Direct fusion of subunits of heterodimeric nitric oxide sensitive guanylyl cyclase leads to functional enzymes with preserved biochemical properties: Evidence for isoform specific activation by ciguates

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Graphical abstractNitric oxide sensitive guanylyl cyclase (NOsGC) is a heterodimeric enzyme consisting of an α and a β subunit. Two heterodimeric enzymes are known to be important for NO-signalling in humans: α11 and α21. No difference had so far been detected with respect to their pharmacological properties, but as we show in the present paper the new drugs cinaciguat and ataciguat activate the α11 form more effectively. Recent evidence suggests that homodimeric complexes of α and β subunits exist in vivo and that these non-heterodimerizing subunits have a separate function from cGMP signaling. To isolate the effect of the α11 or α21 heterodimeric enzyme in overexpression experiments from potential effects of non-heterodimerizing α1, β1 or α2 subunits, we cloned constructs that guarantee a 1:1 stochiometry between α and β subunits and rule out the presence of homodimers. The carboxy-terminus of the β1 subunit was directly fused to the amino-terminus of either the α1 or α2 subunit. The two different “conjoined” NOsGCs faithfully reproduced the biochemical and pharmacological properties of the α11 and α21 heterodimeric enzymes including the differential activation by ciguat-activators. Conjoined NOsGCs can be used for isoform specific overexpression in transgenic animals and therapeutic overexpression may be an application in the future. In both cases possible side effects of homodimeric α or β subunits are avoided. Crystallization with the goal of structure determination may also be easier for conjoined NOsGCs because enzyme preparations are more homogenous and are free of “contaminating” homodimers.

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