Molecular & Biochemical Parasitology 136 (2004) 257–264 A nucleotidase with unique catalytic properties is secreted by Trichinella spiralis  Kleoniki Gounaris a,∗ , Murray E. Selkirk a , Sheila J. Sadeghi a,b a Department of Biological Sciences, Imperial College London, Biochemistry Building, South Kensington Campus, London SW7 2AZ, UK b Department of Cellular and Molecular Pharmacology, UCSF Medical Sciences Building, 513 Parnassus, P.O. Box 0450, San Francisco, CA 94143, USA Received 30 January 2004; received in revised form 1 April 2004; accepted 6 April 2004 Available online 25 May 2004 Abstract We have isolated and expressed a cDNA from the parasitic nematode Trichinella spiralis encoding a novel secreted nucleotidase which catalyses the hydrolysis of nucleoside 5  -diphosphates and 5  -monophosphates, but not 5  -triphosphates. The full length cDNA encodes a protein of 550 amino acids with an N-terminal signal peptide, but lacking a C-terminal signature sequence for addition of a glycosyl phosphatidylinositol (GPI) anchor. Expression in Pichia pastoris resulted in the secretion of an active enzyme with the catalytic properties of both a Mg 2+ -dependent diphosphohydrolase/apyrase and a 5  -nucleotidase. The protein sequence is homologous to 5  -nucleotidases from a wide variety of organisms but contains no sequences specifically conserved in apyrases, suggesting that it is a representative of a new class of secreted nucleotidase. The enzyme was essentially monospecific for AMP among the nucleoside 5  -monophosphates and catalysed the hydrolysis of nucleoside 5  -diphosphates in the order of UDP  ADP. The diphosphatase activity was dependent on the presence of magnesium ions and a reducing agent, while the 5  -nucleotidase activity was enhanced by these additions. Kinetic analyses indicated that the enzyme exhibits allosteric behaviour. Determination of the number of active sites suggested that catalysis of the two different reactions occurs at the same active site. The data are discussed in terms of regulation of host purinergic signalling during infection. © 2004 Elsevier B.V. All rights reserved. Keywords: Nucleotidase; Purinergic signalling; Nematode; Trichinella 1. Introduction Extracellular nucleotides are purinergic signalling molecules with diverse physiological functions in numerous biological systems [1]. Purinergic signalling relies on the interaction of nucleotides or their degradation products with membrane-bound purinergic receptors, which transduce the signal to the interior of the cell. Extracellular nucleotides are degraded by ubiquitous families of nucleotidases, which include the ecto-nucleoside 5  -triphosphate diphos- phohydrolase (E-NTPDase) and 5  -nucleotidase families. Members of the former family, also referred to as nucleo- side 5  -triphosphate diphosphohydrolases or apyrases, were Abbreviations: E-NTPDase, ecto-nucleoside 5  -triphosphate diphos- phohydrolase; DTT, dithiothreitol; GSH, glutathione  Note: Nucleotide sequence data reported in this paper are available in the GenBank TM , EMBL and DDBJ databases under the accession num- ber AY127571. ∗ Corresponding author. Tel.: +44-20-7594-5209; fax: +44-20-7594-5207. E-mail address: k.gounaris@imperial.ac.uk (K. Gounaris). originally named after the lymphocyte surface marker CD39 and have been sub-divided into two groups according to membrane topography [2]. This family of enzymes has a wide phylogenetic distribution, and related enzymes have been identified in organisms ranging from vertebrates to protozoa. The products of their enzymatic action, like the reactants, act on various P2 classes of purinergic receptors. All members of this family exhibit amino acid sequence homology in five conserved domains termed “apyrase con- served regions” and in addition all possess the - and -phosphate binding sequence motif [3,4]. Enzymes be- longing to this family depend on either Mg 2+ or Ca 2+ ions for activity and hydrolyse both nucleoside 5  -triphosphates and 5  -diphosphates, with the exception of the two members denoted NTPDase5 and NTPDase6 (CD39L4 and CD39L2, respectively) which preferentially hydrolyse nucleoside 5  -diphosphates [5,6]. Another family of apyrases has been identified re- cently which are expressed in the salivary glands of haematophagous arthropods. Although these enzymes have similar catalytic properties to the E-NTPDase family, their 0166-6851/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.molbiopara.2004.04.008