Binding activity of duffy binding protein (PkDBPaII) of Plasmodium Knowlesi clinical isolates from Peninsular Malaysia and Malaysian Borneo / Lim Khai Lone
2018
The zoonotic Plasmodium knowlesi is a major cause of human malaria in Malaysia. This
parasite uses the Duffy binding protein (PkDBPαII) to interact with the Duffy antigen
receptor for chemokines (DARC) receptor on human and macaque erythrocytes to initiate
invasion. Previous studies on P. knowlesi have reported distinct Peninsular Malaysia and
Malaysian Borneo PkDBPαII haplotypes. Notably, patients with knowlesi malaria in
Malaysian Borneo often present with hyperparasitemia and more severe disease
compared to patients in Peninsular Malaysia. Therefore, it is our interest to determine the
implication of the genetic variation of PkDBPαII from Peninsular Malaysia and
Malaysian Borneo on binding activity and affinity to erythrocytes. In this study, the
differential binding activity of these haplotypes with human and macaque (Macaca
fascicularis) erythrocytes was investigated through Erythrocyte-binding assays (rosettes
formation assays) and their binding affinities (KD) were determined using Isothermal
Titration Calorimetry (ITC) analysis. The PkDBPαII of Peninsular Malaysia and
Malaysian Borneo were expressed on the surface of COS-7 cells and tested with human
and monkey erythrocytes, with and without anti-Fy6 (anti-Duffy) monoclonal antibody
treatment. Binding activity level was determined by counting the number of rosettes
formed between the transfected COS-7 cells and the erythrocytes. Anti-Fy6 treatment was
shown to completely block the binding of human erythrocytes with the transfected COS7 cells, thus verifying the specific binding of human DARC with PkDBPαII. Interestingly,
the PkDBPαII of Peninsular Malaysia displayed a higher binding activity with human
erythrocytes when compared to the Malaysian Borneo PkDBPαII haplotype (mean
number of rosettes formed = 156.89 ± 6.62 and 46.00 ± 3.57, respectively; P < 0.0001).
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However, no difference in binding activity level was seen in the binding assay using
Macaca fascicularis erythrocytes. This finding was further supported by ITC, whereby
the PkDBPαII of Peninsular Malaysia and Malaysian Borneo were expressed as
recombinant proteins using Escherichia coli expression system. The expressed PkDBPαII
recombinant proteins were affinity purified and refolded back to native state prior to the
biomolecular interaction analysis with human erythrocytes. The result showed that the
binding affinity of PkDBPαII-Peninsular Malaysia (KD value = 7.75 µM) was higher than
PkDBPαII in Malaysian Borneo (KD value = 74.1 µM). This study is the first report of
phenotypic difference between PkDBPαII haplotypes. The biological implication of this
finding is yet to be determined. Therefore, further studies need to be carried out to
determine whether this differential binding level is associated with disease severity of
knowlesi malaria in humans.
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