A nucleotidase with unique catalytic properties is secreted by Trichinella spiralis.
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 Mg2+-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.
Gounaris
Kleoniki
K
Department of Biological Sciences, Imperial College London, Biochemistry Building, South Kensington Campus, London SW7 2AZ, UK. k.gounaris@imperial.ac.uk
Selkirk
Murray E
ME
Sadeghi
Sheila J
SJ
D015720:5'-Nucleotidase / Q000235:genetics* / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002384:Catalysis; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D018509:DNA, Helminth / Q000235:genetics; D015870:Gene Expression; D017238:Genes, Helminth; D007700:Kinetics; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D017160:Trichinella spiralis / Q000201:enzymology* / Q000235:genetics*
D018076:DNA, Complementary; D018509:DNA, Helminth; D011994:Recombinant Proteins; D015720:5'-Nucleotidase