CLONING AND EXPRESSION OF CHITINASES OF ENTAMOEBAE

Entamoeba histolytica (Eh) and Entamoeba dispar (Ed) are protozoan par asites that infect hundreds of millions of persons. In the colonic lum en, amebae form chitin-walled cysts, the infectious stage of the paras ite. Entamoeba invadens (Ei), which infects reptiles and is a model fo r amebic encystation, produces chitin synthase and chitinase during en cystation. Ei cyst formation is blocked by the chitinase-inhibitor all osamidin. Here molecular cloning techniques were used to identify homo logous genes of Eh, Ed, and Ei that encode chitinases (EC 3.2.1.14). T he Eh gene (Eh cht1) predicts a 507-amino acid (aa) enzyme, which has 93 and 74% positional identities with Ed and Ei chitinases, respective ly. The Entamoeba chitinases have signal sequences, followed by acidic and hydrophilic sequences composed of multiple tandemly arranged 7-aa repeats (Eh and Ed) or repeats varying in length (Ei). The aa composi tions of the chitinase repeats are similar to those of the repeats of the Eh and Ed Ser-rich proteins. The COOH-terminus of each chitinase h as a catalytic domain, which resembles those of Brugia malayi (33% pos itional identity) and Manduca sexta (29%). Recombinant Entamoeba chiti nases are precipitated by chitin and show chitinase activity with chit ooligosacharide substrates. Consistent with previous biochemical data, chitinase mRNAs are absent in Ei trophozoites and accumulate to maxim al levels in Ei encysting for 48 h. (C) 1997 Elsevier Science B.V.