SmCB2, a novel tegumental cathepsin B from adult Schistosoma mansoni.
Papain-like cysteine endopeptidases have been recognized as potential targets for chemotherapy and serodiagnostic reagents in infections with the human parasitic helminth Schistosoma. A novel cathepsin B endopeptidase from adult S. mansoni has been isolated and characterized. The enzyme is termed SmCB2 to distinguish it from the first recorded schistosome cathepsin B, SmCB1, also known as Sm31. A rapid and convenient protocol involving anion exchange and affinity chromatography is described for the isolation of SmCB1 and SmCB2 from the same parasite starting material. SmCB2 has been functionally expressed in and purified from Pichia pastoris. Both native and recombinant SmCB2 migrate similarly (33 kDa) by SDS-PAGE. Both display strict acidic pH activity profiles and similar K(m) and k(cat) for dipeptidyl amidomethylcoumarin substrates. We conclude that the recombinant enzyme is properly folded. The S(2) subsite specificity of recombinant SmCB2 exhibits the preferences Phe>Leu>Val>>Arg. By immunoblotting with anti-SmCB2 IgG, a 33 kDa protein was identified in soluble extracts of male schistosomes. By immunohistochemistry, SmCB2 was localized in the tegumental tubercles and parenchyma of males with less product being visualized in the parenchyma of females. The enzyme may be lysosomal and function at the host parasite-interface.
Caffrey
Conor R
CR
Tropical Disease Research Unit, Department of Pathology, Box 0511, University of California San Francisco, San Francisco, CA 94143, USA. caffrey@socrates.ucsf.edu
Salter
Jason P
JP
Lucas
Kimberley D
KD
Khiem
Dustin
D
Hsieh
Ivy
I
Lim
Kee-Chong
KC
Ruppel
Andreas
A
McKerrow
James H
JH
Sajid
Mohammed
M
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002401:Cathepsin B / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D005260:Female; D015801:Helminth Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D008297:Male; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012550:Schistosoma mansoni / Q000201:enzymology* / Q000254:growth & development; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity
D015801:Helminth Proteins; D011994:Recombinant Proteins; C466432:CB2 protein, Schistosoma mansoni; D002401:Cathepsin B