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Sialic acid content of von Willebrand factor subunit
Clinica Chimica Acta 273 (1998) 209–211
Letter to the Editor
Sialic acid content of von Willebrand factor subunit A simple method for quantitative estimation ´ Nair Y. Maeda, Sergio P. Bydlowski, Antonio Augusto B. Lopes* ˜ Paulo, and Pro-Sangue ´ ˜ Foundation, Sao Heart Institute, School of Medicine of the University of Sao Paulo, 05403 -000, Brazil Received 6 October 1997; received in revised form 5 January 1998; accepted 3 February 1998
Keywords: Sialic acid; von Willebrand factor; Glycoproteins; Carbohydrate structure
In pathological conditions, altered sialic acid content of circulating glycoproteins (GP) may be observed [1,2]. Decreased sialic acid content of adhesive molecules may be associated with changes in their adhesion properties. Removal of sialic acid from von Willebrand factor (vWF) in vitro has been demonstrated to enhance platelet vWF interaction. Platelet deposition to vascular subendothelium measured under flow conditions is increased in the presence of perfusates containing asialo vWF. Binding of asialo vWF to GPIb probably provokes exposure of the GPIIb / IIIa receptor in the appropriate configuration to promote platelet spreading and aggregation [3,4]. Thus, measurement of sialic acid content of vWF may be important in vascular disorders since hyposialylation of vWF is probably associated with increased risk of thrombotic events. The aim of this study was to establish a simple method for measuring the sialic acid content of vWF subunit using small amounts of plasma. Peripheral venous blood from healthy volunteers was collected in 1:10 volumes of 3.8% sodium citrate, in the presence of the following protease inhibitors: 5 mmol / l EDTA, 6 mmol / l N-ethylmaleimide, 1 mmol / l phenylmethylsulphonyl fluoride, 0.25 mmol / l leupeptin and 20 U / ml aprotinin. Plasma was separated by centrifugation at 3000 3 g for 20 min. vWF immunoisolation *Corresponding author. Fax: 1 55 11 2822398. 0009-8981 / 98 / $19.00 1998 Elsevier Science B.V. All rights reserved. PII S0009-8981( 98 )00034-5
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N.Y. Maeda et al. / Clinica Chimica Acta 273 (1998) 209 – 211
was carried out at 48C and protease inhibitors were used at the same final concentrations in all buffers. Plasma (eight aliquots, 1 ml each) was mixed 1:1 with 50 mmol / l Tris pH 7.5, 150 mmol / l NaCl, 0.1% Nonidet P-40, 0.25% gelatin, 0.02% sodium azide and incubated for 1 h with affinity purified anti-human vWF polyclonal antibody (108 mg / ml) (Dako Corporation, Carpinteria, CA, USA). The mixture was then incubated for 1 h with protein A-Sepharose CL4B (140 ml of beads per 2 ml tube) (Pharmacia Fine Chemicals, Piscataway, NJ, USA). After washing the beads twice with the same buffer and once with 10 mmol / l Tris pH 7.5, 0.1% Nonidet P-40, vWF was eluted by incubation with 140 ml sample buffer (50 mmol / l Tris pH 6.8, 2% SDS, 100 mmol / l dithiothreitol, 30% glycerol, 0.02% bromophenol blue), for 20 min at 608C. Beads were removed by centrifugation at 12 000 3 g for 2 min. Eight samples (140 ml each) corresponding to the supernatant of immunoprecipitation procedure were subjected to 5% SDS–PAGE. Proteins were either stained with Coomassie Blue or transferred to nitrocellulose sheets as previously described [5]. In gels stained with Coomassie Blue, a major isolated band corresponding to vWF subunit appeared at 225 kDa. This was further confirmed by Western immunoblotting as described [5]. The amount of vWF detected by densitometric scanning of Coomassie Blue-stained gels using commercially available vWF as a standard (Calbiochem–Novabiochem International, San Diego, CA, USA), was 20 mg per gel in average. This corresponds to 2.5 mg vWF subunit per ml plasma. Since no bands were detected in gels stained after blotting, we assume that vWF was completely transferred to the filters. The region of the nitrocellulose sheet containing vWF subunit was excised and incubated in the presence of 0.05 mol / l H 2 SO 4 for 1 h at 808C. Removal of sialic acid from vWF subunit by acid hydrolysis was confirmed by Western blotting with biotinylated wheat germ agglutinin which has high affinity for terminal sialic acid residues (Fig. 1). Sialic acid concentration in the solution was determined by the
Fig. 1. Removal of sialic acid from immobilized vWF. The nitrocellulose sheet blotted with vWF (225 kDa) was reacted with biotinylated wheat germ agglutinin (WGA) without treatment (A) or after acid hydrolysis with 0.05 mol / l H 2 SO 4 at 808C for 1 h (B). Bound WGA was detected using an avidin–biotin–peroxidase complex.
N.Y. Maeda et al. / Clinica Chimica Acta 273 (1998) 209 – 211
211
thiobarbituric acid assay, according to the method of Aminoff [6]. An adequate standard curve (549 nm) was obtained for pure sialic acid (N-acetylneuraminic acid, Sigma, St. Louis, MO, USA) at concentrations ranging from 0.075 to 2.0 mg / ml ( y 5 0.037x, r 5 0.998). In eight independent determinations, the amount of sialic acid recovered from the nitrocellulose filters was 173614 ng (mean6SD). In other words, 21.6 ng sialic acid was recovered from one vWF band in average. The maximum variation relative to the mean value was 13%. In parallel assays, different amounts of vWF were applied to SDS–PAGE (4–27 mg). Recovered sialic acid correlated well with the amount of vWF present in gel (r 5 0.88, P 5 0.002). The slope of the regression line representing the mean value of vWF subunit sialic acid content was 8.1 ng / mg protein. Sialic acid content of pure ( . 95%), commercially available vWF determined by the same method was 9.0 ng / mg protein. The method used in this study is simple and may be applicable without expensive instrumentation for analysis of sialic acid content of glycoproteins using small amounts of plasma. In the case of vWF, reliable results can be obtained by processing 8 ml plasma. The method involves essentially a one-step immunoisolation procedure followed by electrophoretic separation and protein blotting onto nitrocellulose sheets. Sialic acid is then removed from the specific band of interest and measured in solution. In addition, after sialic acid removal, it is possible to subject the same nitrocellulose paper to glycosidase treatment or appropriate lectin probe reaction [7] for further analysis of the carbohydrate chains.
References [1] Ruelland A, Gallou G, Legras B, Paillard F, Cloarec L. LDL sialic acid content in patients with coronary artery disease. Clin Chim Acta 1993;221:127–33. [2] van Kamp GJ, Mulder K, Kuiper M, Wolters EC. Changed transferrin sialylation in Parkinson’s disease. Clin Chim Acta 1995;235:159–67. [3] Batisda E, Monteagudo J, Ordinas A, De Marco L, Castillo R. Asialo von Willebrand factor enhances platelet adhesion to vessel subendothelium. Thromb Haemost 1988;60:30–4. [4] Federici AB, De Romeuf C, De Groot PG et al. Adhesive properties of the carbohydratemodified von Willebrand factor (CHO-vWF). Blood 1988;71:947–52. [5] Lopes AAB, Maeda NY. Abnormal degradation of von Willebrand factor main subunit in pulmonary hypertension. Eur Respir J 1995;8:530–6. [6] Aminoff D. Methods for the quantitative estimation of N-acetylneuraminic acid and their application to hydrolysates of sialomucoids. Biochem J 1961;81:384–92. [7] Kijimoto-Ochiai S, Katagiri YU, Ochiai H. Analysis of N-linked oligosaccharide chains of glycoproteins on nitrocellulose sheets using lectin–peroxidase reagents. Anal Biochem 1985;147:222–9.
Letter to the Editor
Sialic acid content of von Willebrand factor subunit A simple method for quantitative estimation ´ Nair Y. Maeda, Sergio P. Bydlowski, Antonio Augusto B. Lopes* ˜ Paulo, and Pro-Sangue ´ ˜ Foundation, Sao Heart Institute, School of Medicine of the University of Sao Paulo, 05403 -000, Brazil Received 6 October 1997; received in revised form 5 January 1998; accepted 3 February 1998
Keywords: Sialic acid; von Willebrand factor; Glycoproteins; Carbohydrate structure
In pathological conditions, altered sialic acid content of circulating glycoproteins (GP) may be observed [1,2]. Decreased sialic acid content of adhesive molecules may be associated with changes in their adhesion properties. Removal of sialic acid from von Willebrand factor (vWF) in vitro has been demonstrated to enhance platelet vWF interaction. Platelet deposition to vascular subendothelium measured under flow conditions is increased in the presence of perfusates containing asialo vWF. Binding of asialo vWF to GPIb probably provokes exposure of the GPIIb / IIIa receptor in the appropriate configuration to promote platelet spreading and aggregation [3,4]. Thus, measurement of sialic acid content of vWF may be important in vascular disorders since hyposialylation of vWF is probably associated with increased risk of thrombotic events. The aim of this study was to establish a simple method for measuring the sialic acid content of vWF subunit using small amounts of plasma. Peripheral venous blood from healthy volunteers was collected in 1:10 volumes of 3.8% sodium citrate, in the presence of the following protease inhibitors: 5 mmol / l EDTA, 6 mmol / l N-ethylmaleimide, 1 mmol / l phenylmethylsulphonyl fluoride, 0.25 mmol / l leupeptin and 20 U / ml aprotinin. Plasma was separated by centrifugation at 3000 3 g for 20 min. vWF immunoisolation *Corresponding author. Fax: 1 55 11 2822398. 0009-8981 / 98 / $19.00 1998 Elsevier Science B.V. All rights reserved. PII S0009-8981( 98 )00034-5
210
N.Y. Maeda et al. / Clinica Chimica Acta 273 (1998) 209 – 211
was carried out at 48C and protease inhibitors were used at the same final concentrations in all buffers. Plasma (eight aliquots, 1 ml each) was mixed 1:1 with 50 mmol / l Tris pH 7.5, 150 mmol / l NaCl, 0.1% Nonidet P-40, 0.25% gelatin, 0.02% sodium azide and incubated for 1 h with affinity purified anti-human vWF polyclonal antibody (108 mg / ml) (Dako Corporation, Carpinteria, CA, USA). The mixture was then incubated for 1 h with protein A-Sepharose CL4B (140 ml of beads per 2 ml tube) (Pharmacia Fine Chemicals, Piscataway, NJ, USA). After washing the beads twice with the same buffer and once with 10 mmol / l Tris pH 7.5, 0.1% Nonidet P-40, vWF was eluted by incubation with 140 ml sample buffer (50 mmol / l Tris pH 6.8, 2% SDS, 100 mmol / l dithiothreitol, 30% glycerol, 0.02% bromophenol blue), for 20 min at 608C. Beads were removed by centrifugation at 12 000 3 g for 2 min. Eight samples (140 ml each) corresponding to the supernatant of immunoprecipitation procedure were subjected to 5% SDS–PAGE. Proteins were either stained with Coomassie Blue or transferred to nitrocellulose sheets as previously described [5]. In gels stained with Coomassie Blue, a major isolated band corresponding to vWF subunit appeared at 225 kDa. This was further confirmed by Western immunoblotting as described [5]. The amount of vWF detected by densitometric scanning of Coomassie Blue-stained gels using commercially available vWF as a standard (Calbiochem–Novabiochem International, San Diego, CA, USA), was 20 mg per gel in average. This corresponds to 2.5 mg vWF subunit per ml plasma. Since no bands were detected in gels stained after blotting, we assume that vWF was completely transferred to the filters. The region of the nitrocellulose sheet containing vWF subunit was excised and incubated in the presence of 0.05 mol / l H 2 SO 4 for 1 h at 808C. Removal of sialic acid from vWF subunit by acid hydrolysis was confirmed by Western blotting with biotinylated wheat germ agglutinin which has high affinity for terminal sialic acid residues (Fig. 1). Sialic acid concentration in the solution was determined by the
Fig. 1. Removal of sialic acid from immobilized vWF. The nitrocellulose sheet blotted with vWF (225 kDa) was reacted with biotinylated wheat germ agglutinin (WGA) without treatment (A) or after acid hydrolysis with 0.05 mol / l H 2 SO 4 at 808C for 1 h (B). Bound WGA was detected using an avidin–biotin–peroxidase complex.
N.Y. Maeda et al. / Clinica Chimica Acta 273 (1998) 209 – 211
211
thiobarbituric acid assay, according to the method of Aminoff [6]. An adequate standard curve (549 nm) was obtained for pure sialic acid (N-acetylneuraminic acid, Sigma, St. Louis, MO, USA) at concentrations ranging from 0.075 to 2.0 mg / ml ( y 5 0.037x, r 5 0.998). In eight independent determinations, the amount of sialic acid recovered from the nitrocellulose filters was 173614 ng (mean6SD). In other words, 21.6 ng sialic acid was recovered from one vWF band in average. The maximum variation relative to the mean value was 13%. In parallel assays, different amounts of vWF were applied to SDS–PAGE (4–27 mg). Recovered sialic acid correlated well with the amount of vWF present in gel (r 5 0.88, P 5 0.002). The slope of the regression line representing the mean value of vWF subunit sialic acid content was 8.1 ng / mg protein. Sialic acid content of pure ( . 95%), commercially available vWF determined by the same method was 9.0 ng / mg protein. The method used in this study is simple and may be applicable without expensive instrumentation for analysis of sialic acid content of glycoproteins using small amounts of plasma. In the case of vWF, reliable results can be obtained by processing 8 ml plasma. The method involves essentially a one-step immunoisolation procedure followed by electrophoretic separation and protein blotting onto nitrocellulose sheets. Sialic acid is then removed from the specific band of interest and measured in solution. In addition, after sialic acid removal, it is possible to subject the same nitrocellulose paper to glycosidase treatment or appropriate lectin probe reaction [7] for further analysis of the carbohydrate chains.
References [1] Ruelland A, Gallou G, Legras B, Paillard F, Cloarec L. LDL sialic acid content in patients with coronary artery disease. Clin Chim Acta 1993;221:127–33. [2] van Kamp GJ, Mulder K, Kuiper M, Wolters EC. Changed transferrin sialylation in Parkinson’s disease. Clin Chim Acta 1995;235:159–67. [3] Batisda E, Monteagudo J, Ordinas A, De Marco L, Castillo R. Asialo von Willebrand factor enhances platelet adhesion to vessel subendothelium. Thromb Haemost 1988;60:30–4. [4] Federici AB, De Romeuf C, De Groot PG et al. Adhesive properties of the carbohydratemodified von Willebrand factor (CHO-vWF). Blood 1988;71:947–52. [5] Lopes AAB, Maeda NY. Abnormal degradation of von Willebrand factor main subunit in pulmonary hypertension. Eur Respir J 1995;8:530–6. [6] Aminoff D. Methods for the quantitative estimation of N-acetylneuraminic acid and their application to hydrolysates of sialomucoids. Biochem J 1961;81:384–92. [7] Kijimoto-Ochiai S, Katagiri YU, Ochiai H. Analysis of N-linked oligosaccharide chains of glycoproteins on nitrocellulose sheets using lectin–peroxidase reagents. Anal Biochem 1985;147:222–9.