Plasmodium falciparum: chymotryptic-like proteolysis associated with a 101-kDa acidic-basic repeat antigen.
Malaria proteinases appear to function in the release of merozoites from infected erythrocytes and the invasion of merozoites into erythrocytes. Chymostatin, an inhibitor of chymotrypsin-like proteinases, inhibits malaria invasion and also inhibits apparent autoproteolysis of a 101-kDa acidic-basic repeat antigen (p101-ABRA) that is found in the vacuolar space surrounding merozoites in Plasmodium falciparum-infected erythrocytes. After purification by a monoclonal antibody (MAb 3D5), p101-ABRA degrades into smaller fragments in the absence of chymostatin. In this study fluorogenic proteinase substrates of the type peptidyl-7-amino-4-trifluoromethyl coumarin with phenylalanine or tyrosine linked to AFC were used to characterize chymotryptic-like activity associated with p101-ABRA. When p101-ABRA from the cell extract of P. falciparum-schizont-infected erythrocytes was affinity purified on MAb 3D5 beads, chymotryptic-like activity bound to the beads. Seventy-four percent to 96% of the activity detected using MeOSuc-KLF-AFC, Suc-LLVY-AFC, or SY-AFC at a pH optimum of 7.0 was removed from the extract and 6 to 33% was detected on the washed beads. Attempts to recover active enzyme eluted from the beads were not successful. Enzymes cleaving two other substrates (MeOSuc-AAPM-AFC and F-AFC) did not significantly bind to mAB 3D5 beads. Chymotryptic-like activity was also associated with p101-ABRA in fractions from sequential DEAE-Sephacel chromatography, Sephacryl S-200 chromatography, and nondenaturing polyacrylamide gel electrophoresis.
Nwagwu
M
M
Department of Immunology, Walter Reed Army Institute of Research, Washington, DC 20307-5100.
Haynes
J D
JD
Orlandi
P A
PA
Chulay
J D
JD
D000595:Amino Acid Sequence; D000818:Animals; D000953:Antigens, Protozoan / Q000378:metabolism*; D015153:Blotting, Western; D002846:Chromatography, Affinity; D002918:Chymotrypsin; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010963:Plasmodium falciparum / Q000201:enzymology / Q000276:immunology*; D015800:Protozoan Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity
D000953:Antigens, Protozoan; D015800:Protozoan Proteins; C079596:p101-ABRA protein, Plasmodium falciparum; D010450:Endopeptidases; D002918:Chymotrypsin