Identification and characterization of a cyclooxygenase-like enzyme from Entamoeba histolytica.
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. PGE2 produced 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 PGE2 but not into PGD2 or PGF2alpha. 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.
Institute of Parasitology, McGill University, Macdonald Campus, 21,111 Lakeshore Road, Ste. Anne de Bellevue, QC, Canada H9X 3V9.
Indranil
I
Dey
Kathy
K
Keller
Adam
A
Belley
Kris
K
Chadee
D000595:Amino Acid Sequence; D000818:Animals; D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000494:pharmacology; D001241:Aspirin / Q000494:pharmacology; D015152:Blotting, Northern; D015139:Blotting, Southern; D002384:Catalysis; D002467:Cell Nucleus / Q000378:metabolism; D003001:Cloning, Molecular; D051545:Cyclooxygenase 1; D051546:Cyclooxygenase 2; D015232:Dinoprostone / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004748:Entamoeba histolytica / Q000201:enzymology*; D004847:Epithelial Cells / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D016678:Genome; D006801:Humans; D015151:Immunoblotting; D007213:Indomethacin / Q000494:pharmacology; D007527:Isoenzymes / Q000737:chemistry* / Q000378:metabolism; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D009092:Mucous Membrane / Q000378:metabolism; D010802:Phylogeny; D011451:Prostaglandin-Endoperoxide Synthases / Q000737:chemistry* / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D012756:Sheep; D013997:Time Factors
D000894:Anti-Inflammatory Agents, Non-Steroidal; D007527:Isoenzymes; D008565:Membrane Proteins; D012333:RNA, Messenger; D051545:Cyclooxygenase 1; D051546:Cyclooxygenase 2; C496511:PTGS1 protein, human; C496540:PTGS2 protein, human; D011451:Prostaglandin-Endoperoxide Synthases; D015232:Dinoprostone; D001241:Aspirin; D007213:Indomethacin