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J Biol Chem, Vol. 275, Issue 1, 631-641, January 7, 2000
From the Division of Parasitology, National Institute for Medical
Research, Mill Hill, London NW7 1AA, United Kingdom
Plasmodium falciparum subtilisin-like
protease-1 (PfSUB-1) is a protein belonging to the subtilisin-like
superfamily of serine proteases (subtilases). PfSUB-1 undergoes
extensive posttranslational proteolytic processing. The primary
translation product is converted in the parasite endoplasmic reticulum
to p54. This is further processed to p47, which accumulates in
secretory organelles within the merozoite. Here, we present a detailed
study of this processing. In vitro translated PfSUB-1
showed no capacity to undergo autocatalytic processing. However,
parasite extracts contain a protease that cleaves the in
vitro translated proprotein between Asp219 and
Asn220 to form two products of 31 (p31) and 54 kDa; the
latter was indistinguishable from authentic p54 and remained complexed
with p31 in a noncovalent interaction characteristic of that between a
subtilase prodomain and its cognate catalytic domain. Cross-linking
studies showed that this complex also exists in the parasite.
Expression of PfSUB-1 in recombinant baculovirus also resulted in
processing to p54. Mutation of the predicted active site serine
abolished processing. Recombinant p54 was secreted in a complex with
p31, and could be further converted to p47 in vitro.
Conversion required calcium, was an intramolecular autocatalytic
process, and involved a second cleavage between Asp251 and
Ala252. A decapeptide based on sequence flanking
Asp219 was efficiently cleaved by recombinant PfSUB-1. We
conclude that PfSUB-1 is a subtilase with an unusual substrate
specificity and that it is activated by two autocatalytic processing steps.
Maturation and Specificity of Plasmodium
falciparum Subtilisin-like Protease-1, a Malaria Merozoite
Subtilisin-like Serine Protease*
§,¶, and
*
This work was supported by the Medical Research Council of
the United Kingdom and by the UNDP/World Bank/WHO Special Programme for
Research and Training in Tropical Diseases (TDR).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
These authors contributed equally to this work.
§
Present address: Tropical Disease Research Unit, University of
California, San Francisco, VAMC 113B, 4150 Clement St., San Francisco,
CA 94121.
¶
On sabbatical scientific leave from the CNRS-UPR9039 (AFMB),
IBSM 31 Chemin Joseph-Aiguier, 13402 Marseille cedex 20, France. Supported by an EMBO long term fellowship.
To whom correspondence should be addressed. Tel.:
44-181-959-3666, ext. 2127; Fax: 44-181-913-8593; E-mail:
mblackm@nimr.mrc.ac.uk.
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