title: Identification and characterization of a novel calcium-activated apyrase from Cryptosporidium parasites and its potential role in pathogenesis.
abstract: Herein, we report the biochemical and functional characterization of a novel Ca(2+)-activated nucleoside diphosphatase (apyrase), CApy, of the intracellular gut pathogen Cryptosporidium. The purified recombinant CApy protein displayed activity, substrate specificity and calcium dependency strikingly similar to the previously described human apyrase, SCAN-1 (soluble calcium-activated nucleotidase 1). CApy was found to be expressed in both Cryptosporidium parvum oocysts and sporozoites, and displayed a polar localization in the latter, suggesting a possible co-localization with the apical complex of the parasite. In vitro binding experiments revealed that CApy interacts with the host cell in a dose-dependent fashion, implying the presence of an interacting partner on the surface of the host cell. Antibodies directed against CApy block Cryptosporidium parvum sporozoite invasion of HCT-8 cells, suggesting that CApy may play an active role during the early stages of parasite invasion. Sequence analyses revealed that the capy gene shares a high degree of homology with apyrases identified in other organisms, including parasites, insects and humans. Phylogenetic analysis argues that the capy gene is most likely an ancestral feature that has been lost from most apicomplexan genomes except Cryptosporidium, Neospora and Toxoplasma.
authors.affiliation: Department of Microbiology and Immunology, Virginia Commonwealth University, Richmond, Virginia, United States of America.
authors.forename: Patricio A
authors.initials: PA
authors.lastname: Manque
authors.forename: Ute
authors.initials: U
authors.lastname: Woehlbier
authors.forename: Ana M
authors.initials: AM
authors.lastname: Lara
authors.forename: Fernando
authors.initials: F
authors.lastname: Tenjo
authors.forename: João M
authors.initials: JM
authors.lastname: Alves
authors.forename: Gregory A
authors.initials: GA
authors.lastname: Buck
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000913:Antibodies, Protozoan / Q000276:immunology; D001081:Apyrase / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D002118:Calcium / Q000494:pharmacology*; D003457:Cryptosporidiosis / Q000201:enzymology* / Q000469:parasitology*; D003458:Cryptosporidium / Q000187:drug effects / Q000201:enzymology* / Q000276:immunology / Q000502:physiology*; D004789:Enzyme Activation / Q000187:drug effects; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism / Q000469:parasitology; D006031:Glycosylation / Q000187:drug effects; D006153:Guanosine Diphosphate / Q000378:metabolism; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D010271:Parasites / Q000187:drug effects / Q000201:enzymology*; D010802:Phylogeny; D011485:Protein Binding / Q000187:drug effects; D058849:Protein Refolding / Q000187:drug effects; D021381:Protein Transport / Q000187:drug effects; D015800:Protozoan Proteins / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D034101:Sporozoites / Q000187:drug effects / Q000201:enzymology; D013379:Substrate Specificity / Q000187:drug effects; D014530:Uridine Diphosphate / Q000378:metabolism
chemical_list: D000913:Antibodies, Protozoan; D015800:Protozoan Proteins; D011994:Recombinant Proteins; D006153:Guanosine Diphosphate; D014530:Uridine Diphosphate; D001081:Apyrase; D002118:Calcium