Maturation and specificity of Plasmodium falciparum subtilisin-like protease-1, a malaria merozoite subtilisin-like serine protease.
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 Asp(219) and Asn(220) 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 Asp(251) and Ala(252). A decapeptide based on sequence flanking Asp(219) 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.
Sajid
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M
Division of Parasitology, National Institute for Medical Research, Mill Hill, London NW7 1AA, United Kingdom.
Withers-Martinez
C
C
Blackman
M J
MJ
D000595:Amino Acid Sequence; D000818:Animals; D001692:Biological Transport; D004792:Enzyme Precursors / Q000235:genetics / Q000378:metabolism*; D008954:Models, Biological; D008969:Molecular Sequence Data; D010963:Plasmodium falciparum / Q000201:enzymology*; D014176:Protein Biosynthesis; D011499:Protein Processing, Post-Translational; D015800:Protozoan Proteins; D011994:Recombinant Proteins / Q000378:metabolism; D020539:Sequence Analysis, Protein; D013379:Substrate Specificity; D013381:Subtilisins / Q000096:biosynthesis* / Q000235:genetics
D004792:Enzyme Precursors; D015800:Protozoan Proteins; D011994:Recombinant Proteins; D013381:Subtilisins; C403176:subtilisin-like protease 1, Plasmodium falciparum