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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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Non-denaturing electrophoresis of rPfM18AAP. Non-denaturing agarose/polyacrylamide gels (left) and immunoblot (right) showing the monomer and tetramer of rPfM18AAP. The protein was electrophoresed through non-denaturing gels containing 5, 6, 7, and 8% acrylamide. rPfM18AAP was detected with the PentaHis™ HRP Conjugate antibody.
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Figure 2: Non-denaturing electrophoresis of rPfM18AAP. Non-denaturing agarose/polyacrylamide gels (left) and immunoblot (right) showing the monomer and tetramer of rPfM18AAP. The protein was electrophoresed through non-denaturing gels containing 5, 6, 7, and 8% acrylamide. rPfM18AAP was detected with the PentaHis™ HRP Conjugate antibody.

Mentions: rPfM18AAP minus the hexahistidine-tag (~65 kDa) separated as three entities with pI 6.6, 6.7 and 6.9 during isoelectric focusing (data not shown). Electrophoresis through non-denaturing gels (Figure 2) and Ferguson plot analysis (Additional file 2) showed that the rPfM18AAP occurred mainly as a monomer, tetramer and higher oligomeric forms. The rPfM18AAP dimer was present in very small amounts. Western blot analysis confirmed that the lower protein band was the rPfM18AAP monomer and that the upper smear included the dimer and tetramer (Figure 2). The smear extended past the tetramer to the top of the gels indicating that there were higher oligomeric forms (e.g. octamer and dodecamer) of rPfM18AAP present. The lower bands seen on the non-denaturing gels were not detected during Western blot analysis with the PentaHis™ HRP Conjugate antibody, indicating that these proteins were contaminants isolated during the purification procedure. Teuscher et al showed that recombinant PfM18AAP occurred as an octamer in its active native state by assaying HPLC fractions for enzymatic activity [32]. This is the same number of subunits as the human aspartyl aminopeptidase [27]. However, the unpublished crystal structures of Clostridium acetobutylicum, Thermotoga maritima, and Pseudomonas aeruginosa M18 proteases (Protein Data Bank: ), showed that these enzymes occur as dodecamers. To resolve the issue of the subunit composition of the native PfM18AAP, crystallization of the enzyme will be required.


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

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

Non-denaturing electrophoresis of rPfM18AAP. Non-denaturing agarose/polyacrylamide gels (left) and immunoblot (right) showing the monomer and tetramer of rPfM18AAP. The protein was electrophoresed through non-denaturing gels containing 5, 6, 7, and 8% acrylamide. rPfM18AAP was detected with the PentaHis™ HRP Conjugate antibody.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Non-denaturing electrophoresis of rPfM18AAP. Non-denaturing agarose/polyacrylamide gels (left) and immunoblot (right) showing the monomer and tetramer of rPfM18AAP. The protein was electrophoresed through non-denaturing gels containing 5, 6, 7, and 8% acrylamide. rPfM18AAP was detected with the PentaHis™ HRP Conjugate antibody.
Mentions: rPfM18AAP minus the hexahistidine-tag (~65 kDa) separated as three entities with pI 6.6, 6.7 and 6.9 during isoelectric focusing (data not shown). Electrophoresis through non-denaturing gels (Figure 2) and Ferguson plot analysis (Additional file 2) showed that the rPfM18AAP occurred mainly as a monomer, tetramer and higher oligomeric forms. The rPfM18AAP dimer was present in very small amounts. Western blot analysis confirmed that the lower protein band was the rPfM18AAP monomer and that the upper smear included the dimer and tetramer (Figure 2). The smear extended past the tetramer to the top of the gels indicating that there were higher oligomeric forms (e.g. octamer and dodecamer) of rPfM18AAP present. The lower bands seen on the non-denaturing gels were not detected during Western blot analysis with the PentaHis™ HRP Conjugate antibody, indicating that these proteins were contaminants isolated during the purification procedure. Teuscher et al showed that recombinant PfM18AAP occurred as an octamer in its active native state by assaying HPLC fractions for enzymatic activity [32]. This is the same number of subunits as the human aspartyl aminopeptidase [27]. However, the unpublished crystal structures of Clostridium acetobutylicum, Thermotoga maritima, and Pseudomonas aeruginosa M18 proteases (Protein Data Bank: ), showed that these enzymes occur as dodecamers. To resolve the issue of the subunit composition of the native PfM18AAP, crystallization of the enzyme will be required.

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