RESUMEN
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.
Asunto(s)
Proteína C-Reactiva/inmunología , Activación de Complemento/inmunología , Eritrocitos/inmunología , Hemólisis/inmunología , Leishmaniasis Visceral/inmunología , Tuberculosis/inmunología , Adulto , Proteína C-Reactiva/química , Secuencia de Carbohidratos , Membrana Eritrocítica/inmunología , Citometría de Flujo , Fluoresceína-5-Isotiocianato/metabolismo , Glicosilación , Humanos , India , Proteínas de la Membrana/metabolismo , Persona de Mediana Edad , Datos de Secuencia Molecular , Unión Proteica , Resonancia por Plasmón de Superficie , Adulto JovenRESUMEN
BACKGROUND: Deficiencies of the complement-regulatory proteins on RBC (RBC(Mal)) of patients with Plasmodium falciparum were reported. Here, we sought to determine the role of affinity-purified C-reactive protein from patients (CRP(Mal)), in modulating the complement-regulatory proteins and downstream effect on complement-cascade. METHODS: CRP(Mal) was characterized by analytical ultracentrifuge and electrophoretic analysis. Surface plasmon resonance, Western blotting, co-immuno-precipitation, flow-cytometry and ELISA determined the binding of CRP(Mal) with RBC(Mal). Modifications of membranes for RBC(Mal)-CRP(Mal) binding were explored by scanning electron microscopy, osmotic and turbulence fragility, hydrophobicity and oxyhemoglobin release. Flow-cytometry, ELISA, Western blotting and Scatchard analysis monitored the status of complement-regulatory proteins on RBC(Mal). Complement-activation via CRP(Mal) was quantified by C3-deposition and hemolysis. RESULTS: CRP(Mal) binds specifically to RBC(Mal) through distinct molecules. Such binding altered the normal discoid-shape of RBC(Mal) with increased membrane fluidity and hydrophobicity. In the presence of CRP, RBC(Mal) showed reduced complement-regulatory proteins (CR1 or CD35, CD55 and CD59) with decreased affinity. These changes caused enhanced C3-deposition and complement-mediated hemolysis. CONCLUSION: Taken together, we have established the contributory effect of CRP(Mal) causing decreased complement-regulatory proteins, possibly providing a new mechanism of complement-fueled RBC(Mal) destruction refractory to erythrophagocytosis and may account for pathogenesis of anemia.