The M18 aspartyl aminopeptidase of Plasmodium falciparum binds to human erythrocyte spectrin in vitro

• Published in: Malaria Journal
• Main Authors: Coetzer Theresa L, Lauterbach Sonja B
• Format: Article in Journal/Newspaper
• Language: English
• Published: BMC 2008
• Subjects: Arctic medicine. Tropical medicine; RC955-962; Infectious and parasitic diseases; RC109-216; Arctic; Ferguson
• Online Access: https://doi.org/10.1186/1475-2875-7-161; https://doaj.org/article/d975e2075bd44c9d983ce97e517e5486

Abstract Background During erythrocytic schizogony, Plasmodium falciparum interacts with the human erythrocyte membrane when it enters into, grows within and escapes from the erythrocyte. An interaction between the P. falciparum M18 aspartyl aminopeptidase ( Pf M18AAP) and the human erythrocyte membrane protein spectrin was recently identified using phage display technology. In this study, recombinant (r) Pf M18AAP was characterized and the interaction between the enzyme and spectrin, as well as other erythrocyte membrane proteins, analyzed. Methods r Pf M18AAP was produced as a hexahistidine-fusion protein in Escherichia coli and purified using magnetic bead technology. The pI of the enzyme was determined by two-dimensional gel electrophoresis and the number of subunits in the native enzyme was estimated from Ferguson plots. The enzymatic activity over a pH and temperature range was tested by a coupled enzyme assay. Blot overlays were performed to validate the spectrin- Pf M18AAP interaction, as well as identify additional interactions between the enzyme and other erythrocyte membrane proteins. Sequence analysis identified conserved amino acids that are expected to be involved in cofactor binding, substrate cleavage and quaternary structure stabilization. Results r Pf M18AAP has a molecular weight of ~67 kDa and the enzyme separated as three entities with pI 6.6, 6.7 and 6.9. Non-denaturing gel electrophoresis indicated that r Pf M18AAP aggregated into oligomers. An in vitro coupled enzyme assay showed that r Pf M18AAP cleaved an N-terminal aspartate from a tripeptide substrate with maximum enzymatic activity at pH 7.5 and 37°C. The spectrin-binding region of Pf M18AAP is not found in Homo sapiens, Saccharomyces cerevisiae and other Plasmodium species homologues. Amino acids expected to be involved in cofactor binding, substrate cleavage and quaternary structure stabilization, are conserved. Blot overlays with r Pf M18AAP against spectrin and erythrocyte membrane proteins indicated that r Pf M18AAP binds to ...