Human cyclic nucleotide phosphodiesterases possess a much broader substrate-specificity than previously appreciated

Abstract

a b s t r a c tPhosphodiesterases (PDEs) capable of degrading cAMP and cGMP are indispensable for the regulation of cyclic nucleotide-mediated signals. The existence of other cyclic nucleotides such as cCMP and cUMP has been discussed controversially in the literature. Despite publications on PDEs hydrolyzing cCMP or cUMP, the molecular identity of such enzymes remained elusive. Recently, we have provided evidence for a role of cCMP as second messenger in vascular relaxation and inhibition of platelet aggregation. Using an HPLC-MS based assay, here, we show that human PDEs belonging to various families hydrolyze not only cAMP and cGMP but also other cyclic nucleotides.

PDE7A1 hydrolyzes cCMP

“…However, the following arguments support the notion that PDE7A1 hydrolyzes cCMP: First, the activity was observed in two PDE7A1-containing preparations that were very different (Sf9 cell lysate and purified protein) and from two independent commercial suppliers. Second, as shown in this paper and in our preceding publication [5], the cCMP-degrading activity was absent in all other enzyme preparations from these suppliers (except for the low activity observed with PDE6AB). Finally, the cCMP-hydrolyzing activity was eliminated by the PDE7A1-selective inhibitor BRL-50481 with a K i value that corresponds to the literature [23].…”

“…[5]), we identified PDE7A1 as the first PDE that hydrolyzes cCMP. Except for a very low activity of PDE6AB, the other enzymes described in this paper did not hydrolyze cCMP.…”

“…Hydrolysis of cCMP was undetectable in the control lysate and can therefore be attributed to the over-expressed PDEs. Neither PDE1A3, 10A1, 11A1, nor the eight PDEs analyzed in our previous report [5] degraded cCMP. Thus, PDE6AB and PDE7A1 represent the first PDEs that hydrolyze cCMP.…”

“…We studied nucleoside 3 0 ,5 0 -cyclic monophosphate (cNMP) specificity of eight recombinant PDEs (PDE1B, 2A, 3A, 3B, 4B, 5A, 8A and 9A), but, despite their broad substrate specificity, none of these enzymes accepted cCMP as a substrate [5]. In this publication we identified PDE7A as a cCMP-degrading PDE.…”

“…The lack of sensitive detection methods hampered research activity in this field for many years. With the advent of highly sensitive and selective mass spectrometry-based detection systems [3], it became possible to detect even very low concentrations of cyclic pyrimidine nucleotides in cells, tissues and enzymatically digested samples [4,5]. cCMP is synthesized by soluble adenylyl cyclase [4], by soluble guanylyl cyclase [6] and by the Pseudomonas aeruginosa exotoxin ExoY [7].…”