Proteomics of Stored Red Blood Cell Membrane and Storage-Induced Microvesicles Reveals the Association of Flotillin-2 With Band 3 Complexes
The storage of erythrocyte concentrates (ECs) induces lesions that notably affect metabolism, protein activity, deformability of red blood cells (RBCs), as well as the release of oxygen. Band 3 is one of the proteins affected during the ex vivo aging of RBCs. This membrane protein is an anion transp...
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2018-05-01
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| Series: | Frontiers in Physiology |
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| Online Access: | http://journal.frontiersin.org/article/10.3389/fphys.2018.00421/full |
| _version_ | 1811264510559780864 |
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| author | Michel Prudent Michel Prudent Julien Delobel Aurélie Hübner Corinne Benay Niels Lion Niels Lion Jean-Daniel Tissot Jean-Daniel Tissot |
| author_facet | Michel Prudent Michel Prudent Julien Delobel Aurélie Hübner Corinne Benay Niels Lion Niels Lion Jean-Daniel Tissot Jean-Daniel Tissot |
| author_sort | Michel Prudent |
| collection | DOAJ |
| description | The storage of erythrocyte concentrates (ECs) induces lesions that notably affect metabolism, protein activity, deformability of red blood cells (RBCs), as well as the release of oxygen. Band 3 is one of the proteins affected during the ex vivo aging of RBCs. This membrane protein is an anion transporter, an anchor site for the cytoskeleton and other membrane proteins as well as a binding site for glycolytic enzymes and bears blood group antigens. In the present study, band 3 complexes were isolated from RBCs stored for 7 and 42 days in average (n = 3), as well as from microvesicles (n = 3). After extraction of membrane proteins with a deoxycholate containing buffer, band 3 complexes were co-immunoprecipitated on magnetic beads coated with two anti-band 3 antibodies. Both total membrane protein extracts and eluates (containing band 3 complexes) were separated on SDS-PAGE and analyzed by bottom-up proteomics. It revealed that three proteins were present or absent in band 3 complexes stemming from long-stored or short-stored ECs, respectively, whereas the membrane protein contents remained equivalent. These potential markers for storage-induced RBC aging are adenylosuccinate lyase (ADSL), α-adducin and flotillin-2, and were further analyzed using western blots. ADSL abundance tended to increase during storage in both total membrane protein and band 3 complexes, whereas α-adducin mainly tended to stay onto the membrane extract. Interestingly, flotillin-2 was equivalently present in total membrane proteins whereas it clearly co-immunoprecipitated with band 3 complexes during storage (1.6-fold-change, p = 0.0024). Moreover, flotillin-2 was enriched (almost threefold) in RBCs compared to microvesicles (MVs) (p < 0.001) and the amount found in MVs was associated to band 3 complexes. Different types of band 3 complexes are known to exist in RBCs and further studies will be required to better understand involvement of this protein in microvesiculation during the storage of RBCs. |
| first_indexed | 2024-04-12T20:05:35Z |
| format | Article |
| id | doaj.art-486ad2df58894ebd8a742081a9fd1ea1 |
| institution | Directory Open Access Journal |
| issn | 1664-042X |
| language | English |
| last_indexed | 2024-04-12T20:05:35Z |
| publishDate | 2018-05-01 |
| publisher | Frontiers Media S.A. |
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| series | Frontiers in Physiology |
| spelling | doaj.art-486ad2df58894ebd8a742081a9fd1ea12022-12-22T03:18:24ZengFrontiers Media S.A.Frontiers in Physiology1664-042X2018-05-01910.3389/fphys.2018.00421318559Proteomics of Stored Red Blood Cell Membrane and Storage-Induced Microvesicles Reveals the Association of Flotillin-2 With Band 3 ComplexesMichel Prudent0Michel Prudent1Julien Delobel2Aurélie Hübner3Corinne Benay4Niels Lion5Niels Lion6Jean-Daniel Tissot7Jean-Daniel Tissot8Laboratoire de Recherche sur les Produits Sanguins, Recherche et Développement Produits, Transfusion Interrégionale CRS, Épalinges, SwitzerlandFaculté de Biologie et de Médecine, Université de Lausanne, Lausanne, SwitzerlandLaboratoire de Recherche sur les Produits Sanguins, Recherche et Développement Produits, Transfusion Interrégionale CRS, Épalinges, SwitzerlandLaboratoire de Recherche sur les Produits Sanguins, Recherche et Développement Produits, Transfusion Interrégionale CRS, Épalinges, SwitzerlandLaboratoire de Recherche sur les Produits Sanguins, Recherche et Développement Produits, Transfusion Interrégionale CRS, Épalinges, SwitzerlandLaboratoire de Recherche sur les Produits Sanguins, Recherche et Développement Produits, Transfusion Interrégionale CRS, Épalinges, SwitzerlandFaculté de Biologie et de Médecine, Université de Lausanne, Lausanne, SwitzerlandLaboratoire de Recherche sur les Produits Sanguins, Recherche et Développement Produits, Transfusion Interrégionale CRS, Épalinges, SwitzerlandFaculté de Biologie et de Médecine, Université de Lausanne, Lausanne, SwitzerlandThe storage of erythrocyte concentrates (ECs) induces lesions that notably affect metabolism, protein activity, deformability of red blood cells (RBCs), as well as the release of oxygen. Band 3 is one of the proteins affected during the ex vivo aging of RBCs. This membrane protein is an anion transporter, an anchor site for the cytoskeleton and other membrane proteins as well as a binding site for glycolytic enzymes and bears blood group antigens. In the present study, band 3 complexes were isolated from RBCs stored for 7 and 42 days in average (n = 3), as well as from microvesicles (n = 3). After extraction of membrane proteins with a deoxycholate containing buffer, band 3 complexes were co-immunoprecipitated on magnetic beads coated with two anti-band 3 antibodies. Both total membrane protein extracts and eluates (containing band 3 complexes) were separated on SDS-PAGE and analyzed by bottom-up proteomics. It revealed that three proteins were present or absent in band 3 complexes stemming from long-stored or short-stored ECs, respectively, whereas the membrane protein contents remained equivalent. These potential markers for storage-induced RBC aging are adenylosuccinate lyase (ADSL), α-adducin and flotillin-2, and were further analyzed using western blots. ADSL abundance tended to increase during storage in both total membrane protein and band 3 complexes, whereas α-adducin mainly tended to stay onto the membrane extract. Interestingly, flotillin-2 was equivalently present in total membrane proteins whereas it clearly co-immunoprecipitated with band 3 complexes during storage (1.6-fold-change, p = 0.0024). Moreover, flotillin-2 was enriched (almost threefold) in RBCs compared to microvesicles (MVs) (p < 0.001) and the amount found in MVs was associated to band 3 complexes. Different types of band 3 complexes are known to exist in RBCs and further studies will be required to better understand involvement of this protein in microvesiculation during the storage of RBCs.http://journal.frontiersin.org/article/10.3389/fphys.2018.00421/fullband 3flotillin-2immunoprecipitationproteomicsred blood cellsstorage |
| spellingShingle | Michel Prudent Michel Prudent Julien Delobel Aurélie Hübner Corinne Benay Niels Lion Niels Lion Jean-Daniel Tissot Jean-Daniel Tissot Proteomics of Stored Red Blood Cell Membrane and Storage-Induced Microvesicles Reveals the Association of Flotillin-2 With Band 3 Complexes Frontiers in Physiology band 3 flotillin-2 immunoprecipitation proteomics red blood cells storage |
| title | Proteomics of Stored Red Blood Cell Membrane and Storage-Induced Microvesicles Reveals the Association of Flotillin-2 With Band 3 Complexes |
| title_full | Proteomics of Stored Red Blood Cell Membrane and Storage-Induced Microvesicles Reveals the Association of Flotillin-2 With Band 3 Complexes |
| title_fullStr | Proteomics of Stored Red Blood Cell Membrane and Storage-Induced Microvesicles Reveals the Association of Flotillin-2 With Band 3 Complexes |
| title_full_unstemmed | Proteomics of Stored Red Blood Cell Membrane and Storage-Induced Microvesicles Reveals the Association of Flotillin-2 With Band 3 Complexes |
| title_short | Proteomics of Stored Red Blood Cell Membrane and Storage-Induced Microvesicles Reveals the Association of Flotillin-2 With Band 3 Complexes |
| title_sort | proteomics of stored red blood cell membrane and storage induced microvesicles reveals the association of flotillin 2 with band 3 complexes |
| topic | band 3 flotillin-2 immunoprecipitation proteomics red blood cells storage |
| url | http://journal.frontiersin.org/article/10.3389/fphys.2018.00421/full |
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