Biochimie 85 (2003) 241–244 www.elsevier.com/locate/biochi
Uptake of sialic acid by human erythrocyte. Characterization of a transport system Tatiana Bulai a,1, Daniela Bratosin b,2, Vlad Artenie a,1, Jean Montreuil c,* a Facultatea de Biologie, Laboratorul de Biochimie, Universitatea “Alexandru Ioan Cuza”, Bd-ul Copou nr.11, 6600 Iasi-6, Romania Institutul National de Cercetare-Dezvoltare pentru Stiinte Biologice, 296 Sp. Independentei, P.O. Box 17-16, 77748 Bucuresti, Romania c Laboratoire de Chimie Biologique, Unité Mixte de Recherche CNRS/USTL n° 8576, Université des Sciences et Technologies de Lille 1 (USTL), 59655 Villeneuve d’Ascq cedex, France b
Received 28 December 2002; accepted 7 February 2003
Abstract Upon incubation of human red blood cells (RBC) with [4-9-14C] N-acetylneuraminic acid, the cells incorporated this sugar, as demonstrated by the identification of labelled N-acetylmannosamine in the cytosol, as a result of the action of the sialic acid pyruvate-lyase we discovered previously (Biochimie 84 (2002) 655). The mechanism is saturable and indicates the presence of a limited number of transporter molecules in the RBC membrane. This transport process may have relevance to the desialylation of membrane glycoconjugates which occurs during ageing of erythrocytes. © 2003 Éditions scientifiques et médicales Elsevier SAS and Société française de biochimie et biologie moléculaire. All rights reserved. Keywords: Sialate pyruvate-lyase; Erythrocytes; Red blood cells; Sialic acid; Transporter
1. Introduction The research we developed during the last decade on the cellular and molecular mechanisms of programmed cell death of human erythrocytes [1–5] led us to explore the fate of sialic residues present in human red blood cell (RBC) glycoconjugates, since it has been demonstrated for long time that sialic acids play a central role in ageing of RBC and in their capture by macrophages due to the desialylation of membrane glycoconjugates by sialidases, well characterized by Tettamanti’s group [6]. In a previous paper [7], we have described the discovery in human RBC cytosol of a sialate pyruvate-lyase, an enzyme which catalyses specifically and
Abbreviations: Neu5Ac, N-acetylneuraminic acid; ManNAc, N-acetylmannosamine; PBS, phosphate buffer solution, pH 7.4; RBC, red blood cell; TLC, thin-layer chromatography. 1 Tel.: +40-32-20-13-11; fax: +40-32-21-33-30. 2 Tel.: +40-1-220-79-09; fax: +40-1-220-76-95. * Corresponding author. Tel.: +33-3-20-43-48-84; fax: +33-3-20-43-65-55. E-mail addresses:
[email protected] (D. Bratosin),
[email protected] (V. Artenie).,
[email protected] (J. Montreuil).
reversibly the cleavage of free N-acetylneuraminic acid (Neu5Ac) to form pyruvate and N-acetylmannosamine (ManNAc). This enzyme is totally absent in the cell membrane as well as in circulating blood plasma and its enzymatic parameters are similar to those of the microbial and mammalian lyases described up to now. As a conclusion of our article, we wrote: “At the moment, the presence of sialate pyruvate-lyase in the cytosol of the RBCs remains an enigma. However, we are tempted to hypothesize that this enzyme is related to the desialylation process that occurs during RBC ageing and that the role it plays is to destroy the free sialic acid residues liberated by membrane sialidases. Such a mechanism implies the presence of a sialic acid transporter in the RBC membrane, the existence of which remains to be demonstrated”. In the present paper, we show that our hypothesis was sound. 2. Materials and methods 2.1. Materials Human blood type ORh+ collected on heparin was kindly supplied by the Centre Régional de Transfusion Sanguine de Lille. N-acetyl-D-mannosamine and Clostridium perfrin-
© 2003 Éditions scientifiques et médicales Elsevier SAS and Société française de biochimie et biologie moléculaire. All rights reserved. DOI: 1 0 . 1 0 1 6 / S 0 3 0 0 - 9 0 8 4 ( 0 3 ) 0 0 0 5 9 - 2
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gens sialate pyruvate-lyase were from Sigma Chemicals (Saint Louis, MO, USA), Neu5Ac was from Fluka (Buchs, CH), Dionex 50W×2 (200-400 mesh), H+ form from Supelco (Bellefonte, PA, USA), thin-layer chromatography(TLC) aluminium sheets (Silicagel 60) were from Merck Eurolab (Strasbourg, France) and Biomax™ MR films from Kodak (Rochester, NY, USA). [4-9-14C] Neu5Ac (specific radioactivity: 310 mCi/mmol) was specially prepared by Amersham Pharmacia Biotech (Little Chalfont, England). 2.2. Isolation of erythrocytes Heparinized human blood was processed within 1 h of collection. Cells were sedimented by centrifugation (1000 × g; 4 °C; 5 min). After removal of plasma platelets and leukocytes by aspiration, cells were washed three times with phosphate buffer solution (PBS) pH 7.4 containing 0.2 mM PMSF as antiproteases. 2.3. N-acetylneuraminic acid uptake in whole erythrocytes Human erythrocytes were suspended in PBS buffer pH 7.4 (haematocrit: 40%) and incubated under gentle stirring at 37 °C for 30 min in presence of [4-9-14C] Neu5Ac (0.5 µCi/ml, specific radioactivity: 310 mCi/mmol) and of unlabelled Neu5Ac at a concentration varying from 25 to 500 µg/ml. After incubation, cells were isolated by centrifugation (1000 × g; 4 °C; 5 min), washed three times with a PBS buffer pH 7.4 and lysed by treatment with cold water (1 ml for 400 µl of erythrocytes). To the lysate, were added 10 volumes of cold (–20 °C) absolute ethanol and, after removal of the precipitate of proteins by centrifugation, the supernatant was evaporated to dryness in vacuo. The residue was dissolved in water and after addition of scintillation fluid (4 ml/0.4 ml of aqueous solution), the radioactivity was counted in a liquidscintillation counter Beckman LS 6000TA (Beckman Instruments, Fullerton, CA, USA). In a second series of experiments, the entry of Neu5Ac into the erythrocyte was demonstrated unambiguously by the characterization of [14C] ManNAc in the RBC cytosol as follows. The ethanolic residue described above was dissolved in water and purified by ion exchange chromatography (Dowex 50W×2; H+ form; 6 × 40 mm). After washing of the column with water, the effluents were evaporated to dryness using a Speed Vac Concentrator connected to a refrigerated condensation trap (Savant Instruments Inc. Farmingdale, NY, USA). Chromatography of the dry material was carried out on TLC aluminium sheets (Silicagel 60) using an n-butanol/acetic acid/H2O (2:1:1.5 in volume) solvent system for 6 h at 22 °C. Autoradiography was performed with Biomax™ MR films (exposure time from 1 to 5 d). Unlabelled markers of Neu5Ac and ManNAc were revealed with Svennerholm’s reagent [8]. As a control and internal standard, we used the sialate pyruvate-lyase from C. perfringens as follows. To a solution of [14C] Neu5Ac (3.10 Ci/mol) in 50 µl of a potassium phosphate 0.5 M buffer, pH 7.2, 0.02 U of C. perfringens sialate pyruvate-lyase was added. The
Fig. 1. Uptake of [14C]N-acetylneuraminic acid by human red blood cells in presence of increasing concentrations of unlabelled N-acetylneuraminic acid. Uptake is expressed in percent of [14C]N-acetylneuraminic acid present in the incubation medium.
mixture was incubated at 37 °C for 2 h, heated at 100 °C for 3 min and submitted to TLC and autoradiography as described above.
3. Results Fig. 1 demonstrates that free extracellular Neu5Ac can be transported into human RBC. Interestingly, a saturation curve was obtained when RBCs were incubated for 30 min in the presence of various concentrations of free Neu5Ac, from 10 to 500 µg/ml. This result is in favour of the presence of a limited number of transporter molecules in the RBC membrane. It differs from those of Hirschberg et al. [9] for NIL, BHK and 3T3 fibroblasts and from those of Ferwerda et al. [10] for rat hepatocytes and Oetke et al. [11] for human haematopoietic cell lines BJA-B and HL60 who demonstrated that the uptake was unsaturable. Fig. 2 provides strong evidence that free Neu5Ac is taken up by human RBC and found intact in the cytosol. On the other hand, the presence of ManNAc in this cell compartment confirms this view since, as we have demonstrated previously [7], pyruvate-lyase is present only in the RBC cytosol and is totally lacking in the membranes of these cells.
4. Discussion Although only few experiments have been carried out, free sialic acids do not seem to be able to penetrate bacterial and animal cell plasma membranes at high amounts and the significance of the poor uptake of Neu5Ac has still to be evaluated. In the domain of bacteria, evidence for a sialate permease has been presented in C. perfringens by Nees and Schauer in
T. Bulai et al. / Biochimie 85 (2003) 241–244
Fig. 2. Autoradiography of thin layer chromatograms of cytosolic extract of human erythrocytes incubated in presence of [14C]N-acetylneuraminic acid. Chromatography on Silicagel 60 with solvent n-butanol/acetic acid/H2O (2:1:1.5 in volume). Lane 1: [14C]N-acetylneuraminic acid. Lane 2: [14C]ManNAc liberated by incubation of [14C]N-acetylneuraminic acid with sialate pyruvate-lyase from C. perfringens. Lane 3: characterization of [14C]ManNAc and [14C]N-acetylneuraminic acid in the cytosol of erythrocytes incubated in presence of [14C]N-acetylneuraminic acid in the conditions described in Section 2.3. Lane 4: N-acetylneuraminic acid and ManNAc standards revealed with Svennerholm’s reagents [8].
1974 [12], by studying the induction of neuraminidase and sialate pyruvate-lyase with free and glycopeptide bound Neu5Ac. Later, a sialic acid permease was evidenced in E. coli [13–15] and in Streptococus oralis [16]. The transported sialic acids are next degraded in the cytoplasm to ManNAc and pyruvic acid by a sialic acid pyruvate-lyase (EC 4.1.3.3), an inducible enzyme produced only in the presence of sialic acids. This enzyme plays an important role in the regulation of sialic acid in bacteria by controlling the intracellular concentration of sialic acid of biosynthetic or exogenous origin, thereby preventing the accumulation of toxic levels of this sugar (for review, see Ref. [17]). In the field of eukaryotic cells, it is widely accepted that free sialic acids cannot be efficiently taken up from the extracellular space and the uptake of very small amount of radiolabelled Neu5Ac has been demonstrated in hamster and mouse fibroblasts [18], NIL and 3T3 fibroblasts [9], BHK cells [9,19] and rat hepatocytes and hepatoma cells [10]. Recently, evidence for efficient uptake and incorporation of free sialic acid by eukaryotic cells was provided by Oetke et al. [11] using subclones of human hematopoietic cell lines BJA-B and HL60 with constitutive hyposialylation of cell surface glycoconjugates caused by a lack of expression of UDP-N-acetylglucosamine 2-epimerase [20]. These authors demonstrated that medium supplementation with Neu5Ac potently compensates for the deficient sialic acid biosynthesis in endogenously hyposialylated cells. Studies employing radiolabelled Neu5Ac revealed an uptake consistent with this observation providing the existence of an efficient uptake mechanism for Neu5Ac in eukaryotic cells.
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Our studies provide strong evidence that free Neu5Ac can be taken up by human erythrocytes and destroyed by the cytosolic sialate-lyase, since RBCs are unable to incorporate the sialic acid in cell glycoconjugates via CMP-Neu5Ac. Our demonstration is found on the identification of ManNAc in the cytosolic fraction of RBC. In fact, this compound could originate from the action on free Neu5Ac of the sialate pyruvate-lyase which is present in the RBC cytosol only, as we have previously described [7]. In addition, our studies indicate an uptake of radiolabelled Neu5Ac which reached 18.7 fmol per 5 × 106 cells within 30 min of incubation, i.e. 1.3% of total amount of [14C]Neu5Ac present in the incubation medium. Under the same experimental conditions, Oetke et al. [11] obtained similar result with human haematopoietic cell line BJA-B subclone K20 since “at 37 °C uptake of 9-[3H]Neu5Ac reached 11.7 fmol per 5 × 106 cells within 30 min”.
5. Concluding remarks The results of the present study demonstrate that free Neu5Ac can be taken up by human RBC. The uptake of sialic acid has been evidenced by the identification in the erythrocyte cytosol, in addition to free Neu5Ac, of ManNAc produced by the sialic acid pyruvate-lyase present in the cell cytosol only. This is the first report of a mechanism by which Neu5Ac transport occurs in human RBC.
Acknowledgements This work was supported in part by grants from the Université des Sciences et Technologies de Lille, the Centre National de la Recherche Scientifique (UMR USTL/CNRS n°8576; Directeur : Dr. Jean-Claude Michalski), the Établissement Régional de Transfusion Sanguine de Lille (Directeur : Jean-Jacques Huart) and the MacoProductions Company (Mouvaux, France; Chairman: Hervé Dubly and Dr. Francis Goudaliez and Dr. Joël Poplineau). We are grateful to Prs. René Cacan, Henry Debray and Philippe Delannoy, for their invaluable help in the domains of membrane physiology and of use of radiolabelled compounds. This work was initiated during the late Professeur André Verbert’s tenure as Director of the Institute. We wish to express our heartfelt gratitude for his constant, smiling and enthusiastic support. T.B. had a doctoral fellowship from the French Government.
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