Leishmania donovani: expression and characterization of Escherichia coli-expressed recombinant chitinase LdCHT1.
Leishmania parasites produce chitinase activity (EC. 3.2.1.14) thought to be important in parasite-sandfly interactions and transmission of the parasite to the vertebrate host. Previous observations have suggested that parasite chitinases are involved in degradation of the sandfly peritrophic matrix and the chitinous layer of the cardiac valve cuticle. This chitinase activity is thought to produce an incompetent pharyngeal valve sphincter and a route of egress that allow Leishmania promastigotes to be regurgitated into the site of blood feeding. In the studies reported here, enzymatically active L. donovani chitinase LdCHT1 was expressed as a thioredoxin fusion protein in Escherichia coli strain AD494 (DE3). Recombinant LdCHT1 had a predominantly endochitinase activity, in contrast to previous reports of both exo- and endochitinase activities in axenic culture supernatants of diverse Leishmania spp. promastigotes. The predominant endochitinase activity of recombinant LdCHT1 is consistent with the presumed function of the enzyme in disrupting chitinous structures in the sandfly digestive system to allow transmission.
Razek-Desouky
A
A
Department of Pathology, WHO Collaborating Center for Tropical Diseases, University of Texas Medical Branch, 301 University Boulevard, Galveston, Texas, 77555-0609, USA.
Specht
C A
CA
Soong
L
L
Vinetz
J M
JM
D000117:Acetylglucosamine / Q000031:analogs & derivatives* / Q000378:metabolism; D000818:Animals; D002686:Chitin / Q000378:metabolism; D002688:Chitinases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D002850:Chromatography, Gel; D002855:Chromatography, Thin Layer; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005470:Fluorometry; D015971:Gene Expression Regulation, Enzymologic; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D006923:Hymecromone / Q000031:analogs & derivatives* / Q000378:metabolism; D007893:Leishmania donovani / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity
D011994:Recombinant Proteins; D002686:Chitin; C025158:4-methylumbelliferyl 2-acetamido-2-deoxy-beta-D-glucopyranoside; D006923:Hymecromone; D002688:Chitinases; D000117:Acetylglucosamine