title: Functional expression and characterization of Schistosoma mansoni cathepsin B and its trans-activation by an endogenous asparaginyl endopeptidase.
abstract: Peptidases are essential for the establishment and survival of the medically important parasite, Schistosoma mansoni. This helminth expresses a number of gut-associated peptidases that degrade host blood proteins, including hemoglobin, as a means of nutrition. Using irreversible affinity probes, we demonstrate that S. mansoni cathepsin B-like endopeptidase 1 (SmCB1) is the most abundant papain family cysteine peptidase in both the parasite gut and somatic extracts. SmCB1 zymogen (SmCB1pm) was functionally expressed in Pichia pastoris (4-11mgl(-1)). Monospecific and immunoselected antibodies raised against SmCB1pm localized the enzyme exclusively to the gut lumen and surrounding gastrodermis of adult worms. Recombinant SmCB1pm was unable to catalyze its activation, even at low pH. However, recombinant S. mansoni asparaginyl endopeptidase (SmAE), another gut-associated cysteine peptidase, processed and activated SmCB1pm in trans. Consistent with the known specificity of AEs, processing occurred on the carboxyl side of an asparagine residue, two residues upstream of the start of the mature SmCB1 sequence. The remaining pro-region dipeptide was removed by rat cathepsin C (dipeptidyl-peptidase I)-an action conceivably performed by an endogenous cathepsin C in vivo. The activated recombinant SmCB1 is biochemically identical to the native enzyme with respect to dipeptidyl substrate kinetics and pH profiles. Also, the serum proteins, hemoglobin, serum albumin, IgG, and alpha-2 macroglobulin were efficiently degraded. Further, a novel application of an assay to measure the peptidyl carboxypeptidase activity of SmCB1 and other cathepsins B was developed using the synthetic substrate benzoyl-glycinyl-histidinyl-leucine (Bz-Gly-His-Leu). This study characterizes the major digestive cysteine peptidase in schistosomes and defines novel trans-processing events required to activate the SmCB1 zymogen in vitro which may facilitate the digestive process in vivo.
authors.affiliation: Department of Pathology, Tropical Disease Research Unit and Sandler Centre for Basic Research in Parasitic Diseases, University of California-San Francisco, Box 0511, San Francisco, CA 94143, USA. sajid@cgl.ucsf.edu
authors.forename: Mohammed
authors.initials: M
authors.lastname: Sajid
authors.forename: James H
authors.initials: JH
authors.lastname: McKerrow
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Hansell
authors.forename: Mary A
authors.initials: MA
authors.lastname: Mathieu
authors.forename: Kimberley D
authors.initials: KD
authors.lastname: Lucas
authors.forename: Ivy
authors.initials: I
authors.lastname: Hsieh
authors.forename: Doron
authors.initials: D
authors.lastname: Greenbaum
authors.forename: Matthew
authors.initials: M
authors.lastname: Bogyo
authors.forename: Jason P
authors.initials: JP
authors.lastname: Salter
authors.forename: Kee C
authors.initials: KC
authors.lastname: Lim
authors.forename: Christopher
authors.initials: C
authors.lastname: Franklin
authors.forename: Jea-Hyoun
authors.initials: JH
authors.lastname: Kim
authors.forename: Conor R
authors.initials: CR
authors.lastname: Caffrey
mesh_terms: D000818:Animals; D002401:Cathepsin B / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D005753:Gastric Mucosa / Q000378:metabolism; D005786:Gene Expression Regulation; D015801:Helminth Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D010843:Pichia / Q000201:enzymology / Q000235:genetics; D010940:Plant Proteins / Q000378:metabolism*; D012550:Schistosoma mansoni / Q000201:enzymology* / Q000235:genetics; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity; D015533:Transcriptional Activation; D000099240:Asparaginyl Endopeptidase
chemical_list: D002401:Cathepsin B; D003546:Cysteine Endopeptidases; D015801:Helminth Proteins; D010940:Plant Proteins; D000099240:Asparaginyl Endopeptidase