Identification and Characterization of a Cyclooxygenase-like Enzyme from Entamoeba Histolytica

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 100; no. 23; pp. 13561 - 13566
• Main Authors: Dey, Indranil, Keller, Kathy, Belley, Adam, Chadee, Kris
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences 11.11.2003; National Acad Sciences
• Subjects: Amino Acid Sequence; Amino acids; Animals; Anti-Inflammatory Agents, Non-Steroidal - pharmacology; Antibodies; Aspirin - pharmacology; Biological Sciences; Blotting, Northern; Blotting, Southern; Catalysis; Cell Nucleus - metabolism; Cloning, Molecular; Cyclooxygenase 1; Cyclooxygenase 2; Cyclooxygenase inhibitors; Dinoprostone - metabolism; Dose-Response Relationship, Drug; Electrophoresis, Polyacrylamide Gel; Entamoeba histolytica; Entamoeba histolytica - enzymology; Enzymes; Epithelial Cells - metabolism; Escherichia coli; Escherichia coli - metabolism; Genome; Humans; Immunoblotting; Indomethacin - pharmacology; Isoenzymes - chemistry; Isoenzymes - metabolism; Medical research; Membrane Proteins; Messenger RNA; Molecular Sequence Data; Mucous Membrane - metabolism; Parasites; Phylogeny; Polyclonal antibodies; Prostaglandin-Endoperoxide Synthases - chemistry; Prostaglandin-Endoperoxide Synthases - metabolism; Prostaglandins; Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger - metabolism; Sequence Homology, Amino Acid; Sheep; Time Factors
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.1835863100

The intestinal protozoan parasite Entamoeba histolytica remains a significant cause of morbidity and mortality worldwide. However, almost nothing is known about the molecules secreted by the parasite that modulate host immune responses or epithelial barrier function in the colon. Herein, we describe the isolation and characterization of a cyclooxygenase (COX)-like enzyme in E. histolytica that is responsible for the biosynthesis of prostaglandin (PG)E2. PGE2produced by ameba was constitutive but highly dependent on exogenous arachidonic acid substrate. COX-like activity and the immunoreactive protein were localized to the nuclear fraction of E. histolytica. The COX-like protein (72 kDa) was microsequenced and cloned by reverse transcriptase PCR. Ameba COX showed little homology with COX-1/2 enzymes from different species at the nucleotide and amino acid levels. Surprisingly, the arachidonate-binding domain and heme-coordinating and catalytic sites, which are conserved in other species, were absent in ameba. Ameba COX expressed in Escherichia coli demonstrated COX-like enzyme activity in vitro by converting arachidonic acid into PGE2but not into PGD2or PGF2α. COX activity was inhibited with 1 mM aspirin but not with indomethacin or COX-1/2-specific inhibitors. Taken together, these studies reveal that E. histolytica produces PGE2, by means of a previously undescribed ancestral COX-like enzyme, which could play a major role in pathogenesis and immune evasion.