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.

Glycosylation of Erythrocyte Spectrin and Its Modification in Visceral Leishmaniasis

“…Therefore, it may be envisaged that oxidative modification of spectrin affects membrane morphology of the erythrocytes. Enhanced fragility, membrane fluidity and hydrophobicity of RBC VL as compared to RBC N were demonstrated earlier [25]. Hence, the evidence of altered spectrin reported here may provide an explanation for the known-impaired stability of erythrocytes in VL.…”

“…Altered binding of highly sialylated spectrin VL with spectrin-depleted inside-out membrane vesicles of RBC N possibly suggested functional abnormality. Membrane characteristics of RBC VL were observed by enhanced hydrophobicity, fragility, fluidity as compared to RBC N hinting towards membrane damage [25].…”

“…The functional attributes of erythrocytes are determined by the structural integrity of the membrane, which is often described in terms of alterations in the membrane characteristics like osmotic fragility, fluidity and hydrophobicity. Any kind of perturbation in the milieu of the erythrocytes like oxidative changes or ligand specific interaction culminates in changes in the membrane characters generally associated with pathological conditions [25]. Therefore, we considered it worthwhile to unravel the molecular determinants and implications of 9- O -AcSGPs on RBC VL .…”

“…Purified spectrin N , spectrin VL or affinity-purified spectrin VL and gel eluted 60 kDa fragment and were analysed by SDS-PAGE (5 and 7.5%) in a minigel apparatus (Bio-Rad, USA) [25] and the gels were stained. N - or O -linked glycosylation of spectrin VL , spectrin N and 60 kDa fragment was demonstrated after deglycosylation with specific glycosidases using deglycosylation kit (Roche Applied Science, Mannheim, Germany) according to the manufacturer's protocol [26].…”

Redox regulation of protein damage in plasma

“…Using a series of glycosidases to prune the glycoform, the effect of oligosaccharide structure has been described for IgG1, with the glycoform changing effector functions of the molecule in binding complement and Fc receptors [24]. In a different approach, the function of C-reactive protein (CRP) with different glycosylation patterns that was isolated from patients with infectious disease was examined; patient CRP caused greater erythrocyte fragility which the authors attributed to the glycosylation pattern and may explain in part the anaemia associated with tuberculosis and leishmaniasis [25].…”