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Identification of the minimal binding region of a Plasmodium falciparum IgM binding PfEMP1 domain.

Semblat JP, Ghumra A, Czajkowsky DM, Wallis R, Mitchell DA, Raza A, Rowe JA - Mol. Biochem. Parasitol. (2015)

Bottom Line: Here we studied an IgM binding domain from a rosette-mediating PfEMP1 variant, DBL4ζ of TM284var1, and found that the minimal IgM binding region mapped to the central region of the DBL domain, comprising all of subdomain 2 and adjoining parts of subdomains 1 and 3.Site-directed mutagenesis of charged amino acids within subdomain 2, predicted by molecular modelling to form the IgM binding site, showed no marked effect on IgM binding properties.Further work is needed to identify the specific interaction site for IgM within the minimal binding region of PfEMP1.

View Article: PubMed Central - PubMed

Affiliation: Institute of Immunology and Infection Research, Centre for Immunity, Infection and Evolution, School of Biological Sciences, University of Edinburgh, Edinburgh EH9 3FL, United Kingdom.

No MeSH data available.


Related in: MedlinePlus

Binding of DBL4ζ mutants to human IgM by SPR.SPR sensograms showing wild type and mutant DBL4ζ recombinant proteins binding to human IgM. Proteins were diluted two-fold starting at 600 nM. All six dilutions were flowed at the rate of 25 μl/min (200 s contact time and 200 s dissociation) and regenerated with Glycine-HCl (pH 2.5). Values obtained using an uncoated lane were subtracted to give specific binding data. All of the recombinant proteins that contain the R1764E mutation did not fully dissociate, suggesting that some protein may be interacting non-specifically to the chip. Two independent experiments were carried out with similar results.
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fig0020: Binding of DBL4ζ mutants to human IgM by SPR.SPR sensograms showing wild type and mutant DBL4ζ recombinant proteins binding to human IgM. Proteins were diluted two-fold starting at 600 nM. All six dilutions were flowed at the rate of 25 μl/min (200 s contact time and 200 s dissociation) and regenerated with Glycine-HCl (pH 2.5). Values obtained using an uncoated lane were subtracted to give specific binding data. All of the recombinant proteins that contain the R1764E mutation did not fully dissociate, suggesting that some protein may be interacting non-specifically to the chip. Two independent experiments were carried out with similar results.

Mentions: Surface Plasmon Resonance (SPR) was used to assess the binding of single (E1663R and R1764E), double (E1663R, R1764E) and quadruple (E1663R, E1665R, R1764E, E1779K) mutants to immobilized IgM. All mutants bound IgM similarly to the wild type (Fig. 4), with binding affinities (KD) in the nanomolar range for all proteins (Table 3). There were minor differences in KD between some of the proteins, but further investigation with completely pure protein preparations would be required to determine the significance of these minor changes. All mutants containing R1764E showed biphasic association and dissociation curves (Fig. 4). Fitting the data to a model with two independent binding sites gave a KD similar to the value obtained for the single-site model, together with a much tighter complex with no detectable dissociation over the time course of the experiment (koff < 10−6 s−1, the lower limit of the SPR machine). This suggests that a fraction of the R1764E protein preparation bound irreversibly to the chip, probably due to small amounts of aggregates. Critically, however, the rest of the protein bound with similar kinetics to the wild type protein. Overall, the SPR data show that the mutated residues do not play a major role in binding human IgM, although further work would be needed to exclude minor effects.


Identification of the minimal binding region of a Plasmodium falciparum IgM binding PfEMP1 domain.

Semblat JP, Ghumra A, Czajkowsky DM, Wallis R, Mitchell DA, Raza A, Rowe JA - Mol. Biochem. Parasitol. (2015)

Binding of DBL4ζ mutants to human IgM by SPR.SPR sensograms showing wild type and mutant DBL4ζ recombinant proteins binding to human IgM. Proteins were diluted two-fold starting at 600 nM. All six dilutions were flowed at the rate of 25 μl/min (200 s contact time and 200 s dissociation) and regenerated with Glycine-HCl (pH 2.5). Values obtained using an uncoated lane were subtracted to give specific binding data. All of the recombinant proteins that contain the R1764E mutation did not fully dissociate, suggesting that some protein may be interacting non-specifically to the chip. Two independent experiments were carried out with similar results.
© Copyright Policy - CC BY
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4539346&req=5

fig0020: Binding of DBL4ζ mutants to human IgM by SPR.SPR sensograms showing wild type and mutant DBL4ζ recombinant proteins binding to human IgM. Proteins were diluted two-fold starting at 600 nM. All six dilutions were flowed at the rate of 25 μl/min (200 s contact time and 200 s dissociation) and regenerated with Glycine-HCl (pH 2.5). Values obtained using an uncoated lane were subtracted to give specific binding data. All of the recombinant proteins that contain the R1764E mutation did not fully dissociate, suggesting that some protein may be interacting non-specifically to the chip. Two independent experiments were carried out with similar results.
Mentions: Surface Plasmon Resonance (SPR) was used to assess the binding of single (E1663R and R1764E), double (E1663R, R1764E) and quadruple (E1663R, E1665R, R1764E, E1779K) mutants to immobilized IgM. All mutants bound IgM similarly to the wild type (Fig. 4), with binding affinities (KD) in the nanomolar range for all proteins (Table 3). There were minor differences in KD between some of the proteins, but further investigation with completely pure protein preparations would be required to determine the significance of these minor changes. All mutants containing R1764E showed biphasic association and dissociation curves (Fig. 4). Fitting the data to a model with two independent binding sites gave a KD similar to the value obtained for the single-site model, together with a much tighter complex with no detectable dissociation over the time course of the experiment (koff < 10−6 s−1, the lower limit of the SPR machine). This suggests that a fraction of the R1764E protein preparation bound irreversibly to the chip, probably due to small amounts of aggregates. Critically, however, the rest of the protein bound with similar kinetics to the wild type protein. Overall, the SPR data show that the mutated residues do not play a major role in binding human IgM, although further work would be needed to exclude minor effects.

Bottom Line: Here we studied an IgM binding domain from a rosette-mediating PfEMP1 variant, DBL4ζ of TM284var1, and found that the minimal IgM binding region mapped to the central region of the DBL domain, comprising all of subdomain 2 and adjoining parts of subdomains 1 and 3.Site-directed mutagenesis of charged amino acids within subdomain 2, predicted by molecular modelling to form the IgM binding site, showed no marked effect on IgM binding properties.Further work is needed to identify the specific interaction site for IgM within the minimal binding region of PfEMP1.

View Article: PubMed Central - PubMed

Affiliation: Institute of Immunology and Infection Research, Centre for Immunity, Infection and Evolution, School of Biological Sciences, University of Edinburgh, Edinburgh EH9 3FL, United Kingdom.

No MeSH data available.


Related in: MedlinePlus