Sialoglycosylation of RBC in Visceral Leishmaniasis Leads to Enhanced Oxidative Stress, Calpain-Induced Fragmentation of Spectrin and Hemolysis

Abstract

Visceral leishmaniasis (VL) caused by the intracellular parasite Leishmania donovani accounts for an estimated 12 million cases of human infection. It is almost always associated with anemia, which severely complicates the disease course. However, the pathological processes leading to anemia in VL have thus far not been adequately characterized to date. In studying the glycosylation patterns of peripheral blood cells we found that the red blood cells (RBC) of VL patients (RBC_VL) express eight 9-O-acetylated sialoglycoproteins (9-O-AcSGPs) that are not detected in the RBC of healthy individuals (RBC_N). At the same time, the patients had high titers of anti-9-O-AcSGP IgG antibodies in their sera. These two conditions appear to be linked and related to the anemic state of the patients, as exposure of RBC_VL but not RBC_N to anti-9-O-AcSGPs antibodies purified from patient sera triggered a series of responses. These included calcium influx via the P/Q-type but not L-type channels, activation of calpain I, proteolysis of spectrin, enhanced oxidative stress, lipid peroxidation, externalization of phosphatidyl serine with enhanced erythrophagocytosis, enhanced membrane fragility and, finally, hemolysis. Taken together, this study suggests that the enhanced hemolysis is linked to an impairment of membrane integrity in RBC_VL which is mediated by ligand-specific interaction of surface 9-O-AcSGPs. This affords a potential explanation for the structural and functional features of RBC_VL which are involved in the hemolysis related to the anemia which develops in VL patients.

Introduction

Leishmania donovani, the causative organism of visceral leishmaniasis (VL), is an obligatory intracellular parasite that resides and proliferates within the hostile environment of host macrophages [1]. Approximately 12 million humans suffer from VL with an incidence of 0.5 million cases per year and increasing prevalence on the Indian subcontinent [1]. The clinical spectrum of VL ranges from asymptomatic infection to mortality, if untreated.

VL is usually associated with severe anemia which severely complicates the clinical courses and adds to the patients' suffering [2]. In general, the average life span of the erythrocytes of patients with VL (RBC_VL) is significantly reduced [3]. Accordingly the hemoglobin content in blood of these patients is lower than in normal healthy individuals. Despite its profound impact on the patients' fate and chances for recovery, little is known about the pathological processes contributing to the hemolysis and anemia.

Sialic acids (SA) are 9-carbon acidic sugars that constitute a family of monosaccharide and terminal components of glycoproteins and glycolipids that play very crucial roles in intercellular communication and defense against different pathogens under various pathological conditions [4]–[7]. Earlier we have demonstrated exclusive presence of eight VL-associated 9-O-acetylated sialoglycoproteins (9-O-AcSGPs) on RBC_VL not detected on erythrocytes of healthy individuals [8]–[11]. Also the white blood cells of VL patients display different derivatives of sialic acids [12]–[21]. Additionally, high titers of anti-9-O-AcSGP IgG were found in the patients' sera [18], [22] whereas only a fraction of the polyclonal IgG2 purified from normal human serum shows specificity for 9-O-AcSA [23]–[24].

The structural integrity of mammalian erythrocyte is supported by a complex network of different cytoskeleton proteins which comprises of five to seven spectrin subunits linked to actin filaments [25]. Structural and biochemical changes of RBC that cause alterations in the cytoskeleton proteins may lead to degradation of the cell. Modifications like glycation and oxidation of spectrin have been documented in diabetes mellitus and associated to erythrocyte membrane changes [26]–[27]. We recently could demonstrate glycosylation and proteolytic cleavage of spectrin in RBC of VL patients [11].

With the present study we addressed the possible contribution of 9-O-AcSGPs associated with RBC_VL and of anti-9-O-AcSGP IgG_VL antibodies in VL. We report that anti-9-O-AcSGP IgG purified from serum of VL patients trigger a series of pathological events in RBC_VL including (1) altered membrane properties as indicated by increased osmotic fragility, hydrophobicity, morphological changes like vesiculation, cell shrinkage, (2) influx of extracellular calcium ion (Ca²⁺) into the cell through P/Q-type channel and elevation of intracellular Ca²⁺ level, (3) activation of calpain I caused by elevated cytosolic Ca²⁺ accompanied by enhanced fragmentation of human erythrocytic α-spectrin to a 60 kDa 9-O-AcSGP (SGP-60) fragment, (4) enhanced production of reactive oxygen species (ROS) and lipid peroxidation, and (5) externalization of phosphatidyl serine (PS) and erythrophagocytosis of sensitized RBC_VL. This study thus suggests a mechanism of hemolysis in VL patients that may relate to their anemic state.

Materials and Methods

Clinical samples

The study involved clinically confirmed VL patients (Table 1, n = 40; 20 males, 20 females; median age: 30 years) admitted to the School of Tropical Medicine, Kolkata. The diagnosis of VL was based on WHO recommended microscopic demonstration of Leishmania sp. amastigotes in splenic aspirates [28]. Blood was sent to the Indian Institute of Chemical Biology where it was processed immediately and the diagnosis validated by two in-house techniques in which the increased presence of linkage-specific 9-O-AcSGPs on erythrocytes was quantified by an erythrocyte binding assay [9] and anti-9-O-AcSGP antibodies in serum or plasma were detected by ELISA [18], [29]. The serum was also checked for the level of parasite-specific antibodies by ELISA with parasite lysates as coating antigen [22]. The hematological parameters of the patients were indicative of anemia but no other blood cell disorder. Controls included normal healthy individuals from endemic (n = 20) and non-endemic areas (n = 20) of the median age 28 for age matched study. The Institutional Human Ethical Committee had approved the study and samples were taken with the consent of donors, patients.

Table 1. Clinical and laboratory features of patients with visceral leishmaniasis (VL).

Parameters	PatientVL	Normal
Number	Male (n = 20), Female (n = 20)	endemic area (n = 20), non-endemic area (n = 20)
Age (yr)	30±2.25	28±4.32
Height (m)	1.55±0.72	1.65±0.52
Weight (kg)	38.50±4.25	60±5.25
Body Mass Index (BMI)a	16±0.55	22±1.25
Duration of illness (month)	4.02±0.25	Not applicable
RBC count	0.92–2.5×106/µl	4.5–6.2×106/µl
Leukocyte count (/mm3)	3.48–3.55×103	5–11×103
Hemoglobin concn. (g/dl)	5.32±0.32	11.5±2.2
Mean cell hemoglobin concn. (g/dl)	30–31 g/dl	30–36 g/dl
Hematocrit (%)	35	41–53
Reticulocyte count (%)	4.25–5.05	0.5–2.5
Spleen size (cm)	10.72±1.06	Not palpable
Splenic aspirate scoreb	4.05±0.17	Negative
Bilirubin (mg/dl)	1.5–2.5	0.9–1
Albumin (g/dl)	3.0–3.5	3.5–5.5
Aminotransferase	Normal	Normal
Alkalinephosphatase	Normal	Normal
RBC-ELISAc	0.9–1.12	0.2–0.3
BSM-ELISAd	0.77–1.1	0.16–0.23
Parasite-ELISAe	1.1–1.7	0.22–0.32

^aBMI is weight in kilograms divided by height square in meter, normal BMI = 18.5–24.9.

^bSplenic aspirate score is taken to be 4 when the number of parasites per microscopic field ranges from 1–10.

^cRBC-ELISA refers to the antigen-ELISA as described elsewhere [9]. The presence of 9-O-AcSGPs on RBC_VL was determined by exploiting the binding specificity of a lectin, Achatinin-H, which has a restricted specificity towards 9-O-acetylated sialic acids [62] and therefore used as coating antigen. Briefly, Achatinin-H was immobilized in a 96-well plate and allowed to bind with RBC in 4°C for overnight. After washing, bound RBC was lysed by double distilled water. The extent of binding was determined by using a chromogenic substrate 2,7-diamino fluorine dihydrochloride and measuring absorbance values at 620 nm.

^dAnti-9-O-AcSGP antibody was detected by using BSM as coating antigen as described elsewhere [22].

^eParasite specific antibody was detected by using parasite lysate as coating antigen as described elsewhere [8].

Anti-9-O-AcSGP IgG and sensitization of erythrocytes

Antibodies (IgG) specific for 9-O-AcSGPs were affinity purified from pooled sera from three patients (anti-O-AcSGP IgG_VL) and normal healthy (NHS) individuals (anti-O-AcSGP IgG_NHS), respectively, as described by Pal et al. [24]. Briefly, the succession of purification steps was 33% ammonium sulfate fractionation, removal of galactose-binding antibodies by passage through asialo-BSM Sepharose 4B, purification of anti-O-AcSGP by BSM (bovine submandibular mucin) Sepharose 4B affinity chromatograpgy, and isolation of IgG by Protein G affinity chromatography. The yield of anti-9-O-AcSGP IgG purified from 12 ml VL sera was 588±25 µg (49 µg/ml serum) and from 20 ml normal control sera 240±19 µg (12 µg/ml serum). For sensitization with anti-O-AcSGP IgG, RBC (1×10⁷) were suspended in Ringer solution (125 mM NaCl, 5 mM KCl, 1 mM MgSO₄, 32 mM HEPES, 5 mM glucose, 5 mM CaCl₂, pH 7.4) and incubated with the antibodies at 37°C for 30 min. As positive controls, RBC were incubated with calcium ionophore (A23187, 1.0 µM, Sigma) in Ringer solution.

Osmofragility of erythrocytes

RBC (1×10⁷) were incubated in NaCl concentrations from 0.1 to 0.9 g % as indicated for 1 h at 37°C and extent of hemolysis measured spectrophotometrically at 412 nm. Likewise, erythrocytes in Ringer solution were incubated with anti-O-AcSGP IgG antibodies (6 µg/ml) or A23187 (1 µM) or buffer only for 30 min at 37°C. After centrifugation at for 3 min 4000 rpm, the extent of hemolysis in supernatant was determined spectrophotometrically at 412 nm. Hemolysis in distilled water was taken as 100% lysis. Percent hemolysis (%) was calculated as OD_{412 nm} at the given condition/OD_{412 nm} with 100% lysis×100 [20].

Hydrophobicity measurements

Hydrophobicity was detected before and after sensitization of RBC (1×10⁷) with anti-9-O-AcSGP IgG_VL antibodies (6 µg/ml) in phosphate buffered saline (PBS) at 37°C for 30 min. Sensitized erythrocytes were washed with PBS, suspended in PBS and loaded with ANS in PBS (5 µl, 1 mM) for 1 h at 37°C. The binding of ANS to hydrophobic sites on erythrocyte membrane was measured with a spectrofluorimeter (Perkin-Elmer, LS55, Ex_max = 365 nm) as described elsewhere [20], [30]. Fluorescence emission spectra were recorded from 400 to 800 nm with excitation and emission band passes of 5 nm.

Scanning electron microscopy (SEM)

The morphology of RBC before and after sensitization with anti-9-O-AcSGP IgG_VL antibodies (6 µg/ml) at 37°C for 30 min was done by SEM. Cells were fixed overnight with 2.5% glutaraldehyde in PBS followed by an overnight incubation with osmium tetroxide (1%), dehydration in an ethanol series, carbon dioxide by the critical point method, sputter coating with gold and examined with a SEM (Vegaii Lsu, Tescan, Czech Republic) [31]. Micrographs were taken at magnification of 18,000 and about 200 erythrocytes were counted to calculate the percentage of deformed cells.

Measurement of reactive oxygen species

RBC were incubated with 2′,7′-dichlorofluorescein diacetate (H₂DCF-DA,100 µM) in PBS for 30 min at 37°C and sensitized with anti-9-O-AcSGP IgG. ROS generation was detected and quantified by measuring the fluorescence intensity at λ_ex, = 485 nm and λ_em = 538 nm. As controls, ROS generation was determined after treatment of the RBC with N-acetyl cysteine (10 mM), a scavenger of ROS. The results are expressed as fold increase in comparison to untreated erythrocytes [32].

Lipid peroxidation

Lipid peroxidation of erythrocyte membrane was measured by the thiobarbituric acid (TBA) method in which malondialdehyde (MDA), a product of peroxidation reaction of polyunsaturated fatty acids and thioberbituric acid-reactive species (TBA-RS) is used as indicator [33]. In brief, RBC membrane proteins were precipitated in twice volume of trichloro acetic acid (10%) for 30 min at 37°C and centrifuged at 1000× g for 10 min at 4°C. The cleared supernatant was incubated with thiobarbituric acid (0.67% in 7.1% sodium sulphate) at 100°C for 25 min in a water bath. After centrifugation at 1000× g for 10 min the absorbance of the pink color reaction product of MDA with TBA was measured at 535 nm. Calibration standards were generated with 1,1,3,3-tetramethoxypropane. Results are expressed as fold increase in comparison with unsensitized RBC.

Phosphatidyl serine (PS) externalization

RBC (1×10⁶) in annexin V-binding buffer (10 mM HEPES, pH 7.4; 140 mM NaCl, 2.5 mM CaCl₂) were incubated with FITC-labeled annexin V in the dark for 15 min at room temperature and analyzed by flow cytometry. A23187 (1.0 µM) treated and unsensitized RBC served as positive and negative controls, respectively.

Erythrophagocytosis assay

RBC (2×10⁶) with or without sensitization with 6.0 µg/ml anti-O-AcSGP IgG for 30 min at 37°C, were layered over macrophages adhered on cover slips and incubated at 37°C for 1 h. Non-adherent erythrocytes were removed by gentle washing with PBS and cell surface-bound erythrocytes lysed by treatment with Tris-NH₄Cl (140 mM NH₄Cl, 17 mM Tris-HCl, pH 7.6) for 5 min. The slides were stained with diaminobenzidine for erythrocytes and counterstained with Giemsa stain for macrophages. Phagocytosis was calculated as the percentage of macrophages that had ingested one or more erythrocytes [34].

Measurement of cytoplasmic Ca²⁺

Erythrocytes (2×10⁷) were washed in Ringer solution, loaded with Fluo-3/AM (2 µM, Calbiochem, Germany) in Ringer solution for 10 min at 37°C in dark under gentle shaking and washed twice with the same buffer [35]. Fluo 3-loaded erythrocytes were sensitization with buffer only or varying concentrations of anti-9-O-AcSGP IgG (0–40 µg/ml) in Ringer solution for 30 min at 37°C, washed twice and resuspended in same buffer and analyzed by spectrofluorimetry (λ_ex 506 nm, λ_em 530 nm) and flow cytometry. Time dependent increase of intracellular calcium ion was also carried out by incubating RBC with anti-9-O-AcSGP IgG (2.5 µg/ml) for different time point at 37°C followed by flow cytometry. Calibration was done at the end of each experiment. To determine the Ca²⁺ channel type, Fluo 3-loaded RBC_VL (2×10⁷) in Ringer solution containing Ca²⁺ (5 mM) were preincubated without or with the indicated doses of ω-agatoxin TK, a spider venom peptide (P/Q-type Ca²⁺ channel blocker, Calbiochem) or nifedipine (L-type Ca²⁺ channel blocker, Sigma) for 20 min at 37°C, washed, sensitized with anti-9-O-AcSGP IgG (2.5 µg/ml) as above and analyzed by flow cytometry [35]. The Ca²⁺ concentrations were calculated as [Ca²⁺] = K_d [(F−F_min)/(F_max−F)] where K_d is the dissociation constant of the Ca²⁺ Fluo-3 complex (864 nM at 37°C), F fluorescence of anti-9-O-AcSGP IgG-treated RBC, F_max the maximal fluorescence intensity with A23187 treated, F_min corresponds to minimum fluorescence intensity with A23187 and EGTA [36].

Electrophoresis and Western Blotting

RBC (2×10⁷) were incubated or not with anti-9-O-AcSGP IgG_VL (10 µg/ml) for 60 min at 37°C in Ringer solution, washed and lysed by sonication on ice. Cell lysates were centrifuged to remove debris, the proteins separated in SDS-PAGE (10% or 7.5%) and blotted onto nitrocellulose. The Western blots were developed separately with horse reddish-labeled anti-calpain I antibodies (1∶1000, Cell Signaling, USA) and rabbit anti-spectrin antibodies (1∶1000, Sigma, USA). Positive and negative controls were RBC treated with A23187 (1 µM) in Ringer solution for 30 min at 37°C in the absence or presence of EGTA (25 mM), respectively. For confirmation of specificity, RBC were preincubated (15 min, 37°C) or coincubated during sesitization with the calpain inhibitor I N-acetyl-leucyl-leucyl-norleucinal (ALLN; 200 µM, Sigma) in Ringer solution before sensitization or A23187 treatment. The blots were developed with diaminobenzidine and peroxide, scanned densitometrically and analyzed with the Quantity One software (BIO-RAD, USA).

Calpain I assays

Spectrin was purified separately from RBC as described by Ungewickell et al. [37] and confirmed by SDS-PAGE and Western blot analysis as above. Spectrin (3.0 µg) was digested with the indicated doses of active calpain I (Sigma) for 60 min at 24°C in reaction buffer (50 mM HEPES, pH 7.0, 50 mM NaCl, 1 mM NaN₃, 1 mM CaCl₂, 1 mM DTT), the reactions stopped with EGTA (15 mM final concentration) [38] and the reaction product analyzed by SDS-PAGE. For specificity control calpain I (10 µg/ml) was pre-incubated with ALLN (150 µM) for 15 min on ice prior to addition of reaction buffer containing purified spectrin. Gels were stained by Coomassie brilliant blue and band densities were compared by densitometric analysis.

Statistical analysis

Results are reported as mean ± SD. All statistical analyses were done using Excel software (Microsoft Co.). The one or two-tailed t test for significance was performed, P<0.05 was considered significant.

Results

Alterations in the membrane characteristics of RBC in VL

To test the stability of RBC_VL in comparison to RBC_N, the cells were incubated in NaCl solutions with at concentrations ranging from 0 to isoosmotic 0.9 g%, and the degree of hemolysis was determined spectrometrically. The osmofragility thus determined is an indicator of cell stability and alterations in membrane properties. RBC_VL were osmotically more fragile than RBC_N, as indicated by an approximately 3-fold enhancement of lysis at 0.5 g% NaCl (Fig. 1A).

Figure 1. Altered membrane properties and reduced stability of RBC_VL after sensitization with anti-9-O-AcSGP IgG_VL antibodies.

A. The osmotic fragility of RBC_VL (open square) compared to RBC_N (filled square) was measured by degree of hemolysis with increasing osmotic stress by incubation in NaCl (0–0.9%) determined by spectrophotometry. Data are represented as % hemolysis, are means of triplicates ± SD and representative of three independent experiments. B. Sensitization of RBC_VL for hemolysis with anti-9-O-AcSGP IgG_VL antibodies. RBC_VL (open bars) and RBC_N (filled bars) were incubated for 30 min at 37°C with increasing concentration (0.5 µg/ml–12.0 µg/ml) of anti-9-O-AcSGP IgG_VL and anti-9-O-AcSGP IgG_NHS, respectively. The extent of hemolysis was then determined by spectrophotometry. The Spectrophotometric readings of RBC_VL or RBC_N in buffer under similar condition were taken as base values and subtracted from the experimental values. Data are expressed as means ± SD of three independent experiments. C. Kinetics of the hemolysis of RBC_VL and RBC_N after sensitization with anti-9-O-AcSGP IgG_VL antibodies. RBC_VL (open squares) and RBC_N (filled squares) were incubated with equal (6.0 µg/ml) amounts of anti-9-O-AcSGP IgG_VL and anti-9-O-AcSGP IgG_NHS, respectively, for different time points (0–120 min). Base value was routinely subtracted from each experimental value as in Fig. 1B. Data are means ± SD of three independent experiments. D. Hemolysis of RBC_N (filled bars) and RBC_VL (open bars) after sensitization of RBC_VL with anti-9-O-AcSGP IgG_NHS or anti-9-O-AcSGP IgG_VL (6.0 µg/ml), respectively, or incubation with the calcium ionophore A23187 (1 µM) in Ringer solution for 30 min. Data are means ± SD of three independent experiments. E. Enhanced membrane hydrophobicity of RBC_VL after sensitization with anti-9-O-AcSGP IgG_VL. The hydrophobicity was determined spectrofluorimetrically using ANS as indicator. RBC_VL were incubated with anti-9-O-AcSGP IgG_VL antibodies or PBS only for 30 min at 37°C. As controls, RBC_N were incubated with anti-9-O-AcSGP IgG_NHS. The cells were washed with PBS, loaded with ANS and incubated for 1 h at 37°C. The fluorescence emission spectra were recorded from 400 to 850 nm with excitation at 365 nm. The graphs are representatives of the results obtained from three independent experiments. F. Morphological changes of RBC_VL after sensitization with anti-9-O-AcSGP IgG_VL antibodies. RBC_N and RBC_VL were not sensitized or sensitized with anti-9-O-ACSGP IgG_NHS or anti-9-O-AcSGP IgG_VL antibodies, respectively, (6.0 µg/ml) for 30 min at 37°C, and then processed for SEM as described in Materials and Methods. Representative images from three independent experiments are shown. G. Decrease of RBC_VL cell size after sensitization detected by low-angle scattering light flow cytometry (forward scatter, FCS). FCS was measured of RBC_N (upper left panel) and of RBC_VL without sensitization (upper right panel) or with sensitization with anti-9-O-AcSGP IgG_VL (2.5 µg/ml) (lower left panel) or treatment with A23187 (1.0 µM) (lower right panel). Figure 1F is excluded from this article's CC-BY license. See the accompanying retraction notice for more information.

Both RBC_VL and RBC_N were sensitized for 30 min, at 37°C with increasing concentrations of anti-9-O-AcSGP IgG_VL and anti-9-O-AcSGP IgG_NHS, respectively. Sensitization of RBC_VL with 6.0 or 10.0 µg/ml of anti-9-O-AcSGP IgG_VL resulted in a high percentage of lysis, i.e. 50.25±2.0% and 61±3.0%, respectively (Fig. 1B). An increase in the lysis of sensitized RBC_VL with 6.0 µg/ml of anti-9-O-AcSGP IgG_VL was observed over time compared to sensitized RBC_N (Fig. 1C). Sensitization of RBC_VL using 6.0 µg/ml of anti-9-O-AcSGP IgG_VL for 30 min at 37°C resulted in a 4.5 fold higher lysis than un-sensitized RBC_VL i.e. 63±4.0% vs. 14±2.0% (Fig. 1D). Sensitized RBC_VL displayed a 5.7-fold higher lysis compared to sensitized RBC_N. A23187 in the presence of Ca²⁺ induced the maximum possible hemolysis of the erythrocytes.

RBC_VL exhibited higher membrane hydrophobicity than RBC_N, as indicated by increased fluorescence due to enhanced ANS binding. Sensitized RBC_VL demonstrated a further increase in ANS binding and enhanced hydrophobicity compared to un-sensitized erythrocytes (Fig. 1E). A blue shift of the emission maxima from 544 to 500 nm in sensitized RBC_VL suggested that sensitization increased the membrane hydrophobicity resulting in a greater number of accessible sites for the binding of ANS. Unsensitized RBC_VL exhibited a higher hydrophobicity than sensitized RBC_N (emission maxima at 560 nm). Unsensitized RBC_N displayed only a negligible reading.

SEM revealed sensitized RBC_VL to have a greater number of ultra structural morphological changes compared to un-sensitized RBC_VL, suggesting a stressed condition in these erythrocytes (Fig. 1F). The presence of shrunken RBC_VL, as reflected by morphometric analyses indicating membrane alterations due to a sensitization of the 9-O-AcSGPs. The sensitized RBC_N did not exhibit any noteworthy alterations of the membrane, retaining a normal discoid shape.

Altered cell morphology in RBC_VL

For further demonstration of changes in cell size, we used flow cytometry to monitor the forward light scattering of RBC_VL before and after sensitization. The forward scattering data (FSC) showed there were only 27% un-sensitized RBC_VL in M1 as compared to 73% cells in M2 (Fig. 1G). In contrast, sensitized RBC_VL exhibited a higher percentage (59%) of cells in M1. RBC_N exhibited only 17% cells in M1. A considerably enhanced (80%) percentage of cells were observed in the A23187-treated RBC_VL.

Sensitized RBC_VL exhibit enhanced ROS generation, lipid peroxidation and externalization of PS

The generation of ROS is usually linked with membrane damage, specifically lipid-peroxidation, which may lead to membrane alterations such as PS externalization which are associated with cell death. As mature RBC is devoid of intracellular components such as nuclei and mitochondria, alterations may also result in these cells in a stressed condition. To address the question whether anti-9-O-AcSGP IgG_VL is capable of inducing stress, we measured ROS generation, lipid peroxidation and PS-externalization in sensitized RBC_VL. 5.7 and 3-fold higher levels of ROS and TBA-RS were observed in sensitized RBC_VL compared to un-sensitized erythrocytes (Fig. 2A–B). RBC_N displayed negligible ROS generation and insignificant lipid peroxidation. Sensitized RBC_VL also showed enhanced annexin-V binding (32±5%) compared to un-sensitized (0.2±0.01%) erythrocytes, indicating a rapid externalization of PS, whereas sensitized and unsensitized RBC_N demonstrated only a negligible percentage of annexin-V positive cells (Fig. 2C–D). A23187 treated RBC_VL exhibited an increase in annexin-V positivity and served as a positive control.

Figure 2. Enhanced ROS, lipid peroxidation and externalization of phosphatidyl serine (PS) in RBC_VL after sensitization with anti-9-O-AcSGP IgG_VL.

A. Induction of ROS in sensitized RBC_VL. Prior to sensitization with anti-9-O-AcSGP IgG_VL or anti-9-O-AcSGP IgG_NHS RBC_VL and RBC_N were incubated with the hydroperoxide indicator H₂DCF-DA in PBS for 30 min at 37°C. Then the antibodies were added, the cell incubated as before and ROS generation determined by fluorimetry. As controls, ROS generation was determined after prior treatment of the RBC with N-acetyl cysteine, a quencher of hydroperoxides. The results are expressed as means ± S.D. (n = 5) of fold increases in comparison to the fluorescence levels detected with untreated erythrocytes. B. Increased lipid peroxidation in sensitized RBC_VL. Lipid peroxidation of erythrocyte membrane was detected and quantified with thiobarbituric acid (TBA) and the TBA-reactive species (TBA-RS) measured with a spectrophotometer at 532 nm. The results are mean ± S.D of fold increase in comparison with unsensitized RBC of five independent measurments. C–D. Enhanced externalization of phosphatidylserine on sensitized RBC_VL. Sensitized or unsensitized erythrocytes were incubated in annexin-V binding buffer with FITC-annexin-V and analyzed by flow cytometry. A23187-treated RBC in the presence of Ca²⁺ served as positive control. The results are shown as representative histogram of three independent experiments (C) and as comparative flow-cytometric analysis (D).

Sensitized RBC_VL exhibit increased erythrophagocytosis

The exposure of PS on the outer leaflet of the plasma membrane is one of the signals that induce macrophages to bind and ingest apoptotic cells [39]. Sensitization of 9-O-AcSGPs on RBC_VL by anti-O-AcSGP IgG_VL antibodies resulted in an increase in phagocytosis of RBC_VL compared to unsensitized, as evidenced by the increase in the percentage of positive macrophages that ingested one or more erythrocytes, from 3.0±1.0 to 52.0±5.0 (Table 2). The extent of phagocytosis may be dependent on the externalization of PS, as suggested by the good correlation (r = 0.92) with annexin-V positivity. In contrast, RBC_N demonstrated negligible uptake by macrophages under identical conditions.

Table 2. Erythrophagocytosis Assay.

	RBCN	RBCVL
a Group	bPositive macrophages (%) uptaking diseased erythrocytes
RBC	2±1	3±1
Sensitized RBC	3±2	52±5

^aRBC_VL and RBC_N (2×10⁶), without or with sensitization by anti-O-AcSGP IgG_VL and anti-O-AcSGP IgG_NHS antibodies and processed for erythrophagocytosis assay as described in Materials and Methods. Results represent the mean ± S.D. of five separate determinations.

^bPositive macrophages (%) are the percentage of macrophages that ingested one or more erythrocytes and was used as the index of phagocytosis. Results represent the mean ± S.D. of five separate determinations.

Intracellular accumulation of Ca²⁺ in sensitized RBC_VL

Spectrofluorimetric data suggested that the cytosolic Ca²⁺ ion content of un-sensitized RBC_VL is slightly higher than RBC_N, suggesting a stressed condition in VL (Fig. 3A). However, the sensitization of 9-O-AcSGPs on Fluo-3-loaded RBC_VL showed a significant increase in fluorescence with increasing concentrations of anti-9-O-AcSGP IgG_VL antibodies, indicating an enhanced Ca²⁺ influx as compared to the un-sensitized erythrocytes (Fig. 3B).

Figure 3. Calcium influx into RBC upon sensitization with anti-9-O-AcSGP IgG_VL antibodies.

For all experiments, erythrocytes were washed and loaded with the Ca²⁺-indicator fluorochrome Fluo-3/AM in Ringer solution. A23187-treated Fluo-3/AM-loaded RBC_VL served as positive controls (black line) and A23187-treated cells in the presence of EGTA as negative controls (gray background). A. Fluo-3/AM loaded RBC_VL and RBC_N (2×10⁷) were left unsensitized or sensitized with varying concentrations of anti-9-O-AcSGP IgG_VL or anti-9-O-AcSGP IgG_NHS, respectively, (0–40 µg/ml) in Ringer solution for 30 min at 37°C. After washing the cells twice with same solution the fluorescence intensities of the Ca²⁺-Fluo-3 complexes was determined by spectrofluorimetry and the levels of intracellular calcium calculated as described in Materials and Methods. B. Cells treated as in A with 0.5 µg/ml (red line), 1.0 µg/ml (green line) and 2.5 µg/ml (pink line) anti-9-O-AcSGP IgG_VL were analyzed by flow cytometry to determine the fraction of responding RBC. C. Similarly, RBC_N were analyzed without and with sensitization with 5 µg/ml anti-9-O-AcSGP IgG_NHS (blue line). D–E. Time dependent increase of intracellular Ca²⁺ in RBC_VL after sensitization with anti-9-O-AcSGP IgG_VL (2.5 µg/ml) for 5 min (red line), 10 min (green line), 20 min (pink line) and 30 min (blue line) at 37°C. Cells were washed and analyzed by flow cytometry. D. Histogram representation; E. Mean fluorescence intensities calculated from D. F–G. Inhibition of the Ca²⁺ influx into RBC sensitized with anti-9-O-AcSGP IgG_VL. Prior to sensitization with anti-9-O-AcSGP-IgG_VL (2.5 µg/ml), the cells were incubated without (green line) or with the P/Q-type channel blocker ω-agatoxin TK (1 µM, pink line, F), or without (green line) or with the L-type channel blocker nifedipine (10 µM, blue line, G) and analyzed by flow cytometry.

Sensitized Fluo-3-loaded RBC_VL also displayed enhanced fluorescence with an increasing concentration of anti-9-O-AcSGP IgG_VL antibodies as compared to un-sensitized erythrocytes, as determined by flow cytometry (Fig. 3C). It is noteworthy that not all of the cells responded equally to stimulation at the lower doses of the antibody (1.0 µg/ml) compared to 2.5 µg/ml, at which almost all of the cells showed higher fluorescence. In contrast, Fluo-3-loaded sensitized RBC_N displayed only a minimal increase in fluorescence, even at higher doses, as compared to un-sensitized RBC_N, suggesting the absence of 9-O-AcSGPs (Fig. 3B, 3D). However, a lack of signaling for other, undetermined reasons cannot be ruled out.

A time dependent increase in intracellular Ca²⁺ was observed in sensitized RBC_VL (Fig. 3E–F). Almost all of the cells exhibited higher Fluo-3 fluorescence after 30 min of stimulation. As expected, Fluo-3-loaded RBC_VL and RBC_N incubated with A23187 exhibited maximum fluorescence and were used as positive control, which effect was decreased in the presence of EGTA, confirming the assay specificity.

P/Q-type channel mediated Influx of Ca²⁺ ions

In order to understand the Ca²⁺ influx pathway of sensitized RBC_VL, we used different concentrations (0.01 µM, 0.05 µM, 0.1 µM, 0.5 µM, 1.0 µM and 2.0 µM) of ω-agatoxin TK, a P/Q-type Ca²⁺-ion channel blocker (Fig. 3G). At a concentration of 1 µM, ω-agatoxin TK strongly inhibited the influx of Ca²⁺ ions by reducing the MFI from 210 arbitrary units to the background value of 43 arbitrary units, suggesting the involvement of P/Q-type calcium channels in the Ca²⁺ ion influx in sensitized RBC_VL. In contrast, nifedipine, an L-type channel blocker, even at a 10 µM concentration, did not inhibit the influx of Ca²⁺ (Fig. 3H). No inhibition could be detected at up to a 50 µM concentration of nifedipine.

Enhanced cytoplasmic Ca²⁺ activated calpain I in sensitized RBC_VL

Increased intracellular Ca²⁺ activates the Ca²⁺-dependent protease calpain I [40]. Therefore, we investigated the involvement of enhanced Ca²⁺ in activating calpain I in sensitized/unsensitized RBC_VL (Fig. 4A). Sensitized RBC_VL displayed an approximate 2-fold increase of the 75 kDa active form of calpain I as a result of Ca²⁺-dependent autoproteolysis of the inactive membrane localized 80 kDa native form, indicating that an increased cytosolic Ca²⁺ level was sufficient for protease activation. It also suggested that the activation of calpain I was dependent on Ca²⁺ through sensitized 9-O-AcSGPs. A23187-treated RBC_VL or RBC_N produced an intense 75 kDa band. Unsensitized RBC_VL inherently contain a less intense 75 kDa active form of calpain I which was completely absent in RBC_N. Sensitization of RBC_N with A23187 in the presence of EGTA resulted in only the native 80 kDa form.

Figure 4. Activation of calpain I in RBC_VL or RBC_N.

A. Cells (2×10⁷) were incubated with or without anti-9-O-AcSGP IgG_VL, anti-9-O-AcSGP IgG_NHS (10 µg/ml) or the Ca²⁺ ionophore A23187 in the absence or presence of EGTA (25 mM) for 60 min at 37°C in Ringer solution, washed with same buffer and lysed by sonication on ice. After removal of cell debris by centrifugation the lysates were separated by SDS-PAGE, and calpain I was detected by Western blot analysis with an anti-calpain-I antibody. B. Enhanced degradation of α-spectrin in sensitized RBC_VL. RBC_VL and RBC_N were suspended separately in Ringer solution and sensitized with anti-9-O-AcSGP IgG_VL, anti-9-O-AcSGP IgG_NHS or A23187 in the absence or presence of EGTA for 30 min at 37°C. The cells were then lysed as before and the lysates subjected to SDS-PAGE and Western blot analysis with a spectrin-specific antibody. C. Involvement of calpain I in the degradation of spectrin in RBC_VL. RBC_VL and RBC_N were pre-incubated with the calpain I inhibitor ALLN in Ringer solution for 30 min at 37°C before sensitization or co-incubated together with anti-9-O-AcSGP IgG_VL or A23187 as positive controls. The cells were processed as before and spectrin detected by Western blot after SDS-PAGE of the cellulaproteins. D. Dependence of the hemolysis of RBC_N on the activation of calpain I. RBC_N were incubated without or with A23187 and with A23187 plus EGTA or ALLN for 1 h at 37°C, and the degree of hemolysis determined spetrophotometrically as in Figure 1. The results are shown as representative bar graphs of three independent experiments.

Activated calpain I induced proteolysis of spectrin_VL in sensitized RBC_VL

Active calpain I cleaves cytoskeleton proteins such as spectrin [40]. The enhancement of the Ca²⁺ influxes upon sensitization suggests an underlying relationship of the 9-O-AcSGPs on RBC_VL and the elevated level of calcium. A four-fold more intense band of the SGP-60 kDa protein and reduced intensity of the α-spectrin band in sensitized RBC_VL suggests an enhanced fragmentation of spectrin_VL compared to un-sensitized RBC_VL (Fig. 4B). EGTA reduced the proteolysis of spectrin_VL to SGP-60, indicating a role for calcium-dependant proteolysis in the fragmentation of spectrin. In support of this notion, A23187/Ca²⁺-treated RBC_VL/RBC_N exhibited an intense SGP-60 band and complete loss of the α-spectrin band, and effect which was reversed by the addition of EGTA (Fig. 4B). Similar treatment of RBC_VL in the presence of ALLN did not exhibit any enhancement of the SGP-60 band (Fig. 4C). The SGP-60 band was absent in sensitized RBC_N in the presence of ALLN (Fig. 4C).

Activated calpain I induced hemolysis in RBC_N

The activation of calpain I caused a degradation of spectrin in RBC. Does this degradation lead to RBC hemolysis? We checked the percentage of hemolysis in A23187-treated RBC_N (Fig. 4D). Approximately 85% of the RBC hemolysis was observed after the activation of calpain I by the A23187 treatment, suggesting a positive correlation of spectrin degradation with cells hemolysis. Calpain I-specific inhibitor (ALLN)-treated RBC or the chelation of cytosolic Ca²⁺ by EGTA reduced the percentage of hemolysis to 12–14%, confirming the assay specificity. RBC_N exhibited only 8.4% hemolysis.

Activated calpain I cleaved purified spectrin

Purified spectrin_VL showed bands of 280, 246 and 60 kDa (Fig. 5A). SGP-60 was not present in spectrin_N. Purified spectrin_VL digested with active calpain I displayed a similar enhancement of fragmented spectrin_VL, as evidenced by the increased presence of the SGP-60 band and reduced intensity of α-spectrin compared to undigested spectrin_VL, suggesting the presence of active calpain in the patient's erythrocytes may be responsible for such proteolysis (Fig. 5B). Similar treatment in the presence of ALLN exhibited a reduced intensity of the SGP-60 band, thus showing the specificity of the reaction (Fig. 5C). Spectrin_N digested with active calpain I also displayed an increase in the intensity of SGP-60 and corresponding decrease in α-spectrin in a calpain I dose-dependent manner (Fig. 5B). The SGP-60 band was absent in both undigested spectrin_N and in the case of treatment with active calpain I in the presence of ALLN (Fig. 5C).

Figure 5. Degradation of purified α-spectrin by activated calpain I.

A. Characterization of purified spectrins. Spectrin_VL and spectrin_N were purified from RBC_VL and RBC_N as described in Materials and Methods, and analyzed on SDS-PAGE (7.5%). The purified spectrins were Western blotted and detected with polyclonal rabbit anti-spectrin antibodies. B. Proteolysis of purified spectrin_VL and spectrin_N by active calpain I. Spectrin_N and Spectrin_VL (3.0 µg) were digested with different doses of active calpain I as indicated in reaction buffer, the reaction was stopped with EGTA and the products analyzed by SDS-PAGE. C. Inhibition of proteolysis by the calpain I inhibitor ALLN. Calpain I was preincubated with ALLN for 15 min on ice prior to addition of reaction buffer containing purified spectrin_VL or spectrin_N and processed as before.

Discussion

Studies reported by our group initially demonstrated the presence of 9-O-AcSGPs and anti-9-O-AcSGP antibodies in VL [22], [29]. Earlier we have reported the presence of only two 9-O-AcSGPs of molecular weight 36 kDa and 144 kDa on PBMC_N respectively [41]–[43]. In contrast, several other distinct VL–associated newly induced 9-O-AcSGPs (19, 56, 65 kDa) were demonstrated on PBMC_VL [21], [41], [44]. Interestingly, almost 40% of the membrane proteins present on the RBC_VL were 9-O-acetylated, that were totally absent on RBC_N which further signified their link with disease pathogenesis [8]–[11]. However, little progress has been made determining the extent of the contribution of 9-O-AcSGPs to the enhanced hemolysis of RBC in VL in the active disease state. Accordingly, our aim was to investigate the specific role of 9-O-AcSGPs in RBC_VL hemolysis and their ligand-specific interaction with anti-9-O-AcSGP IgG_VL. The major achievement of the study was to demonstrate the involvement of VL-associated 9-O-AcSGPs in triggering the altered cellular and membrane biochemical characteristics leading to the phagocytosis of these altered erythrocytes.

Sensitization of 9-O-AcSGP using anti-O-AcSGP IgG_VL antibodies led to an alteration of the membrane characteristics, as evidenced by enhanced osmotic fragility and hydrophobicity, suggesting a mechanism for the membrane damage which developed in RBC_VL in contrast with RBC_N. Moreover, profound ultrastructural changes in morphology from the normal discoid shape and oxidative stress induced in the sensitized RBC_VL indicate definite alterations in their membranes, suggesting a key role for 9-O-AcSGPs. Similar alterations in erythrocyte membrane organization have been documented in Fanconi's anemia [45] and acute childhood lymphoblastic leukemia [33].

The physiological concentration of anti-9-O-AcSGPs antibodies in normal serum against two 9-O-AcSGPs (36 kDa and 144 kDa) present on PBMC_N is only 11–13 µg/ml [13], [18], [22], [46]. However, the total anti-9-O-AcSGPs (54 µg/ml) in VL-serum are developed against several 9-O-AcSGPs (112, 107, 103, 57, 51, and 48 kDa) newly induced both on PBMC_VL and RBC_VL. Therefore, it may be envisages that the enhanced anti-9-O-AcSGP antibody found in the VL serum is definitely different from the antibody present in normal human serum. Hence, affinity purified anti-9-O-AcSGP_VL antibody used to sensitize 9-O-AcSGPs on RBC_VL certainly has a distinct identity, specific and active. Accordingly, even a lower concentration (6 µg/ml) of anti-9-O-AcSGP IgG_VL antibody used for sensitization is capable of inducing 5.7-fold higher degree of RBC_VL hemolysis compared to sensitized RBC_N. This observation signifies that even lower doses of the anti-9-O-AcSGP IgG_VL antibody through the ligand-specific interaction could play an important role in hemolysis of RBC_VL leading to anemia.

In contrast, no significant increase in the hemolysis (%) of RBC_N was observed even at higher concentration of the anti-9-O-AcSGP IgG_N antibody because of the negligible presence of 9-O-AcSGPs on normal cells. This further indicated that this change in red cell morphology due to this specific ligand-mediated interaction was VL-associated.

ROS are linked to cell death signaling in a variety of cell types. Loss of membrane PS asymmetry has been reported in human erythrocytes, sickle cell disease, thalassemia and diabetes [39], [47]–[48]. Increased oxidative stress in erythrocytes of Leishmania-infected hamsters has been reported [49]. A 6-fold higher ROS generation and a 4-fold increased lipid peroxidation in sensitized RBC_VL suggested that the signaling through 9-O-AcSGPs was indeed VL-associated.

PS exposure on the outer leaflet of the cell membrane serves as a signal for the removal of apoptotic cells from the circulation [50]. Sensitized RBC_VL demonstrated display an enhanced externalization of PS, an event which is reported to be correlated with membrane damage in other diseases [46], [47]–[48], [50]–[51]. A 17-fold higher erythrophagocytosis of sensitized RBC_VL was demonstrated which indicated their efficient removal from the circulation, suggesting a probable cause for the anemia- associated VL patients.

Sensitization of RBC_VL with anti-9-O-AcSGP IgG_VL resulted in an increase in the cytosolic Ca²⁺ level. However, the uptake of Ca²⁺ was not equal in all of the cells, suggesting that the 9-O-AcSGP content of the cells is also not equal, with possibly a few cells having a higher number of 9-O-AcSGPs stimulated earlier at a lower dose of antibody and earlier time point. The increased cytosolic Ca²⁺ causes the activation of calpain I, which in turn meditated an enhanced proteolysis of spectrin_VL. Hence, the possible mechanism of a destabilization of RBC by damaging cytoskeleton proteins which in turn leads to hemolysis has been established in VL, with a cell-specific role for 9-O-AcSGPs.

Calcium must be taken up from the extracellular compartment into the inside of the cell, as erythrocytes are devoid of any Ca²⁺ storage organelles such as the endoplasmic reticulum and mitochondria. We performed a channel-inhibition experiment in order to characterize the specific type of channel utilized in the course of ion influx. Inhibition of Ca²⁺ influx by ω-agatoxin TK confirmed the involvement of the P/Q-type channel. In contrast, even at higher doses, an L-type channel blocker (nifedipine) was unable to block the influx of Ca²⁺ during the sensitization process, clearly showing that this influx was not through the L-type channel.

The sensitization of RBC_VL caused activation of the Ca²⁺ channel and along with an enhanced influx of the Ca²⁺ ion, which may have further caused the activation of the Na⁺/K⁺ ion channel [52]. Activation of the Na⁺/K⁺ ion channel opens up the Gardos channel followed by an efflux of water from the cells, as reflected by the shift of a significant population of sensitized cells towards a lower FSC. The RBC_VL cell size was typically lower than RBC_N, possibly due to the higher level of intracellular Ca²⁺ in the VL condition.

Spectrin, a cytoskeleton membrane protein which is crucial for the maintenance of the structural integrity of the cell, is thought to be a target of calpain-mediated proteolysis [53]. Supporting this idea, we observed increased cytosolic Ca²⁺ activated calpain I in sensitized RBC_VL, as evidenced by the appearance of the active form mediating the proteolysis of spectrin_VL. Calpain is activated by autoproteolysis and calpain I relocates from the cytoplasm to the inner surface of the plasma membrane, where it may cause damage to the cytoskeleton structure [40]. It degrades cytoskeletal proteins in neuronal cells during cerebral malaria, traumatic/post-traumatic neurodegeneration [54]–[57], aneurysmal subarachnoid hemorrhage [58] and spinal cord ischemia [59]. Calcium and phenylhydrazine-induced proteolysis of spectrin in rat and human erythrocytes has been documented [60]–[61].

The enhanced presence of SGP-60 in sensitized RBC_VL which indicates the enhanced degradation of spectrin may be due to Ca²⁺-mediated proteolysis of cytoskeleton proteins destabilizing RBC_VL. However, SGP-60 is already present in unsensitized RBC_VL and its lack in RBC_N suggested an association of degraded spectrin with the active disease state. The identification of SGP-60 as a fragment of erythrocytic α-I spectrin was confirmed by sequencing [11]. The fragmentation of purified spectrin_VL/spectrin_N by active calpain I displayed a similar pattern of spectrin-proteolysis as in RBC_VL, suggesting an enhanced activation of calpain in RBC_VL in vivo. Erythrocytes may have lost their membrane integrity as a consequence of spectrin degradation, as reflected in the enhanced hemolysis of the A23187-sensitized RBC. A higher degree of RBC_N hemolysis may occur through the activation of calpain I, as chelating the cellular Ca²⁺ by EGTA suppressed the hemolysis. This was corroborated when a specific inhibitor of calpain I which blocks spectrin proteolysis (ALLN) was used, resulting in a decrease in the degree of hemolysis.

Taken together, the evidence suggests that 9-O-acetylated sialoglycoproteins have an important role in Ca²⁺ influx, activating calpain-I, which in turn cleaves spectrin, causing destabilization of RBC_VL and ultimately their removal through phagocytosis by macrophages. Hence the study findings have yielded important insight into the pathophysiological role of 9-O-AcSGPs on RBC_VL, including potential cell-biological mechanisms which result in anemia (Fig. S1).

Supporting Information

Figure S1

Overview of the proposed mechanism. A hypothetical model has been shown in describing the role of 9-O-AcSGPs for the hemolysis of erythrocytes in VL. The model highlights the possible events inside the RBC_VL including activation of calpain I followed by spectrin degradation and phosphatidyl serine exposure after sensitization of 9-O-AcSGPs by anti-9-O-AcSGP IgG_VL.

(TIF)

Funding Statement

This work received financial support from the CSIR-IICB, Department of Biotechnology (GAP235) (http://dbtindia.nic.in/index.asp), Indian Council of Medical Research (GAP266) (http://www.icmr.nic.in/), New Delhi, J.C. Bose Fellowship, Department of Science and Technology (P90807) (http://www.dst.gov.in/), Govt. of India. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.