Journal of Biochemistry Advance Access published April 9, 2015
Regular paper Field: Biochemistry Topic: Glycobiology and Carbohydrate Biochemistry Title: Diet-induced hypercholesterolemia imparts structure-function changes to erythrocyte
Kiran G1, Srikanth C.B2, Salimath P. V1 and Nandini C. D2* 1
Department of Biochemistry and Nutrition, 2Department of Molecular Nutrition
CSIR-Central Food Technological Research Institute, Mysore – 570 020, Karnataka, India
*Corresponding Author Dr. Nandini C. D E-mail address: [email protected]; [email protected]
Tel: +0091-821-2514876 Fax: +0091-821-2517233 Abbreviations: 2AB - 2Aminobenzamide, CS/DS - chondroitin sulphate/dermatan sulphate, DMMB - 1,9-dimethyl methylene blue, ECM - extracellular matrix, GAG - glycosaminoglycan, HDLc - high density lipoprotein cholesterol, LDLc - low density lipoprotein cholesterol, PBS - phosphate buffer saline, TCA - trichloroacetic acid, SFC - starch fed control, SFH - starch fed hypercholesterolemic.
© The Authors 2015. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.
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chondroitin sulfate/dermatan sulfate
Summary Hypercholesterolemia is one of the factors contributing to cardiovascular problems. Erythrocytes are known to contribute its cholesterol to atherosclerotic plaque. Our earlier study showed that erythrocytes overexpress chondroitin sulfate/dermatan sulfate (CS/DS), a linear co-polymer, during diabetes which resulted in increased cytoadherence to extracellular matrix (ECM) components. The present study was carried out to determine if diet-induced hypercholesterolemia had any effect on
hypercholesterolemia did not show quantitative changes in erythrocyte CS/DS but showed difference in proportion of un-sulfated and 4-O-sulfated disaccharides. Erythrocytes from hypercholesterolemic rats showed increased adhesion to ECM components which was abrogated to various extents when subjected to chondroitinase ABC digestion. However, isolated CS/DS chains showed a different pattern of binding to ECM components indicating that orientation of CS/DS chains could be playing a role in binding. Key words Chondroitin sulphate/dermatan sulphate, erythrocytes, extracellular matrix, glycosaminoglycans, hypercholesterolemia.
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erythrocyte CS/DS and impacted cytoadherence to ECM components. Unlike in diabetes, diet-induced
Erythrocyte, a component of the vascular system, plays an important role in normal physiology of body. The behaviour of erythrocytes under various pathological conditions is garnering renewed attention wherein microenvironment has been seen to play a vital role (1). Pathological condition brings about changes in erythrocyte membrane components and in various instance results in increased adhesion of erythrocytes to endothelium leading to micro-vascular occlusion (vasoocclusion) (2). Adhesion of erythrocytes to vascular endothelium has been linked to pathophysiology
was brought to light that changes in endothelial glycocalyx are reflected by changes in erythrocyte glycocalyx hinting at interlinking between the two (8). Erythrocytes also harbour molecules such as Ag332 (9), sequestrin (10), modified band 3 protein (11) and P. falciparum erythrocyte membrane protein (PFEMP-1) (12) on their membrane and have been implicated in cytoadherence during malarial parasite infection. Furthermore, heparan sulphate (HS) class of glycosaminoglycans (GAGs) has been implicated in the rosette formation of erythrocytes infected by malarial parasite which was sensitive to heparinase digestion (13). Erythrocytes have also been detected in atherosclerotic plaques indicating its role in cardiovascular diseases (14, 15). Recently, we demonstrated the presence of chondroitin sulphate/dermatan sulphate (CS/DS) class of GAGs in rat erythrocytes which gets overexpressed in diabetic condition and concomitantly showed increased adhesion to various extracellular matrix components (16) Furthermore, erythrocytes in the combined milieu of high glucose and high cholesterol showed GAG-dependent cytoadherence (17) . CS/DS is an important class of heteropolysaccharides which are long, linear, unbranched and highly negatively charged consisting of repeating disaccharide units – D-glucuronic acid and Nacetyl-D-galactosamine (GalNAc). Virtually all cells in the body express these molecules. Hypercholesterolemia, one of the causes of dyslipidemia can have serious implications on health of an individual. It is marked by increase in total cholesterol levels in blood as well as 3
Downloaded from http://jb.oxfordjournals.org/ at SUNY Health Science Center at Brooklyn on April 12, 2015
of diseases such as malaria (3, 4), sickle cell anaemia (5, 6), and diabetes (7). Moreover, recently, it
erythrocytes. Erythrocytes are known to contribute cholesterol to atherosclerotic plaques (15). Furthermore, erythrocytes in the milieu of hypercholesterolemia have been recorded to undergo changes in osmotic fragility indicating changes in its structure (18). In a clinical study, it was observed that total cholesterol content of erythrocyte membrane was increased in patients with acute coronary syndrome (19).
GAGs play an important role in lipoprotein metabolism along with key enzymes; lipoprotein
also rich in the form of glycocalyx on the endothelial cells lining the arterial walls which plays an important role in binding of oxidized LDL thereby increasing the atherogenicity (23). In view of the above, and based on our recent work on erythrocyte GAGs, our focus in this paper has been to study the impact of diet-induced hypercholesterolemia on erythrocyte GAGs and its likely effect on their adhesion to various extracellular matrix components.
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lipase (20) and hepatic lipase (21). It also plays a key role in uptake of lipoproteins in liver (22). It is
Materials and methods Materials Heparin, type IV collagen, laminin, fibronectin, 2-aminobenzamide (2AB), 1,9-dimethylmethylene blue (DMMB), papain from Carica papaya, anti-chondroitin sulfate (mouse IgM isotype, monoclonal) primary antibody, anti-mouse IgM (µ-chain specific)-FITC conjugated secondary
USA. Standard chondroitin sulphate unsaturated disaccharides, chondroitin sulfate B, and heparan sulphate was from Associates of Cape Cod, USA. Pre-packed disposable PD-10 columns containing Sephadex G-25 (medium), Superdex 200 was from GE Healthcare, USA. Anti-laminin, anti-type IV collagen and anti-fibronectin were obtained from AbCAM, USA. Bovine serum albumin (BSA) was from Genei, Bangalore. All other chemicals and reagents used were of analytical grade. Cholesterol, triglycerides, HDL and LDL kits were procured from Agape, India..
Animals and Diet Male albino Wistar rats [OUTB-Wistar-IND cftri (2c)] weighing between 120-140 g were used for the study which had clearance from Institute Animal Ethical Committee (IAEC NO 244/12). They were kept in individual cages with facilities to keep diet cup and water bottle and were fed ad libitum with standard diet. The animals were grouped based on body weight into control and hypercholesterolemic group. A total of 24 rats were divided into two groups as control (SFC) and two groups as hypercholesterolemia (SFH) containing 6 rats each. After 48 hours of acclimatization with standard diet, rats in hypercholesterolemic group were fed with AIN–93 diet supplemented with 0.5% cholesterol and 0.125% bile salt to induce hypercholesterolemia whereas control rats were fed with normal AIN– 93 diets devoid of cholesterol. The experimental duration was for a period of four months.
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antibody and chondroitinase ABC from Proteus vulgaris (EC 4.2.2.4) were obtained from Sigma,
Isolation of GAGs from erythrocytes This was carried out as detailed earlier (16). Briefly, erythrocytes were separated from platelet-rich plasma and leukocytes according to established protocols (24, 25). Packed erythrocytes were washed thrice in 5 mM PBS (pH 8.0) and were immediately hemolysed by rapid and thorough mixing with chilled 5 mM phosphate buffer, pH 8.0. The erythrocyte ghost pellet obtained after centrifugation was washed sequentially with 2.5 mM and 1.25 mM phosphate buffer, pH 8 (26). It
subjected to papain digestion for 48 h by adding aliquots of freshly prepared enzyme solution at regular intervals of 12 hours (27). Digested samples were then subjected to TCA and alcohol precipitation sequentially so as to get glycosaminoglycans (GAGs). The precipitate was collected, dried using nitrogen gas and reconstituted in minimal volume of water. They were then desalted on a PD10 column using pyridine-acetate buffer and dried. They were reconstituted in minimal water and stored at -20 oC until use. GAG was isolated from erythrocytes from two independent experiments tentatively named as SFC A and SFC B for control animals and as SFH A and SFH B for hypercholesterolemic animals
Biochemical measurements Total cholesterol, triglycerides, LDLc and HDLc were determined in plasma by established methods using kits from Agape pvt ltd. India. Amount of sulphated GAGs were estimated by DMMB assay using chondroitin sulphate B as a reference standard (28).
Cellulose acetate membrane electrophoresis To identify the type of GAGs, samples were subjected to cellulose acetate membrane electrophoresis. This was carried out as described earlier (16).
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was subjected to papain digestion after reconstituting it with 0.1 M phosphate buffer, pH 6.5 and
Flow Cytometry For flow analysis of erythrocytes for CS/DS, initially 2 µl of properly mixed anticoagulated (EDTA) blood was diluted in 1 ml of phosphate buffer saline (stock). From the stock 20 µl of blood sample was again diluted to 1 ml by mixing with phosphate buffer saline (PBS) containing 1 % BSA and was labeled with CS-56 primary antibody at two different antibody dilution for 30 min. After incubation cells were washed twice with PBS containing 1% BSA at 180 g. The pellet was again
30 min in dark. After incubation excess secondary antibody was removed by washing twice with PBS containing 1% BSA at 180 g. Finally the pellet was resuspended in 500 µl PBS and analysed in FACS-Calibur instrument. Disaccharide composition analysis by reverse phase HPLC
Disaccharide composition analysis of CS/DS isolated and purified from erythrocytes of control and hypercholesterolemic rats was carried out by digesting 2 µg (as sulfated GAGs) with chondroitinase ABC for 1 h at 37o C (29). The digested samples were derivatized with 2AB (30). Excess 2AB was removed by paper chromatography using solvent system consisting of butanol: ethanol: water (4:1:1) (31). The 2AB-labeled disaccharides were diluted to 100 µl with 0.1 M Ammonium acetate and an aliquot analyzed by reverse phase HPLC on a Zorbax Eclipse XDB-C18 column over a period of 72 min employing fluorescence detection with excitation and emission wavelengths set at 330 and 420 nm, respectively (32).
Cytoadherence assay Binding of erythrocytes to ECM proteins was evaluated by adherence assay. Varying amounts of type IV collagen, laminin and fibronectin (0-200 ng/well) were coated onto a 96-welled flatbottomed polystyrene microtiter plates (Nunc-Immnuoplate, USA) after diluting them in coating buffer. Excess Poly-L-Ornithine or ECM components, which remained unbound, were removed by
7
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reconstituted in PBS containing 1% BSA and labeled with FITC conjugated secondary antibody for
washing the wells thrice with PBS after overnight standing. To prevent non-specific binding, the sites were blocked with 2 % BSA diluted in PBS for 2 h. Erythrocytes (1X108 cells /100 µl/well), washed in saline containing calcium chloride (CaCl2, 25 mM) were added and incubated overnight at 4 oC. Unbound erythrocytes were removed by subjecting them to a gentle wash with saline containing CaCl2. The erythrocytes were then treated with 0.25% glutaraldehyde exercising utmost care for 10 min at 4 oC. Later the wells were rigorously washed three times and the extent of specifically bound
haemolysis was quantified by measuring the absorbance at 405 nm (33). To determine if CS/DS is involved in adherence, erythrocytes were subjected to chondroitinase ABC (5 mIU units) at 37 oC for 1 h and then seeded into the wells. Other procedures are as detailed. Solid phase immunobinding assay Isolated GAGs were tested for binding to various ECM components such as laminin, type IV collagen and fibronectin. Initially, the wells were coated with poly-L-lysine (400 ng) overnight. Excess poly-L-lysine was removed by repeated washing of the wells. Heparin (500 ng) was immobilized overnight and blocked with 2% BSA. ECM components laminin, type IV collagen and fibronectin, were tested for binding in absence or presence of heparin and isolated GAGs by competition. Binding was detected by specific primary antibodies directed against ECM components followed by ALP-conjugated secondary antibody using pNPP as a substrate. The colour obtained was read at 405 nm.
Statistical analysis All the values are expressed as mean ± SEM or SD of duplicates or triplicates of two or three independent experiments. Statistical analysis was done using Instat software. Significance between
8
Downloaded from http://jb.oxfordjournals.org/ at SUNY Health Science Center at Brooklyn on April 12, 2015
erythrocytes was quantified by cell lysis with double distilled water (100 µl /well) and the degree of
the groups was determined by performing one-way ANOVA with Tukey post t-test. A value of p
Regular paper Field: Biochemistry Topic: Glycobiology and Carbohydrate Biochemistry Title: Diet-induced hypercholesterolemia imparts structure-function changes to erythrocyte
Kiran G1, Srikanth C.B2, Salimath P. V1 and Nandini C. D2* 1
Department of Biochemistry and Nutrition, 2Department of Molecular Nutrition
CSIR-Central Food Technological Research Institute, Mysore – 570 020, Karnataka, India
*Corresponding Author Dr. Nandini C. D E-mail address: [email protected]; [email protected]
Tel: +0091-821-2514876 Fax: +0091-821-2517233 Abbreviations: 2AB - 2Aminobenzamide, CS/DS - chondroitin sulphate/dermatan sulphate, DMMB - 1,9-dimethyl methylene blue, ECM - extracellular matrix, GAG - glycosaminoglycan, HDLc - high density lipoprotein cholesterol, LDLc - low density lipoprotein cholesterol, PBS - phosphate buffer saline, TCA - trichloroacetic acid, SFC - starch fed control, SFH - starch fed hypercholesterolemic.
© The Authors 2015. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.
1
Downloaded from http://jb.oxfordjournals.org/ at SUNY Health Science Center at Brooklyn on April 12, 2015
chondroitin sulfate/dermatan sulfate
Summary Hypercholesterolemia is one of the factors contributing to cardiovascular problems. Erythrocytes are known to contribute its cholesterol to atherosclerotic plaque. Our earlier study showed that erythrocytes overexpress chondroitin sulfate/dermatan sulfate (CS/DS), a linear co-polymer, during diabetes which resulted in increased cytoadherence to extracellular matrix (ECM) components. The present study was carried out to determine if diet-induced hypercholesterolemia had any effect on
hypercholesterolemia did not show quantitative changes in erythrocyte CS/DS but showed difference in proportion of un-sulfated and 4-O-sulfated disaccharides. Erythrocytes from hypercholesterolemic rats showed increased adhesion to ECM components which was abrogated to various extents when subjected to chondroitinase ABC digestion. However, isolated CS/DS chains showed a different pattern of binding to ECM components indicating that orientation of CS/DS chains could be playing a role in binding. Key words Chondroitin sulphate/dermatan sulphate, erythrocytes, extracellular matrix, glycosaminoglycans, hypercholesterolemia.
2
Downloaded from http://jb.oxfordjournals.org/ at SUNY Health Science Center at Brooklyn on April 12, 2015
erythrocyte CS/DS and impacted cytoadherence to ECM components. Unlike in diabetes, diet-induced
Erythrocyte, a component of the vascular system, plays an important role in normal physiology of body. The behaviour of erythrocytes under various pathological conditions is garnering renewed attention wherein microenvironment has been seen to play a vital role (1). Pathological condition brings about changes in erythrocyte membrane components and in various instance results in increased adhesion of erythrocytes to endothelium leading to micro-vascular occlusion (vasoocclusion) (2). Adhesion of erythrocytes to vascular endothelium has been linked to pathophysiology
was brought to light that changes in endothelial glycocalyx are reflected by changes in erythrocyte glycocalyx hinting at interlinking between the two (8). Erythrocytes also harbour molecules such as Ag332 (9), sequestrin (10), modified band 3 protein (11) and P. falciparum erythrocyte membrane protein (PFEMP-1) (12) on their membrane and have been implicated in cytoadherence during malarial parasite infection. Furthermore, heparan sulphate (HS) class of glycosaminoglycans (GAGs) has been implicated in the rosette formation of erythrocytes infected by malarial parasite which was sensitive to heparinase digestion (13). Erythrocytes have also been detected in atherosclerotic plaques indicating its role in cardiovascular diseases (14, 15). Recently, we demonstrated the presence of chondroitin sulphate/dermatan sulphate (CS/DS) class of GAGs in rat erythrocytes which gets overexpressed in diabetic condition and concomitantly showed increased adhesion to various extracellular matrix components (16) Furthermore, erythrocytes in the combined milieu of high glucose and high cholesterol showed GAG-dependent cytoadherence (17) . CS/DS is an important class of heteropolysaccharides which are long, linear, unbranched and highly negatively charged consisting of repeating disaccharide units – D-glucuronic acid and Nacetyl-D-galactosamine (GalNAc). Virtually all cells in the body express these molecules. Hypercholesterolemia, one of the causes of dyslipidemia can have serious implications on health of an individual. It is marked by increase in total cholesterol levels in blood as well as 3
Downloaded from http://jb.oxfordjournals.org/ at SUNY Health Science Center at Brooklyn on April 12, 2015
of diseases such as malaria (3, 4), sickle cell anaemia (5, 6), and diabetes (7). Moreover, recently, it
erythrocytes. Erythrocytes are known to contribute cholesterol to atherosclerotic plaques (15). Furthermore, erythrocytes in the milieu of hypercholesterolemia have been recorded to undergo changes in osmotic fragility indicating changes in its structure (18). In a clinical study, it was observed that total cholesterol content of erythrocyte membrane was increased in patients with acute coronary syndrome (19).
GAGs play an important role in lipoprotein metabolism along with key enzymes; lipoprotein
also rich in the form of glycocalyx on the endothelial cells lining the arterial walls which plays an important role in binding of oxidized LDL thereby increasing the atherogenicity (23). In view of the above, and based on our recent work on erythrocyte GAGs, our focus in this paper has been to study the impact of diet-induced hypercholesterolemia on erythrocyte GAGs and its likely effect on their adhesion to various extracellular matrix components.
4
Downloaded from http://jb.oxfordjournals.org/ at SUNY Health Science Center at Brooklyn on April 12, 2015
lipase (20) and hepatic lipase (21). It also plays a key role in uptake of lipoproteins in liver (22). It is
Materials and methods Materials Heparin, type IV collagen, laminin, fibronectin, 2-aminobenzamide (2AB), 1,9-dimethylmethylene blue (DMMB), papain from Carica papaya, anti-chondroitin sulfate (mouse IgM isotype, monoclonal) primary antibody, anti-mouse IgM (µ-chain specific)-FITC conjugated secondary
USA. Standard chondroitin sulphate unsaturated disaccharides, chondroitin sulfate B, and heparan sulphate was from Associates of Cape Cod, USA. Pre-packed disposable PD-10 columns containing Sephadex G-25 (medium), Superdex 200 was from GE Healthcare, USA. Anti-laminin, anti-type IV collagen and anti-fibronectin were obtained from AbCAM, USA. Bovine serum albumin (BSA) was from Genei, Bangalore. All other chemicals and reagents used were of analytical grade. Cholesterol, triglycerides, HDL and LDL kits were procured from Agape, India..
Animals and Diet Male albino Wistar rats [OUTB-Wistar-IND cftri (2c)] weighing between 120-140 g were used for the study which had clearance from Institute Animal Ethical Committee (IAEC NO 244/12). They were kept in individual cages with facilities to keep diet cup and water bottle and were fed ad libitum with standard diet. The animals were grouped based on body weight into control and hypercholesterolemic group. A total of 24 rats were divided into two groups as control (SFC) and two groups as hypercholesterolemia (SFH) containing 6 rats each. After 48 hours of acclimatization with standard diet, rats in hypercholesterolemic group were fed with AIN–93 diet supplemented with 0.5% cholesterol and 0.125% bile salt to induce hypercholesterolemia whereas control rats were fed with normal AIN– 93 diets devoid of cholesterol. The experimental duration was for a period of four months.
5
Downloaded from http://jb.oxfordjournals.org/ at SUNY Health Science Center at Brooklyn on April 12, 2015
antibody and chondroitinase ABC from Proteus vulgaris (EC 4.2.2.4) were obtained from Sigma,
Isolation of GAGs from erythrocytes This was carried out as detailed earlier (16). Briefly, erythrocytes were separated from platelet-rich plasma and leukocytes according to established protocols (24, 25). Packed erythrocytes were washed thrice in 5 mM PBS (pH 8.0) and were immediately hemolysed by rapid and thorough mixing with chilled 5 mM phosphate buffer, pH 8.0. The erythrocyte ghost pellet obtained after centrifugation was washed sequentially with 2.5 mM and 1.25 mM phosphate buffer, pH 8 (26). It
subjected to papain digestion for 48 h by adding aliquots of freshly prepared enzyme solution at regular intervals of 12 hours (27). Digested samples were then subjected to TCA and alcohol precipitation sequentially so as to get glycosaminoglycans (GAGs). The precipitate was collected, dried using nitrogen gas and reconstituted in minimal volume of water. They were then desalted on a PD10 column using pyridine-acetate buffer and dried. They were reconstituted in minimal water and stored at -20 oC until use. GAG was isolated from erythrocytes from two independent experiments tentatively named as SFC A and SFC B for control animals and as SFH A and SFH B for hypercholesterolemic animals
Biochemical measurements Total cholesterol, triglycerides, LDLc and HDLc were determined in plasma by established methods using kits from Agape pvt ltd. India. Amount of sulphated GAGs were estimated by DMMB assay using chondroitin sulphate B as a reference standard (28).
Cellulose acetate membrane electrophoresis To identify the type of GAGs, samples were subjected to cellulose acetate membrane electrophoresis. This was carried out as described earlier (16).
6
Downloaded from http://jb.oxfordjournals.org/ at SUNY Health Science Center at Brooklyn on April 12, 2015
was subjected to papain digestion after reconstituting it with 0.1 M phosphate buffer, pH 6.5 and
Flow Cytometry For flow analysis of erythrocytes for CS/DS, initially 2 µl of properly mixed anticoagulated (EDTA) blood was diluted in 1 ml of phosphate buffer saline (stock). From the stock 20 µl of blood sample was again diluted to 1 ml by mixing with phosphate buffer saline (PBS) containing 1 % BSA and was labeled with CS-56 primary antibody at two different antibody dilution for 30 min. After incubation cells were washed twice with PBS containing 1% BSA at 180 g. The pellet was again
30 min in dark. After incubation excess secondary antibody was removed by washing twice with PBS containing 1% BSA at 180 g. Finally the pellet was resuspended in 500 µl PBS and analysed in FACS-Calibur instrument. Disaccharide composition analysis by reverse phase HPLC
Disaccharide composition analysis of CS/DS isolated and purified from erythrocytes of control and hypercholesterolemic rats was carried out by digesting 2 µg (as sulfated GAGs) with chondroitinase ABC for 1 h at 37o C (29). The digested samples were derivatized with 2AB (30). Excess 2AB was removed by paper chromatography using solvent system consisting of butanol: ethanol: water (4:1:1) (31). The 2AB-labeled disaccharides were diluted to 100 µl with 0.1 M Ammonium acetate and an aliquot analyzed by reverse phase HPLC on a Zorbax Eclipse XDB-C18 column over a period of 72 min employing fluorescence detection with excitation and emission wavelengths set at 330 and 420 nm, respectively (32).
Cytoadherence assay Binding of erythrocytes to ECM proteins was evaluated by adherence assay. Varying amounts of type IV collagen, laminin and fibronectin (0-200 ng/well) were coated onto a 96-welled flatbottomed polystyrene microtiter plates (Nunc-Immnuoplate, USA) after diluting them in coating buffer. Excess Poly-L-Ornithine or ECM components, which remained unbound, were removed by
7
Downloaded from http://jb.oxfordjournals.org/ at SUNY Health Science Center at Brooklyn on April 12, 2015
reconstituted in PBS containing 1% BSA and labeled with FITC conjugated secondary antibody for
washing the wells thrice with PBS after overnight standing. To prevent non-specific binding, the sites were blocked with 2 % BSA diluted in PBS for 2 h. Erythrocytes (1X108 cells /100 µl/well), washed in saline containing calcium chloride (CaCl2, 25 mM) were added and incubated overnight at 4 oC. Unbound erythrocytes were removed by subjecting them to a gentle wash with saline containing CaCl2. The erythrocytes were then treated with 0.25% glutaraldehyde exercising utmost care for 10 min at 4 oC. Later the wells were rigorously washed three times and the extent of specifically bound
haemolysis was quantified by measuring the absorbance at 405 nm (33). To determine if CS/DS is involved in adherence, erythrocytes were subjected to chondroitinase ABC (5 mIU units) at 37 oC for 1 h and then seeded into the wells. Other procedures are as detailed. Solid phase immunobinding assay Isolated GAGs were tested for binding to various ECM components such as laminin, type IV collagen and fibronectin. Initially, the wells were coated with poly-L-lysine (400 ng) overnight. Excess poly-L-lysine was removed by repeated washing of the wells. Heparin (500 ng) was immobilized overnight and blocked with 2% BSA. ECM components laminin, type IV collagen and fibronectin, were tested for binding in absence or presence of heparin and isolated GAGs by competition. Binding was detected by specific primary antibodies directed against ECM components followed by ALP-conjugated secondary antibody using pNPP as a substrate. The colour obtained was read at 405 nm.
Statistical analysis All the values are expressed as mean ± SEM or SD of duplicates or triplicates of two or three independent experiments. Statistical analysis was done using Instat software. Significance between
8
Downloaded from http://jb.oxfordjournals.org/ at SUNY Health Science Center at Brooklyn on April 12, 2015
erythrocytes was quantified by cell lysis with double distilled water (100 µl /well) and the degree of
the groups was determined by performing one-way ANOVA with Tukey post t-test. A value of p
