Enzymatic characterization of the Plasmodium vivax chitinase, a potential malaria transmission-blocking target.
The chitinase (EC 3.2.1.14) of the human malaria parasite Plasmodium falciparum, PfCHT1, has been validated as a malaria transmission-blocking vaccine (TBV). The present study aimed to delineate functional characteristics of the P. vivax chitinase PvCHT1, whose primary structure differs from that of PfCHT1 by having proenzyme and chitin-binding domains. The recombinant protein rPvCHT1 expressed with a wheat germ cell-free system hydrolyzed 4-methylumbelliferone (4MU) derivatives of chitin oligosaccharides (beta-1,4-poly-N-acetyl glucosamine (GlcNAc)). An anti-rPvCHT1 polyclonal antiserum reacted with in vitro-obtained P. vivax ookinetes in anterior cytoplasm, showing uneven patchy distribution. Enzymatic activity of rPvCHT1 shared the exclusive endochitinase property with parallelly expressed rPfCHT1 as demonstrated by a marked substrate preference for 4MU-GlcNAc(3) compared to shorter GlcNAc substrates. While rPvCHT1 was found to be sensitive to the general family-18 chitinase inhibitor, allosamidin, its pH (maximal in neutral environment) and temperature (max. at approximately 25 degrees C) activity profiles and sensitivity to allosamidin (IC50=6 microM) were different from rPfCHT1. The results in this first report of functional rPvCHT1 synthesis indicate that the P. vivax chitinase is enzymatically close to long form Plasmodium chitinases represented by P. gallinaceum PgCHT1.
Cell-Free Science and Technology Research Center, Ehime University, 3 Bunkyo-cho, Matsuyama, Ehime 790-8577, Japan.
Satoru
S
Takeo
Daisuke
D
Hisamori
Shusaku
S
Matsuda
Joseph
J
Vinetz
Jetsumon
J
Sattabongkot
Takafumi
T
Tsuboi
D000117:Acetylglucosamine / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D002686:Chitin / Q000378:metabolism; D002688:Chitinases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004792:Enzyme Precursors / Q000737:chemistry; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006923:Hymecromone / Q000031:analogs & derivatives / Q000378:metabolism; D008288:Malaria / Q000517:prevention & control* / Q000635:transmission*; D010966:Plasmodium vivax / Q000201:enzymology* / Q000235:genetics; D015800:Protozoan Proteins; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013696:Temperature; D014312:Trisaccharides / Q000494:pharmacology
D004791:Enzyme Inhibitors; D004792:Enzyme Precursors; D015800:Protozoan Proteins; D011994:Recombinant Proteins; D014312:Trisaccharides; C054851:allosamidin; D002686:Chitin; D006923:Hymecromone; D002688:Chitinases; C402444:pfCHT1, chitinase; D000117:Acetylglucosamine