Purification, Cloning, and Expression of an Apyrase from the Bed Bug Cimex lectularius
A NEW TYPE OF NUCLEOTIDE-BINDING ENZYME*
- From the Medical Entomology Section, Laboratory of Parasitic Diseases, NIAID, National Institutes of Health, Bethesda, Maryland 20892-0425, and ‡National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, Maryland 20894
Abstract
An enzyme that hydrolyzes the phosphodiester bonds of nucleoside tri- and diphosphates, but not monophosphates, thus displaying apyrase (EC 3.6.1.5) activity, was purified from salivary glands of the bed bug, Cimex lectularius. The purifiedC. lectularius apyrase was an acidic protein with a pI of 5.1 and molecular mass of ∼40 kDa that inhibited ADP-induced platelet aggregation and hydrolyzed platelet agonist ADP with specific activity of 379 units/mg protein. Amplification of C. lectulariuscDNA corresponding to the N-terminal sequence of purified apyrase produced a probe that allowed identification of a 1.3 kilobase pair cDNA clone coding for a protein of 364 amino acid residues, the first 35 of which constituted the signal peptide. The processed form of the protein was predicted to have a molecular mass of 37.5 kDa and pI of 4.95. The identity of the product of the cDNA clone with nativeC. lectularius apyrase was proved by immunological testing and by expressing the gene in a heterologous host. Immune serum made against a synthetic peptide with sequence corresponding to the C-terminal region of the predicted cDNA clone recognized bothC. lectularius apyrase fractions eluted from a molecular sieving high pressure liquid chromatography and the apyrase active band from chromatofocusing gels. Furthermore, transfected COS-7 cells secreted a Ca2+-dependent apyrase with a pI of 5.1 and immunoreactive material detected by the anti-apyrase serum.C. lectularius apyrase has no significant sequence similarity to any other known apyrases, but homologous sequences have been found in the genome of the nematode C. elegansand in mouse and human expressed sequence tags from fetal and tumor EST libraries.
Footnotes
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↵* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵§ To whom correspondence should be addressed: Medical Entomology Section, Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, Bldg. 4, Rm. 126, 4 Center Dr., MSC 0425, NIH, Bethesda, MD 20892-0425. Tel.: 301-496-3066; Fax: 301-402-4941; E-mail: JRibeiro{at}atlas.niaid.nih.gov.
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↵2 T. Tatusov and T. L. Madden, unpublished; available at the National Center for Biotechnology Information World Wide Web site at http://www.ncbi.nlm.nih.gov/BLAST.
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↵3 D. E. Champagne and J. M. C. Ribeiro, unpublished data (GenBankTM accession numberU70037).
- Abbreviations:
- HPLC
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high pressure liquid chromatography
- BSA
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bovine serum albumin
- PCR
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polymerase chain reaction
- ELISA
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enzyme-linked immunosorbent assay
- EST
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expressed sequence tag.
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- Received August 4, 1998.
- The American Society for Biochemistry and Molecular Biology, Inc.
