Scripps VIVO scripps research logo

  • Index
  • Log in
  • Home
  • People
  • Organizations
  • Research
  • Events
Search form
As of April 1st VIVO Scientific Profiles will no longer updated for faculty, and the link to VIVO will be removed from the library website. Faculty profile pages will continue to be updated via Interfolio. VIVO will continue being used behind the scenes to update graduate student profiles. Please contact helplib@scripps.edu if you have questions.
How to download citations from VIVO | Alternative profile options

Active site mapping, biochemical properties and subcellular localization of rhodesain, the major cysteine protease of Trypanosoma brucei rhodesiense

Academic Article
uri icon
  • Overview
  • Research
  • Identity
  • Additional Document Info
  • View All
scroll to property group menus

Overview

authors

  • Caffrey, C. R.
  • Hansell, E.
  • Lucas, K. D.
  • Brinen, L. S.
  • Hernandez, A. A.
  • Cheng, J. N.
  • Gwaltney, S. L.
  • Roush, William
  • Stierhof, Y. D.
  • Bogyo, M.
  • Steverding, D.
  • McKerrow, J. H.

publication date

  • November 2001

journal

  • Molecular and Biochemical Parasitology  Journal

abstract

  • Cysteine protease activity of African trypanosome parasites is a target for new chemotherapy using synthetic protease inhibitors. To support this effort and further characterize the enzyme, we expressed and purified rhodesain, the target protease of Trypanosoma brucei rhodesiense (MVAT4 strain), in reagent quantities from Pichia pastoris. Rhodesain was secreted as an active, mature protease. Site-directed mutagenesis of a cryptic glycosylation motif not previously identified allowed production of rhodesain suitable for crystallization. An invariable ER(A/V)FNAA motif in the pro-peptide sequence of rhodesain was identified as being unique to the genus Trypanosoma. Antibodies to rhodesain localized the protease in the lysosome and identified a 40-kDa protein in long slender forms of T. b. rhodesiense and all life-cycle stages of T. b. brucei. With the latter parasite, protease expression was five times greater in short stumpy trypanosomes than in the other stages. Radiolabeled active site-directed inhibitors identified brucipain as the major cysteine protease in T. b. brucei. Peptidomimetic vinyl sulfone and epoxide inhibitors designed to interact with the S2, S1 and S' subsites of the active site cleft revealed differences between rhodesain and the related trypanosome protease cruzain. Using fluorogenic dipeptidyl substrates, rhodesain and cruzain had acid pH optima, but unlike some mammalian cathepsins retained significant activity and stability up to pH 8.0, consistent with a possible extracellular function. S2 subsite mapping of rhodesain and cruzain with fluorogenic peptidyl substrates demonstrates that the presence of alanine rather than glutamate at S2 prevents rhodesain from cleaving substrates in which P2 is arginine.

subject areas

  • Animals
  • Binding Sites
  • Cysteine Endopeptidases
  • Epoxy Compounds
  • Lysosomes
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Protease Inhibitors
  • Recombinant Proteins
  • Sequence Analysis, DNA
  • Sulfones
  • Trypanosoma brucei rhodesiense
scroll to property group menus

Research

keywords

  • cathepsin
  • recombinant expression
  • rhodesain
  • sleeping sickness
  • trypanosome
scroll to property group menus

Identity

International Standard Serial Number (ISSN)

  • 0166-6851

Digital Object Identifier (DOI)

  • 10.1016/s0166-6851(01)00368-1

PubMed ID

  • 11704274
scroll to property group menus

Additional Document Info

start page

  • 61

end page

  • 73

volume

  • 118

issue

  • 1

©2022 The Scripps Research Institute | Terms of Use | Powered by VIVO

  • About
  • Contact Us
  • Support