Nucleotidyl Cyclase Activity of Particulate Guanylyl Cyclase A: Comparison with Particulate Guanylyl Cyclases E and F, Soluble Guanylyl Cyclase and Bacterial Adenylyl Cyclases Cyaa and Edema Factor

Kerstin Y Beste,Roland Seifert,Yuequan Shen,Corinna M Spangler,Heike Burhenne,Wei-Jen Tang,Volkhard Kaever,Karl-Wilhelm Koch,Paolo Carloni

doi:10.1371/journal.pone.0070223

Abstract

Guanylyl cyclases (GCs) regulate many physiological processes by catalyzing the synthesis of the second messenger cGMP. The GC family consists of seven particulate GCs (pGCs) and a nitric oxide-activated soluble GC (sGC). Rat sGC α1β1 possesses much broader substrate specificity than previously assumed. Moreover, the exotoxins CyaA from Bordetella pertussis and edema factor (EF) from Bacillus anthracis possess nucleotidyl cyclase (NC) activity. pGC-A is a natriuretic peptide-activated homodimer with two catalytic sites that act cooperatively. Here, we studied the NC activity of rat pGC-A in membranes of stably transfected HEK293 cells using a highly sensitive and specific HPLC-MS/MS technique. GTP and ITP were effective, and ATP and XTP were only poor, pGC-A substrates. In contrast to sGC, pGC-A did not use CTP and UTP as substrates. pGC-E and pGC-F expressed in bovine rod outer segment membranes used only GTP as substrate. In intact HEK293 cells, pGC-A generated only cGMP. In contrast to pGCs, EF and CyaA showed very broad substrate-specificity. In conclusion, NCs exhibit different substrate-specificities, arguing against substrate-leakiness of enzymes and pointing to distinct physiological functions of cyclic purine and pyrimidine nucleotides.

Highlights

Guanosine 39,59-cyclic monophosphate is a wellestablished second messenger that is involved in the regulation of many physiological processes like blood homeostasis, neurotransmission, intestinal secretion, and vision [1]. cGMP is generated from GTP by guanylyl cyclases (GCs) that are expressed in two isoforms: the soluble and particulate or membranebound form
We examined the influence of ATP/Mn2+ (0.5 mM) on GC activity in the presence of additional 4 mM MnCl2
UC- and CC activity of certain nucleotidyl cyclase (NC) cannot be considered as trivial substrate leakiness of enzymes since some NCs such as the particulate GCs (pGCs) studied possess a higher substrate-specificity than soluble GC (sGC), edema factor (EF) and CyaA

Summary

Introduction

Guanosine 39,59-cyclic monophosphate (cGMP) is a wellestablished second messenger that is involved in the regulation of many physiological processes like blood homeostasis, neurotransmission, intestinal secretion, and vision [1]. cGMP is generated from GTP by guanylyl cyclases (GCs) that are expressed in two isoforms: the soluble (sGC) and particulate or membranebound (pGC) form. Biochemical studies with pGC-A and pGC-B revealed that both isoforms possess separate and distinct catalytic and allosteric binding sites that bind GTP and ATP, respectively, under physiological conditions [4]. PGC-B mediates vascular regeneration, cell growth, and endochondral ossification through binding of C-type natriuretic peptide and by activating cGMP-dependent protein kinase II [1]. PGC-C is expressed in the intestinal epithelium [1] This pGC is activated by the peptides guanylin and uroguanylin, and bacterial heat-stable enterotoxins regulating electrolyte and water transport in the intestine, and epithelial cell growth and differentiation. This pGC is activated by the peptides guanylin and uroguanylin, and bacterial heat-stable enterotoxins regulating electrolyte and water transport in the intestine, and epithelial cell growth and differentiation. pGC-D is located in the olfactory neuroepithelium playing an important role in odor recognition and modulating the sensitivity of sensory neurons [1]

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