title: Disease-associated glycosylated molecular variants of human C-reactive protein activate complement-mediated hemolysis of erythrocytes in tuberculosis and Indian visceral leishmaniasis.
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 (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.
authors.affiliation: Infectious disease and Immunology Division, Indian Institute of Chemical Biology, 4, Raja S.C Mullick Road, Jadavpur, Kolkata, 700 032, India.
authors.forename: Waliza
authors.initials: W
authors.lastname: Ansar
authors.forename: Sumi
authors.initials: S
authors.lastname: Mukhopadhyay
authors.forename: S K Hasan
authors.initials: SK
authors.lastname: Habib
authors.forename: Shyamasree
authors.initials: S
authors.lastname: Basu
authors.forename: Bibhuti
authors.initials: B
authors.lastname: Saha
authors.forename: Asish Kumar
authors.initials: AK
authors.lastname: Sen
authors.forename: C N
authors.initials: CN
authors.lastname: Mandal
authors.forename: Chitra
authors.initials: C
authors.lastname: Mandal
mesh_terms: 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
chemical_list: D008565:Membrane Proteins; D002097:C-Reactive Protein; D016650:Fluorescein-5-isothiocyanate