Disease-associated glycosylated molecular variants of human C-reactive protein activate complement-mediated hemolysis of erythrocytes in tuberculosis and Indian visceral leishmaniasis.
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 (10(4)-10(5) M(-1)) and number of binding sites (10(6)-10(7)sites/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 microg/ml). Thus, it may be postulated that CRP have a protective role towards the clearance of damaged-erythrocytes in these two diseases.
Ansar
Waliza
W
Infectious disease and Immunology Division, Indian Institute of Chemical Biology, 4, Raja S.C Mullick Road, Jadavpur, Kolkata, 700 032, India.
Mukhopadhyay
Sumi
S
Habib
S K Hasan
SK
Basu
Shyamasree
S
Saha
Bibhuti
B
Sen
Asish Kumar
AK
Mandal
C N
CN
Mandal
Chitra
C
D000328:Adult; D002097:C-Reactive Protein / Q000737:chemistry / Q000276:immunology*; D002240:Carbohydrate Sequence; D003167:Complement Activation / Q000276:immunology*; D004910:Erythrocyte Membrane / Q000276:immunology; D004912:Erythrocytes / Q000276:immunology*; D005434:Flow Cytometry; D016650:Fluorescein-5-isothiocyanate / Q000378:metabolism; D006031:Glycosylation; D006461:Hemolysis / Q000276:immunology*; D006801:Humans; D007194:India; D007898:Leishmaniasis, Visceral / Q000276:immunology*; D008565:Membrane Proteins / Q000378:metabolism; D008875:Middle Aged; D008969:Molecular Sequence Data; D011485:Protein Binding; D020349:Surface Plasmon Resonance; D014376:Tuberculosis / Q000276:immunology*; D055815:Young Adult
D008565:Membrane Proteins; D002097:C-Reactive Protein; D016650:Fluorescein-5-isothiocyanate