Cloning and expression of chitinases of Entamoebae.
Entamoeba histolytica (Eh) and Entamoeba dispar (Ed) are protozoan parasites that infect hundreds of millions of persons. In the colonic lumen, amebae form chitin-walled cysts, the infectious stage of the parasite. Entamoeba invadens (Ei), which infects reptiles and is a model for amebic encystation, produces chitin synthase and chitinase during encystation. Ei cysts formation is blocked by the chitinase-inhibitor allosamidin. Here molecular cloning techniques were used to identify homologous genes of Eh, Ed, and Ei that encode chitinases (EC 3.2.1.14). The Eh gene (Eh cht1) predicts a 507-amino acid (aa) enzyme, which has 93 and 74% positional identities with Ed and Ei chitinases, respectively. 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 compositions 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 has a catalytic domain, which resembles those of Brugia malayi (33% positional identity) and Manduca sexta (29%). Recombinant entamoeba chitinases are precipitated by chitin and show chitinase activity with chitooligosacharide substrates. Consistent with previous biochemical data, chitinase mRNAs are absent in Ei trophozoites and accumulate to maximal levels in Ei encysting for 48 h.
de la Vega
H
H
Center for Cancer Research, Massachusetts Institute of Technology, Cambridge 02139, USA.
Specht
C A
CA
Semino
C E
CE
Robbins
P W
PW
Eichinger
D
D
Caplivski
D
D
Ghosh
S
S
Samuelson
J
J
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002688:Chitinases / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics*; D003001:Cloning, Molecular; D004747:Entamoeba / Q000201:enzymology / Q000235:genetics*; D004748:Entamoeba histolytica / Q000201:enzymology* / Q000235:genetics* / Q000254:growth & development; D015870:Gene Expression; D017125:Genes, Protozoan / Q000235:genetics; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D012091:Repetitive Sequences, Nucleic Acid; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid
D011994:Recombinant Proteins; D002688:Chitinases