Molecular cloning and analysis of the Cryptosporidium parvum aminopeptidase N gene

• Published in: International journal for parasitology Vol. 32; no. 2; pp. 187 - 197
• Main Authors: PADDA, Ranjit S, TSAI, An, CHAPPELL, Cynthia L, OKHUYSEN, Pablo C
• Format: Journal Article
• Language: English
• Published: Oxford Elsevier Science 01-02-2002
• Subjects: Amino Acid Sequence; Animals; Base Sequence; Biological and medical sciences; Blotting, Southern; CD13 Antigens - chemistry; CD13 Antigens - genetics; Cells, Cultured; Cloning, Molecular; Cryptosporidium parvum; Cryptosporidium parvum - enzymology; Cryptosporidium parvum - genetics; Cryptosporidium parvum - growth & development; DNA, Protozoan - chemistry; DNA, Protozoan - genetics; DNA, Protozoan - isolation & purification; Fundamental and applied biological sciences. Psychology; Gene Library; Humans; Life cycle. Host-agent relationship. Pathogenesis; Molecular Sequence Data; Protozoa; Reverse Transcriptase Polymerase Chain Reaction; Sequence Homology, Amino Acid; Human; Protozoa; Host parasite relation; Apicomplexa; Protozoal disease; Nucleotide sequence; Digestive system; Enzyme; Aminopeptidases; Gut; Parasite; Parasitosis; Gene expression; In vitro; Infection; Cryptosporidiosis; Peptidases; Cryptosporidium parvum; Mitochondrial RNA; Hydrolases; Epithelial cell; Molecular cloning; Aminoacid sequence; Membrane alanyl aminopeptidase
• ISSN: 0020-7519; 1879-0135
• DOI: 10.1016/s0020-7519(01)00317-4

Cryptosporidium parvum proteases have been associated with release of infective sporozoites from oocysts, and their specific inhibition blocks parasite excystation in vitro. Additionally, proteases have been implicated in the processing of parasite adhesion molecules found on the surface of sporozoites and merozoites. In this study, we cloned and expressed the C. parvum aminopeptidase N gene by screening a large insert, P1 artificial chromosome library with a probe identified from a Cryptosporidium genome survey-sequencing project. Analysis of the predicted protein encoded by the 2.3 kb gene demonstrated a high degree of homology with prokaryotic and eukaryotic aminopeptidases. The 783 amino acid sequence predicted a M(r) of approximately 89,000. The active site sequence was found to be highly conserved when compared with other Apicomplexan aminopeptidases. Motifs commonly found in aminopeptidases of this class and a unique single Arg-Gly-Asp (RGD) tripeptide motif predictive of cell adhesion were identified. The aminopeptidase N mRNA was expressed in infective sporozoites and during the infection of human HCT-8 enterocytes as revealed by reverse transcription PCR.