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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

Characterization of recombinant DBL4ζ mutant proteins.(A) SDS-PAGE of purified wild type DBL4ζ and mutant proteins on 4–12% bis-Tris polyacrylamide gels. Five micrograms of proteins were used per lane and the broad range pre-stained protein marker (NEB) was used as a reference. Predominant bands at approximately 56 kDa were observed under non-reducing conditions, which shifted upon reduction, which is typical of DBL domains and characteristic of folded proteins with disulphide bonds. Lanes were as follows: (1) Wild type DBL4ζ, (2) E1663R, (3) R1764E, (4) E1663R, R1764E and (5) E1663R, E1665R, R1784E, E1779K. (B) CD spectra of purified recombinant wild type and mutant DBL4ζ proteins. Minima near 208 and 222 nm and maximum near 190 nm indicate the presence of significant α-helical content.
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fig0015: Characterization of recombinant DBL4ζ mutant proteins.(A) SDS-PAGE of purified wild type DBL4ζ and mutant proteins on 4–12% bis-Tris polyacrylamide gels. Five micrograms of proteins were used per lane and the broad range pre-stained protein marker (NEB) was used as a reference. Predominant bands at approximately 56 kDa were observed under non-reducing conditions, which shifted upon reduction, which is typical of DBL domains and characteristic of folded proteins with disulphide bonds. Lanes were as follows: (1) Wild type DBL4ζ, (2) E1663R, (3) R1764E, (4) E1663R, R1764E and (5) E1663R, E1665R, R1784E, E1779K. (B) CD spectra of purified recombinant wild type and mutant DBL4ζ proteins. Minima near 208 and 222 nm and maximum near 190 nm indicate the presence of significant α-helical content.

Mentions: Therefore, to test the role in IgM binding of the charged residues predicted by the model, we expressed mutant recombinant proteins in E. coli and tested the ability of each mutant to bind human IgM. In each mutant the key residue(s) were mutated to residues showing the opposite charge. The proteins tested were single amino acid mutants E1663R and R1764E, double mutant E1663R, R1764E and quadruple mutant E1663R, E1665R, R1764E, E1779K. All proteins were expressed as soluble his-tagged proteins in E. coli and were purified using nickel affinity chromatography followed by size exclusion chromatography. The proteins were assessed by SDS-PAGE and in each case showed a major band about 56 kDa under non-reducing conditions, with a shift upon reduction consistent with the presence of disulfide bonds in the recombinant proteins (Fig. 3A). All proteins showed complex size exclusion chromatograms (an example is shown in Fig. S2) and contained some smaller fragments in addition to the major species (Fig. 3A), possibly due to proteolytic degradation [23]. Western blotting with an anti-his mAb showed that all the proteins of approx. 50 kDa and larger retained the N-terminal his-tag. However, the 27 and 23 kDa fragments were not recognised by the anti-his mAb, and therefore could be bacterial impurities or DBL fragments cleaved at the N-terminus (Fig. S3).


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)

Characterization of recombinant DBL4ζ mutant proteins.(A) SDS-PAGE of purified wild type DBL4ζ and mutant proteins on 4–12% bis-Tris polyacrylamide gels. Five micrograms of proteins were used per lane and the broad range pre-stained protein marker (NEB) was used as a reference. Predominant bands at approximately 56 kDa were observed under non-reducing conditions, which shifted upon reduction, which is typical of DBL domains and characteristic of folded proteins with disulphide bonds. Lanes were as follows: (1) Wild type DBL4ζ, (2) E1663R, (3) R1764E, (4) E1663R, R1764E and (5) E1663R, E1665R, R1784E, E1779K. (B) CD spectra of purified recombinant wild type and mutant DBL4ζ proteins. Minima near 208 and 222 nm and maximum near 190 nm indicate the presence of significant α-helical content.
© Copyright Policy - CC BY
Related In: Results  -  Collection

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

fig0015: Characterization of recombinant DBL4ζ mutant proteins.(A) SDS-PAGE of purified wild type DBL4ζ and mutant proteins on 4–12% bis-Tris polyacrylamide gels. Five micrograms of proteins were used per lane and the broad range pre-stained protein marker (NEB) was used as a reference. Predominant bands at approximately 56 kDa were observed under non-reducing conditions, which shifted upon reduction, which is typical of DBL domains and characteristic of folded proteins with disulphide bonds. Lanes were as follows: (1) Wild type DBL4ζ, (2) E1663R, (3) R1764E, (4) E1663R, R1764E and (5) E1663R, E1665R, R1784E, E1779K. (B) CD spectra of purified recombinant wild type and mutant DBL4ζ proteins. Minima near 208 and 222 nm and maximum near 190 nm indicate the presence of significant α-helical content.
Mentions: Therefore, to test the role in IgM binding of the charged residues predicted by the model, we expressed mutant recombinant proteins in E. coli and tested the ability of each mutant to bind human IgM. In each mutant the key residue(s) were mutated to residues showing the opposite charge. The proteins tested were single amino acid mutants E1663R and R1764E, double mutant E1663R, R1764E and quadruple mutant E1663R, E1665R, R1764E, E1779K. All proteins were expressed as soluble his-tagged proteins in E. coli and were purified using nickel affinity chromatography followed by size exclusion chromatography. The proteins were assessed by SDS-PAGE and in each case showed a major band about 56 kDa under non-reducing conditions, with a shift upon reduction consistent with the presence of disulfide bonds in the recombinant proteins (Fig. 3A). All proteins showed complex size exclusion chromatograms (an example is shown in Fig. S2) and contained some smaller fragments in addition to the major species (Fig. 3A), possibly due to proteolytic degradation [23]. Western blotting with an anti-his mAb showed that all the proteins of approx. 50 kDa and larger retained the N-terminal his-tag. However, the 27 and 23 kDa fragments were not recognised by the anti-his mAb, and therefore could be bacterial impurities or DBL fragments cleaved at the N-terminus (Fig. S3).

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