1 antibody (Figure 2A, middle panel) and using specific markers for the plasma membrane (anti-Na+/K+-ATPase antibody, top left panel in Figure 2A) and for the endoplasmic reticulum (anti-calnexin, top right panel) we obtained a staining pattern in the overlay image (Figure 2A, bottom panels) in agreement with published results (Peshenko et al., 2008; Zägel et al., 2013). The localization of all investigated mutants was very similar to the cellular localization of the WT. Using western blotting we further estimated that the amount of ROS-GC1 WT and mutants was nearly the same in a suspension of cells having a similar cell density (Figure 2B, also Zägel et al., 2013).


Dysfunction of outer segment guanylate cyclase caused by retinal disease related mutations.

Zägel P, Koch KW - Front Mol Neurosci (2014)

Cellular localization of WT and ROS-GC1 mutants in HEK293 cells. (A) Cells were stably transfected with human ROS-GC1 constructs and were probed with different antibodies; the corresponding ROS-GC1 construct used for transfection is indicated in the corner of the upper left panel. Anti-Na+/K+-ATPase was used as plasma membrane marker (upper left panel, red staining); anti-calnexin antibodies as marker for the endoplasmic reticulum (top right panel, red staining); anti-ROS-GC1 antibody (middle panels, blue staining); overlay of ROS-GC1 staining and membrane specific localization (lower panels). Secondary antibodies were indicated in the Methods section. Visualization was done in an Olympus fluorescence microscope. Cells were fixed either with paraformaldehyde (localization of plasma membrane) or with paraformaldehyde-methanol (endoplasmic reticulum). Scale bar = 10 μm. (B) Western blot of HEK293 cells expressing human ROS-GC1, WT and the mutants P575L, H1019P, and P1069R. Blots were probed with a primary antibody against ROS-GC1 (GC1#3, dilution 1:2000) and a secondary anti-rabbit antibody from Dianova (dilution 1:4000) as described (Zägel et al., 2013).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3935488&req=5

Figure 2: Cellular localization of WT and ROS-GC1 mutants in HEK293 cells. (A) Cells were stably transfected with human ROS-GC1 constructs and were probed with different antibodies; the corresponding ROS-GC1 construct used for transfection is indicated in the corner of the upper left panel. Anti-Na+/K+-ATPase was used as plasma membrane marker (upper left panel, red staining); anti-calnexin antibodies as marker for the endoplasmic reticulum (top right panel, red staining); anti-ROS-GC1 antibody (middle panels, blue staining); overlay of ROS-GC1 staining and membrane specific localization (lower panels). Secondary antibodies were indicated in the Methods section. Visualization was done in an Olympus fluorescence microscope. Cells were fixed either with paraformaldehyde (localization of plasma membrane) or with paraformaldehyde-methanol (endoplasmic reticulum). Scale bar = 10 μm. (B) Western blot of HEK293 cells expressing human ROS-GC1, WT and the mutants P575L, H1019P, and P1069R. Blots were probed with a primary antibody against ROS-GC1 (GC1#3, dilution 1:2000) and a secondary anti-rabbit antibody from Dianova (dilution 1:4000) as described (Zägel et al., 2013).
Mentions: Previous work has shown that ROS-GC1 can be expressed heterologously in HEK293 cells in sufficient quantity to allow subsequent biochemical studies (Hwang et al., 2003; Koch and Helten, 2008; Zägel et al., 2013). In HEK293 cells the enzyme was found to be present in cell membranes and mainly co-localized with the endoplasmic reticulum. Probing the cells with an anti-ROS-GC1 antibody (Figure 2A, middle panel) and using specific markers for the plasma membrane (anti-Na+/K+-ATPase antibody, top left panel in Figure 2A) and for the endoplasmic reticulum (anti-calnexin, top right panel) we obtained a staining pattern in the overlay image (Figure 2A, bottom panels) in agreement with published results (Peshenko et al., 2008; Zägel et al., 2013). The localization of all investigated mutants was very similar to the cellular localization of the WT. Using western blotting we further estimated that the amount of ROS-GC1 WT and mutants was nearly the same in a suspension of cells having a similar cell density (Figure 2B, also Zägel et al., 2013).

Bottom Line: The mutation H1019P caused the cyclase to become more labile.The different biochemical consequences of these mutations seem to reflect the different clinical symptoms.In contrast, a strong reduction in cGMP synthesis due to an inactive or structurally unstable ROS-GC1 would trigger more severe forms of retinal diseases.

View Article: PubMed Central - PubMed

Affiliation: Biochemistry Group, Department of Neurosciences, Carl von Ossietzky University Oldenburg Oldenburg, Germany.

ABSTRACT
Membrane bound guanylate cyclases are expressed in rod and cone cells of the vertebrate retina and mutations in several domains of rod outer segment guanylate cyclase 1 (ROS-GC1 encoded by the gene GUCY2D) correlate with different forms of retinal degenerations. In the present work we investigated the biochemical consequences of three point mutations, one is located in position P575L in the juxtamembrane domain close to the kinase homology domain and two are located in the cyclase catalytic domain at H1019P and P1069R. These mutations correlate with various retinal diseases like autosomal dominant progressive cone degeneration, e.g., Leber Congenital Amaurosis and a juvenile form of retinitis pigmentosa. Wildtype and mutant forms of ROS-GC1 were heterologously expressed in HEK cells, their cellular distribution was investigated and activity profiles in the presence and absence of guanylate cyclase-activating proteins were measured. The mutant P575L was active under all tested conditions, but it displayed a twofold shift in the Ca(2) (+)-sensitivity, whereas the mutant P1069R remained inactive despite normal expression levels. The mutation H1019P caused the cyclase to become more labile. The different biochemical consequences of these mutations seem to reflect the different clinical symptoms. The mutation P575L induces a dysregulation of the Ca(2) (+)-sensitive cyclase activation profile causing a slow progression of the disease by the distortion of the Ca(2) (+)-cGMP homeostasis. In contrast, a strong reduction in cGMP synthesis due to an inactive or structurally unstable ROS-GC1 would trigger more severe forms of retinal diseases.

No MeSH data available.


Related in: MedlinePlus