TY - JOUR
T1 - Maturation and substrate processing topography of the Plasmodium falciparum invasion/egress protease plasmepsin X
AU - Mukherjee, Sumit
AU - Nguyen, Suong
AU - Sharma, Eashan
AU - Goldberg, Daniel E.
N1 - Funding Information:
This work was supported by grant AI138447 from the National Institute of Allergy and Infectious Diseases (NIAID) to D.E.G. We thank Dr. Wandy Beatty (WUSTL) for immune-electron microscopy, the Molecular Microbiology Imaging Facility at WUSTL for confocal Airyscan microscopy and the Washington University Center for Cellular Imaging (WUCCI) for SR-SIM microscopy, the Washington University nano research facility for Dynamic Light Scattering experiment, the Proteomics & Mass Spectrometry Facility at the Danforth Plant Science Center for LC/MS data acquisition and analysis, Dr. Bob Krantz (WUSTL) and Dr. David Sibley (WUSTL) for usage of the JASCO-J715 polarimeter and the Synergy HTX microplate reader respectively, Dr. Michael J. Blackman (The Francis Crick Institute, London) for anti-SUB1 and SERA5 antibodies, Dr. Jean-François Dubremetz (University of Montpellier, France) for anti-RON4 antibody and Dr. Eizo Takashima (Ehime University, Japan) for anti-Rh5 antibody. For rPM X production, the codon-engineered PM X gene and protocols were provided by Don Lorimer (UCB Pharma). We also thank Barb Vaupel for assistance with cloning, Dr. Darya Urusova (WUSTL) for gel filtration assays, Dr. Eva Istvan, Dr. Sebastian Nasamu and Dr. Alexander Polino for useful suggestions.
Funding Information:
This work was supported by grant AI138447 from the National Institute of Allergy and Infectious Diseases (NIAID) to D.E.G. We thank Dr. Wandy Beatty (WUSTL) for immune-electron microscopy, the Molecular Microbiology Imaging Facility at WUSTL for confocal Airyscan microscopy and the Washington University Center for Cellular Imaging (WUCCI) for SR-SIM microscopy, the Washington University nano research facility for Dynamic Light Scattering experiment, the Proteomics & Mass Spectrometry Facility at the Danforth Plant Science Center for LC/MS data acquisition and analysis, Dr. Bob Krantz (WUSTL) and Dr. David Sibley (WUSTL) for usage of the JASCO-J715 polarimeter and the Synergy HTX microplate reader respectively, Dr. Michael J. Blackman (The Francis Crick Institute, London) for anti-SUB1 and SERA5 antibodies, Dr. Jean-François Dubremetz (University of Montpellier, France) for anti-RON4 antibody and Dr. Eizo Takashima (Ehime University, Japan) for anti-Rh5 antibody. For rPM X production, the codon-engineered PM X gene and protocols were provided by Don Lorimer (UCB Pharma). We also thank Barb Vaupel for assistance with cloning, Dr. Darya Urusova (WUSTL) for gel filtration assays, Dr. Eva Istvan, Dr. Sebastian Nasamu and Dr. Alexander Polino for useful suggestions.
Publisher Copyright:
© 2022, The Author(s).
PY - 2022/12
Y1 - 2022/12
N2 - The malaria parasite Plasmodium invades a host erythrocyte, multiplies within a parasitophorous vacuole (PV) and then ruptures the PV and erythrocyte membranes in a process known as egress. Both egress and invasion are controlled by effector proteins discharged from specialized secretory organelles. The aspartic protease plasmepsin X (PM X) regulates activity for many of these effectors, but it is unclear how PM X accesses its diverse substrates that reside in different organelles. PM X also autoprocesses to generate different isoforms. The function of this processing is not understood. We have mapped the self-cleavage sites and have constructed parasites with cleavage site mutations. Surprisingly, a quadruple mutant that remains full-length retains in vitro activity, is trafficked normally, and supports normal egress, invasion and parasite growth. The N-terminal half of the prodomain stays bound to the catalytic domain even after processing and is required for proper intracellular trafficking of PM X. We find that this enzyme cleaves microneme and exoneme substrates before discharge, while the rhoptry substrates that are dependent on PM X activity are cleaved after exoneme discharge into the PV. The data give insight into the temporal, spatial and biochemical control of this unusual but important aspartic protease.
AB - The malaria parasite Plasmodium invades a host erythrocyte, multiplies within a parasitophorous vacuole (PV) and then ruptures the PV and erythrocyte membranes in a process known as egress. Both egress and invasion are controlled by effector proteins discharged from specialized secretory organelles. The aspartic protease plasmepsin X (PM X) regulates activity for many of these effectors, but it is unclear how PM X accesses its diverse substrates that reside in different organelles. PM X also autoprocesses to generate different isoforms. The function of this processing is not understood. We have mapped the self-cleavage sites and have constructed parasites with cleavage site mutations. Surprisingly, a quadruple mutant that remains full-length retains in vitro activity, is trafficked normally, and supports normal egress, invasion and parasite growth. The N-terminal half of the prodomain stays bound to the catalytic domain even after processing and is required for proper intracellular trafficking of PM X. We find that this enzyme cleaves microneme and exoneme substrates before discharge, while the rhoptry substrates that are dependent on PM X activity are cleaved after exoneme discharge into the PV. The data give insight into the temporal, spatial and biochemical control of this unusual but important aspartic protease.
UR - http://www.scopus.com/inward/record.url?scp=85135412712&partnerID=8YFLogxK
U2 - 10.1038/s41467-022-32271-7
DO - 10.1038/s41467-022-32271-7
M3 - Article
C2 - 35927261
AN - SCOPUS:85135412712
SN - 2041-1723
VL - 13
JO - Nature Communications
JF - Nature Communications
IS - 1
M1 - 4537
ER -