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Unique insertions within Plasmodium falciparum subtilisin-like protease-1 are crucial for enzyme maturation and

Létitia Jean1, Chrislaine Withers-Martinez, Fiona Hackett

  • 1Division of Parasitology, National Institute for Medical Research, Mill Hill, London NW7 1AA, UK.

Molecular and Biochemical Parasitology
|September 27, 2005
PubMed

Insights

Plasmodium falciparum subtilisin-like protease-1 (PfSUB-1) undergoes critical maturation steps involving unique surface loops. Mutations in these loops impact its processing and activity, revealing distinct requirements for its two autocatalytic maturation stages.

Area of Science:

  • Malaria parasite biology
  • Protease biochemistry
  • Structural biology

Background:

  • Parasite serine proteases are crucial for Plasmodium falciparum's asexual erythrocytic stage.
  • Plasmodium falciparum subtilisin-like protease-1 (PfSUB-1) expression timing suggests a role in erythrocyte invasion.
  • PfSUB-1 maturation involves two autocatalytic processing events, converting an 82 kDa precursor to 54 kDa and then to 47 kDa forms.

Purpose of the Study:

  • To investigate the role of unique surface-associated loops in PfSUB-1 structure and function.
  • To compare PfSUB-1 with related subtilases and identify conserved features.
  • To elucidate the distinct structural requirements for PfSUB-1's two autocatalytic maturation steps.

Main Methods:

  • Comparative sequence analysis of PfSUB-1 and orthologues with bacterial subtilases.
  • Site-directed mutagenesis of six surface-associated loops in PfSUB-1.
  • Expression of PfSUB-1 mutants in an insect cell system.
  • Analysis of protein maturation, stability, and proteolytic activity of mutants.

Main Results:

  • PfSUB-1 contains six surface-associated loops conserved in Plasmodium orthologues but absent in bacterial subtilases.
  • Deletion of loop III did not affect maturation, while deletions of other loops impacted maturation and/or stability.
  • Specific mutations in loop II inhibited maturation and enzyme activity.
  • Mutations in loops V and VI specifically impaired the second autocatalytic processing step.

Conclusions:

  • The six surface-associated loops are important for PfSUB-1 maturation and stability, with distinct roles for individual loops.
  • The two autocatalytic processing steps of PfSUB-1 have different structural requirements.
  • Conversion to the p47 form is not necessary for PfSUB-1's trans-proteolytic activity.

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