Disease-associated glycosylated molecular variants of human C-reactive protein activate complement-mediated hemolysis of erythrocytes in tuberculosis and Indian visceral leishmaniasis |
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Authors: | Waliza Ansar Sumi Mukhopadhyay SK Hasan Habib Shyamasree Basu Bibhuti Saha Asish Kumar Sen CN Mandal Chitra Mandal |
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Institution: | (1) Infectious disease and Immunology Division, Indian Institute of Chemical Biology, 4, Raja S.C Mullick Road, Jadavpur, Kolkata, 700 032, India;(2) Structural Biology and Bioinformatics Division, Indian Institute of Chemical Biology, 4, Raja S.C Mullick Road, Jadavpur, Kolkata, 700 032, India;(3) Department of Organic Chemistry, Indian Institute of Chemical Biology, 4, Raja S.C Mullick Road, Jadavpur, Kolkata, 700 032, India;(4) Department of Tropical Medicine, School of Tropical Medicine, Chittaranjan Avenue, Kolkata, 700073, India;(5) Present address: School of Tropical Medicine, Kolkata, India; |
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Abstract: | Human C-reactive protein (CRP), as a mediator of innate immunity, removed damaged cells by activating the classical complement
pathway. Previous studies have successfully demonstrated that CRPs are differentially induced as glycosylated molecular variants
in certain pathological conditions. Affinity-purified CRPs from two most prevalent diseases in India viz. tuberculosis (TB) and visceral leishmaniasis (VL) have differential glycosylation in their sugar composition and linkages. As anemia is a common manifestation in TB and
VL, we assessed the contributory role of glycosylated CRPs to influence hemolysis via CRP-complement-pathway as compared to
healthy control subjects. Accordingly, the specific binding of glycosylated CRPs with erythrocytes was established by flow-cytometry
and ELISA. Significantly, deglycosylated CRPs showed a 7–8-fold reduced binding with erythrocytes confirming the role of glycosylated
moieties. Scatchard analysis revealed striking differences in the apparent binding constants (104–105 M−1) and number of binding sites (106–107sites/erythrocyte) for CRP on patients’ erythrocytes as compared to normal. Western blotting along with immunoprecipitation
analysis revealed the presence of distinct molecular determinants on TB and VL erythrocytes specific to disease-associated
CRP. Increased fragility, hydrophobicity and decreased rigidity of diseased-erythrocytes upon binding with glycosylated CRP
suggested membrane damage. Finally, the erythrocyte-CRP binding was shown to activate the CRP-complement-cascade causing hemolysis,
even at physiological concentration of CRP (10 μg/ml). Thus, it may be postulated that CRP have a protective role towards
the clearance of damaged-erythrocytes in these two diseases. |
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