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

Lauterbach SB, Coetzer TL - Malar. J. (2008)

Bottom Line: An in vitro coupled enzyme assay showed that rPfM18AAP cleaved an N-terminal aspartate from a tripeptide substrate with maximum enzymatic activity at pH 7.5 and 37 degrees C.The spectrin-binding region of PfM18AAP is not found in Homo sapiens, Saccharomyces cerevisiae and otherPlasmodium species homologues.Amino acids expected to be involved in cofactor binding, substrate cleavage and quaternary structure stabilization, are conserved.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Molecular Medicine and Haematology, National Health Laboratory Service, School of Pathology, University of the Witwatersrand, Parktown, Johannesburg 2193, Republic of South Africa.

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 (PfM18AAP) and the human erythrocyte membrane protein spectrin was recently identified using phage display technology. In this study, recombinant (r) PfM18AAP was characterized and the interaction between the enzyme and spectrin, as well as other erythrocyte membrane proteins, analyzed.

Methods: rPfM18AAP 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-PfM18AAP 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: rPfM18AAP 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 rPfM18AAP aggregated into oligomers. An in vitro coupled enzyme assay showed that rPfM18AAP cleaved an N-terminal aspartate from a tripeptide substrate with maximum enzymatic activity at pH 7.5 and 37 degrees C. The spectrin-binding region of PfM18AAP is not found in Homo sapiens, Saccharomyces cerevisiae and otherPlasmodium species homologues. Amino acids expected to be involved in cofactor binding, substrate cleavage and quaternary structure stabilization, are conserved. Blot overlays with rPfM18AAP against spectrin and erythrocyte membrane proteins indicated that rPfM18AAP binds to spectrin, as well as to protein 4.1, protein 4.2, actin and glyceraldehyde 3-phosphate dehydrogenase.

Conclusion: Studies characterizing rPfM18AAP showed that this enzyme interacts with erythrocyte spectrin and other membrane proteins. This suggests that, in addition to its proposed role in hemoglobin digestion, PfM18AAP performs other functions in the erythrocyte host and can utilize several substrates, which highlights the multifunctional role of malaria enzymes.

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Affinity purification of rPfM18AAP. SDS-polyacrylamide gel (left) and immunoblot (right) showing the purification of rPfM18AAP. Lane M – erythrocyte membrane proteins; W – induced E. coli whole cell extract; S – soluble protein fraction after cell lysis; Ub – protein fraction that did not bind to the HIS-Select™ Magnetic Agarose Beads; 1–5 – consecutive 20 mM imidazole washes; E – 200 mM imidazole elution of rPfM18AAP.
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Figure 1: Affinity purification of rPfM18AAP. SDS-polyacrylamide gel (left) and immunoblot (right) showing the purification of rPfM18AAP. Lane M – erythrocyte membrane proteins; W – induced E. coli whole cell extract; S – soluble protein fraction after cell lysis; Ub – protein fraction that did not bind to the HIS-Select™ Magnetic Agarose Beads; 1–5 – consecutive 20 mM imidazole washes; E – 200 mM imidazole elution of rPfM18AAP.

Mentions: Only a small proportion of rPfM18AAP was expressed as soluble protein after induction in Overnight Express™ Instant TB Medium and therefore only ~1 μg of soluble protein was obtained from a one liter E. coli culture. The N-terminal hexahistidine-tag present in rPfM18AAP was detected by Western blot analysis (Figure 1) and purification of rPfM18AAP from E. coli extracts yielded a protein sample of more than 85% purity as determined by scanning densitometry. The rPfM18AAP sample generally contained low molecular weight contaminants (Figure 1). SDS-polyacrylamide gels indicated that rPfM18AAP migrated at ~67 kDa (Figure 1), which approximates the calculated molecular weight (66.9 kDa) of the recombinant protein.


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

Lauterbach SB, Coetzer TL - Malar. J. (2008)

Affinity purification of rPfM18AAP. SDS-polyacrylamide gel (left) and immunoblot (right) showing the purification of rPfM18AAP. Lane M – erythrocyte membrane proteins; W – induced E. coli whole cell extract; S – soluble protein fraction after cell lysis; Ub – protein fraction that did not bind to the HIS-Select™ Magnetic Agarose Beads; 1–5 – consecutive 20 mM imidazole washes; E – 200 mM imidazole elution of rPfM18AAP.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2543045&req=5

Figure 1: Affinity purification of rPfM18AAP. SDS-polyacrylamide gel (left) and immunoblot (right) showing the purification of rPfM18AAP. Lane M – erythrocyte membrane proteins; W – induced E. coli whole cell extract; S – soluble protein fraction after cell lysis; Ub – protein fraction that did not bind to the HIS-Select™ Magnetic Agarose Beads; 1–5 – consecutive 20 mM imidazole washes; E – 200 mM imidazole elution of rPfM18AAP.
Mentions: Only a small proportion of rPfM18AAP was expressed as soluble protein after induction in Overnight Express™ Instant TB Medium and therefore only ~1 μg of soluble protein was obtained from a one liter E. coli culture. The N-terminal hexahistidine-tag present in rPfM18AAP was detected by Western blot analysis (Figure 1) and purification of rPfM18AAP from E. coli extracts yielded a protein sample of more than 85% purity as determined by scanning densitometry. The rPfM18AAP sample generally contained low molecular weight contaminants (Figure 1). SDS-polyacrylamide gels indicated that rPfM18AAP migrated at ~67 kDa (Figure 1), which approximates the calculated molecular weight (66.9 kDa) of the recombinant protein.

Bottom Line: An in vitro coupled enzyme assay showed that rPfM18AAP cleaved an N-terminal aspartate from a tripeptide substrate with maximum enzymatic activity at pH 7.5 and 37 degrees C.The spectrin-binding region of PfM18AAP is not found in Homo sapiens, Saccharomyces cerevisiae and otherPlasmodium species homologues.Amino acids expected to be involved in cofactor binding, substrate cleavage and quaternary structure stabilization, are conserved.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Molecular Medicine and Haematology, National Health Laboratory Service, School of Pathology, University of the Witwatersrand, Parktown, Johannesburg 2193, Republic of South Africa.

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 (PfM18AAP) and the human erythrocyte membrane protein spectrin was recently identified using phage display technology. In this study, recombinant (r) PfM18AAP was characterized and the interaction between the enzyme and spectrin, as well as other erythrocyte membrane proteins, analyzed.

Methods: rPfM18AAP 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-PfM18AAP 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: rPfM18AAP 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 rPfM18AAP aggregated into oligomers. An in vitro coupled enzyme assay showed that rPfM18AAP cleaved an N-terminal aspartate from a tripeptide substrate with maximum enzymatic activity at pH 7.5 and 37 degrees C. The spectrin-binding region of PfM18AAP is not found in Homo sapiens, Saccharomyces cerevisiae and otherPlasmodium species homologues. Amino acids expected to be involved in cofactor binding, substrate cleavage and quaternary structure stabilization, are conserved. Blot overlays with rPfM18AAP against spectrin and erythrocyte membrane proteins indicated that rPfM18AAP binds to spectrin, as well as to protein 4.1, protein 4.2, actin and glyceraldehyde 3-phosphate dehydrogenase.

Conclusion: Studies characterizing rPfM18AAP showed that this enzyme interacts with erythrocyte spectrin and other membrane proteins. This suggests that, in addition to its proposed role in hemoglobin digestion, PfM18AAP performs other functions in the erythrocyte host and can utilize several substrates, which highlights the multifunctional role of malaria enzymes.

Show MeSH
Related in: MedlinePlus