Apoptosis

, Volume 18, Issue 4, pp 373–384 | Cite as

Apoptotic cells selectively uptake minor glycoforms of vitronectin from serum

  • Nadia Malagolini
  • Mariangela Catera
  • Hugo Osorio
  • Celso A. Reis
  • Mariella Chiricolo
  • Fabio Dall’Olio
Original Paper

Abstract

Apoptosis profoundly alters the carbohydrate layer coating the membrane of eukaryotic cells. Previously we showed that apoptotic cells became reactive with the α2,6-sialyl-specific lectin from Sambucus nigra agglutinin (SNA), regardless of their histological origin and the nature of the apoptotic stimulus. Here we reveal the basis of the phenomenon by showing that in apoptotic cancer cell lines SNA reactivity was mainly associated with a 67 kDa glycoprotein which we identified by MALDI-TOF/TOF and immunoblot analysis as bovine vitronectin (bVN). bVN was neither present in non-apoptotic cells, nor in cells induced to apoptosis in serum-free medium, indicating that its uptake from the cell culture serum occurred only during apoptosis. The bVN molecules associated with apoptotic cancer cell lines represented minor isoforms, lacking the carboxyterminal sequence and paradoxically containing a few α2,6-linked sialic acid residues. Despite their poor α2,6-sialylation, these bVN molecules were sufficient to turn apoptotic cells to SNA reactivity, which is a late apoptotic event occurring in cells positive to both annexin-V and propidium iodide. Unlike in cancer cell lines, the major bVN form taken up by apoptotic neutrophils and mononuclear cells was a 80 kDa form. In apoptotic SW948 cells we also detected the α2,6-sialylated forms of the stress-70 mitochondrial precursor (mortalin) and of tubulin-β2C. These data indicate that the acquisition of vitronectin isoforms from the environment is a general, although cell specific phenomenon, potentially playing an important role in post-apoptotic events and that the α2,6-sialylation of intracellular proteins is a new kind of posttranslational modification associated with apoptosis.

Keywords

Apoptosis Vitronectin Mortalin Tubulin Sialylation Glycosylation 

Abbreviations

An-V

Annexin-V

bVN

Bovine vitronectin

FCS

Fetal calf serum

IEF

Isoelectrofocusing

PARP

Poly (ADP-ribose) polymerase

PBMC

Peripheral blood mononuclear cells

PI

Propidium iodide

PMN

Polymorphonuclear neutrophils

PNGase F

Peptide N-glycanase F

SNA

Sambucus nigra agglutinin

TPEN

N,N,N′,N′-tetrakis-(2-pyridylmethyl)ethylenediamine

Supplementary material

10495_2013_812_MOESM1_ESM.doc (46 kb)
Supplementary material 1 (DOC 46 kb)
10495_2013_812_MOESM2_ESM.ppt (138 kb)
Supplementary material 2 (PPT 138 kb)
10495_2013_812_MOESM3_ESM.ppt (404 kb)
Supplementary material 3 (PPT 404 kb)
10495_2013_812_MOESM4_ESM.ppt (311 kb)
Supplementary material 4 (PPT 311 kb)

References

  1. 1.
    Erwig LP, Henson PM (2008) Clearance of apoptotic cells by phagocytes. Cell Death Differ 15:243–250PubMedCrossRefGoogle Scholar
  2. 2.
    Ravichandran KS, Lorenz U (2007) Engulfment of apoptotic cells: signals for a good meal. Nat Rev Immunol 7:964–974PubMedCrossRefGoogle Scholar
  3. 3.
    Fadok VA, Voelker DR, Campbell PA, Cohen JJ, Bratton DL, Henson PM (1992) Exposure of phosphatidylserine on the surface of apoptotic lymphocytes triggers specific recognition and removal by macrophages. J Immunol 148:2207–2216PubMedGoogle Scholar
  4. 4.
    Kinchen JM, Ravichandran KS (2007) Journey to the grave: signaling events regulating removal of apoptotic cells. J Cell Sci 120:2143–2149PubMedCrossRefGoogle Scholar
  5. 5.
    Vandivier RW, Henson PM, Douglas IS (2006) Burying the dead: the impact of failed apoptotic cell removal (efferocytosis) on chronic inflammatory lung disease. Chest 129:1673–1682PubMedCrossRefGoogle Scholar
  6. 6.
    Bilyy R, Kit Y, Hellman U, Tryndyak V, Kaminskyy V, Stoika R (2005) In vivo expression and characteristics of novel alpha-d-mannose-rich glycoprotein markers of apoptotic cells. Cell Biol Int 29:920–928PubMedCrossRefGoogle Scholar
  7. 7.
    Bilyy R, Stoika R (2007) Search for novel cell surface markers of apoptotic cells. Autoimmunity 40:249–253PubMedCrossRefGoogle Scholar
  8. 8.
    Franz S, Herrmann K, Fuhrnrohr B, Sheriff A, Frey B, Gaipl US, Voll RE, Kalden JR, Jack HM, Herrmann M (2007) After shrinkage apoptotic cells expose internal membrane-derived epitopes on their plasma membranes. Cell Death Differ 14:733–742PubMedCrossRefGoogle Scholar
  9. 9.
    Hall SE, Savill JS, Henson PM, Haslett C (1994) Apoptotic neutrophils are phagocytosed by fibroblasts with participation of the fibroblast vitronectin receptor and involvement of a mannose/fucose-specific lectin. J Immunol 153:3218–3227PubMedGoogle Scholar
  10. 10.
    Batisse C, Marquet J, Greffard A, Fleury-Feith J, Jaurand MC, Pilatte Y (2004) Lectin-based three-color flow cytometric approach for studying cell surface glycosylation changes that occur during apoptosis. Cytometry A 62:81–88PubMedCrossRefGoogle Scholar
  11. 11.
    Brockhausen I, Lehotay M, Yang JM, Qin W, Young D, Lucien J, Coles J, Paulsen H (2002) Glycoprotein biosynthesis in porcine aortic endothelial cells and changes in the apoptotic cell population. Glycobiology 12:33–45PubMedCrossRefGoogle Scholar
  12. 12.
    Franz S, Frey B, Sheriff A, Gaipl US, Beer A, Voll RE, Kalden JR, Herrmann M (2006) Lectins detect changes of the glycosylation status of plasma membrane constituents during late apoptosis. Cytometry A 69:230–239PubMedGoogle Scholar
  13. 13.
    Rapoport E, Le Pendu J (1999) Glycosylation alterations of cells in late phase apoptosis from colon carcinomas. Glycobiology 9:1337–1345PubMedCrossRefGoogle Scholar
  14. 14.
    Rapoport EM, Sapot’ko YB, Pazynina GV, Bojenko VK, Bovin NV (2005) Sialoside-binding macrophage lectins in phagocytosis of apoptotic bodies. Biochemistry (Mosc) 70:330–338CrossRefGoogle Scholar
  15. 15.
    Sarter K, Mierke C, Beer A, Frey B, Fuhrnrohr BG, Schulze C, Franz S (2007) Sweet clearance: involvement of cell surface glycans in the recognition of apoptotic cells. Autoimmunity 40:345–348PubMedCrossRefGoogle Scholar
  16. 16.
    Malagolini N, Chiricolo M, Marini M, Dall’Olio F (2009) Exposure of α2,6-sialylated lactosaminic chains marks apoptotic and necrotic death in different cell types. Glycobiology 19:172–181PubMedCrossRefGoogle Scholar
  17. 17.
    Shibuya N, Goldstein IJ, Broekaert WF, Nsimba-Lubaki M, Peeters B, Peumans WJ (1987) The elderberry (Sambucus nigra L.) bark lectin recognizes the Neu5Ac(α 2–6)Gal/GalNAc sequence. J Biol Chem 262:1596–1601PubMedGoogle Scholar
  18. 18.
    Weinstein J, de Souza-e-Silva U, Paulson JC (1982) Purification of a Gal β 1,4GlcNAc α 2,6 sialyltransferase and a Gal β 1,3(4)GlcNAc α 2,3 sialyltransferase to homogeneity from rat liver. J Biol Chem 257:13835–13844PubMedGoogle Scholar
  19. 19.
    Weinstein J, Lee EU, McEntee K, Lai PH, Paulson JC (1987) Primary structure of β-galactoside α 2,6-sialyltransferase. Conversion of membrane-bound enzyme to soluble forms by cleavage of the NH2-terminal signal anchor. J Biol Chem 262:17735–17743PubMedGoogle Scholar
  20. 20.
    Dall’Olio F (2000) The sialyl-α2,6-lactosaminyl-structure: biosynthesis and functional role. Glycoconj J 17:669–676PubMedCrossRefGoogle Scholar
  21. 21.
    Deutsch HF (1954) Fetuin: the mucoprotein of fetal calf serum. J Biol Chem 208:669–678PubMedGoogle Scholar
  22. 22.
    Barnes DW, Silnutzer J (1983) Isolation of human serum spreading factor. J Biol Chem 258:12548–12552PubMedGoogle Scholar
  23. 23.
    Spik G, Bayard B, Fournet B, Strecker G, Bouquelet S, Montreuil J (1975) Studies on glycoconjugates. LXIV. Complete structure of two carbohydrate units of human serotransferrin. FEBS Lett 50:296–299PubMedGoogle Scholar
  24. 24.
    Chiricolo M, Malagolini N, Bonfiglioli S, Dall’Olio F (2006) Phenotypic changes induced by expression of β-galactoside α2,6 sialyltransferase I in the human colon cancer cell line SW948. Glycobiology 16:146–154PubMedCrossRefGoogle Scholar
  25. 25.
    Dall’Olio F, Chiricolo M, Lollini P, Lau JT (1995) Human colon cancer cell lines permanently expressing α2,6-sialylated sugar chains by transfection with rat β-galactoside α 2,6 sialyltransferase cDNA. Biochem Biophys Res Commun 211:554–561PubMedCrossRefGoogle Scholar
  26. 26.
    Preissner KT, Reuning U (2011) Vitronectin in vascular context: facets of a multitalented matricellular protein. Semin Thromb Hemost 37:408–424PubMedCrossRefGoogle Scholar
  27. 27.
    Kitagaki-Ogawa H, Yatohgo T, Izumi M, Hayashi M, Kashiwagi H, Matsumoto I, Seno N (1990) Diversities in animal vitronectins. Differences in molecular weight, immunoreactivity and carbohydrate chains. Biochim Biophys Acta 1033:49–56PubMedCrossRefGoogle Scholar
  28. 28.
    Nakashima N, Miyazaki K, Ishikawa M, Yatohgo T, Ogawa H, Uchibori H, Matsumoto I, Seno N, Hayashi M (1992) Vitronectin diversity in evolution but uniformity in ligand binding and size of the core polypeptide. Biochim Biophys Acta 1120:1–10PubMedCrossRefGoogle Scholar
  29. 29.
    Stepanek O, Brdicka T, Angelisova P, Horvath O, Spicka J, Stockbauer P, Man P, Horejsi V (2011) Interaction of late apoptotic and necrotic cells with vitronectin. PLoS ONE 6:e19243PubMedCrossRefGoogle Scholar
  30. 30.
    Ogawa H, Yoneda A, Seno N, Hayashi M, Ishizuka I, Hase S, Matsumoto I (1995) Structures of the N-linked oligosaccharides on human plasma vitronectin. Eur J Biochem 230:994–1000PubMedCrossRefGoogle Scholar
  31. 31.
    Stockmann A, Hess S, Declerck P, Timpl R, Preissner KT (1993) Multimeric vitronectin. Identification and characterization of conformation-dependent self-association of the adhesive protein. J Biol Chem 268:22874–22882PubMedGoogle Scholar
  32. 32.
    Sano K, Asanuma-Date K, Arisaka F, Hattori S, Ogawa H (2007) Changes in glycosylation of vitronectin modulate multimerization and collagen binding during liver regeneration. Glycobiology 17:784–794PubMedCrossRefGoogle Scholar
  33. 33.
    Seales EC, Jurado GA, Brunson BA, Wakefield JK, Frost AR, Bellis SL (2005) Hypersialylation of β1 integrins, observed in colon adenocarcinoma, may contribute to cancer progression by up-regulating cell motility. Cancer Res 65:4645–4652PubMedCrossRefGoogle Scholar
  34. 34.
    Semel AC, Seales EC, Singhal A, Eklund EA, Colley KJ, Bellis SL (2002) Hyposialylation of integrins stimulates the activity of myeloid fibronectin receptors. J Biol Chem 277:32830–32836PubMedCrossRefGoogle Scholar
  35. 35.
    Sanchez-Ruderisch H, Detjen KM, Welzel M, Andre S, Fischer C, Gabius HJ, Rosewicz S (2011) Galectin-1 sensitizes carcinoma cells to anoikis via the fibronectin receptor α5β1-integrin. Cell Death Differ 18:806–816PubMedCrossRefGoogle Scholar
  36. 36.
    Savill J, Dransfield I, Hogg N, Haslett C (1990) Vitronectin receptor-mediated phagocytosis of cells undergoing apoptosis. Nature 343:170–173PubMedCrossRefGoogle Scholar
  37. 37.
    Fadok VA, Savill JS, Haslett C, Bratton DL, Doherty DE, Campbell PA, Henson PM (1992) Different populations of macrophages use either the vitronectin receptor or the phosphatidylserine receptor to recognize and remove apoptotic cells. J Immunol 149:4029–4035PubMedGoogle Scholar
  38. 38.
    Fadok VA, Warner ML, Bratton DL, Henson PM (1998) CD36 is required for phagocytosis of apoptotic cells by human macrophages that use either a phosphatidylserine receptor or the vitronectin receptor (αvβ3). J Immunol 161:6250–6257PubMedGoogle Scholar
  39. 39.
    Meesmann HM, Fehr EM, Kierschke S, Herrmann M, Bilyy R, Heyder P, Blank N, Krienke S, Lorenz HM, Schiller M (2010) Decrease of sialic acid residues as an eat-me signal on the surface of apoptotic lymphocytes. J Cell Sci 123:3347–3356PubMedCrossRefGoogle Scholar
  40. 40.
    Bilyy RO, Shkandina T, Tomin A, Munoz LE, Franz S, Antonyuk V, Kit YY, Zirngibl M, Furnrohr BG, Janko C, Lauber K, Schiller M, Schett G, Stoika RS, Herrmann M (2012) Macrophages discriminate glycosylation patterns of apoptotic cell-derived microparticles. J Biol Chem 287:496–503PubMedCrossRefGoogle Scholar
  41. 41.
    Bae HB, Zmijewski JW, Deshane JS, Zhi D, Thompson LC, Peterson CB, Chaplin DD, Abraham E (2012) Vitronectin inhibits neutrophil apoptosis through activation of integrin-associated signaling pathways. Am J Respir Cell Mol Biol 46:790–796PubMedCrossRefGoogle Scholar
  42. 42.
    Seiffert D (1997) Constitutive and regulated expression of vitronectin. Histol Histopathol 12:787–797PubMedGoogle Scholar
  43. 43.
    Deocaris CC, Kaul SC, Wadhwa R (2009) The versatile stress protein mortalin as a chaperone therapeutic agent. Protein Pept Lett 16:517–529PubMedCrossRefGoogle Scholar
  44. 44.
    Garrido C, Solary E (2003) A role of HSPs in apoptosis through “protein triage”? Cell Death Differ 10:619–620PubMedCrossRefGoogle Scholar
  45. 45.
    Creagh EM, Carmody RJ, Cotter TG (2000) Heat shock protein 70 inhibits caspase-dependent and -independent apoptosis in Jurkat T cells. Exp Cell Res 257:58–66PubMedCrossRefGoogle Scholar
  46. 46.
    Beere HM (2004) “The stress of dying”: the role of heat shock proteins in the regulation of apoptosis. J Cell Sci 117:2641–2651PubMedCrossRefGoogle Scholar
  47. 47.
    Bhattacharyya T, Karnezis AN, Murphy SP, Hoang T, Freeman BC, Phillips B, Morimoto RI (1995) Cloning and subcellular localization of human mitochondrial hsp70. J Biol Chem 270:1705–1710PubMedCrossRefGoogle Scholar
  48. 48.
    Gasnier F, Ardail D, Lerme F, Simonot C, Vaganay E, Louisot P, Gateau-Roesch O (1994) Further characterization of mitochondrial outer membrane: evidence for the presence of two endogenous sialylated glycoproteins. J Biochem (Tokyo) 116:643–648Google Scholar
  49. 49.
    Avidan A, Perlmutter M, Tal S, Oraki O, Kapp T, Krelin Y, Elkabets M, Dotan S, Apte RN, Lichtenstein RG (2009) Differences in the sialylation patterns of membrane stress proteins in chemical carcinogen-induced tumors developed in BALB/c and IL-1alpha deficient mice. Glycoconj J 26:1181–1195PubMedCrossRefGoogle Scholar
  50. 50.
    Carre M, Andre N, Carles G, Borghi H, Brichese L, Briand C, Braguer D (2002) Tubulin is an inherent component of mitochondrial membranes that interacts with the voltage-dependent anion channel. J Biol Chem 277:33664–33669PubMedCrossRefGoogle Scholar
  51. 51.
    Ireland CM, Pittman SM (1995) Tubulin alterations in taxol-induced apoptosis parallel those observed with other drugs. Biochem Pharmacol 49:1491–1499PubMedCrossRefGoogle Scholar
  52. 52.
    Hino M, Kijima-Suda I, Nagai Y, Hosoya H (2003) Glycosylation of the α- and b-tubulin by sialyloligosaccharides. Zool Sci 20:709–715PubMedCrossRefGoogle Scholar
  53. 53.
    Cassatella MA, Cappelli R, Della BV, Grzeskowiak M, Dusi S, Berton G (1988) Interferon-gamma activates human neutrophil oxygen metabolism and exocytosis. Immunology 63:499–506PubMedGoogle Scholar
  54. 54.
    Chai F, Truong-Tran AQ, Evdokiou A, Young GP, Zalewski PD (2000) Intracellular zinc depletion induces caspase activation and p21 Waf1/Cip1 cleavage in human epithelial cell lines. J Infect Dis 182(Suppl 1):S85–S92PubMedCrossRefGoogle Scholar
  55. 55.
    Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275PubMedGoogle Scholar
  56. 56.
    Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  • Nadia Malagolini
    • 1
  • Mariangela Catera
    • 1
  • Hugo Osorio
    • 2
    • 3
  • Celso A. Reis
    • 2
    • 3
    • 4
  • Mariella Chiricolo
    • 1
  • Fabio Dall’Olio
    • 1
  1. 1.Department of Experimental, Diagnostic and Specialty MedicineDIMES, University of BolognaBolognaItaly
  2. 2.Institute of Molecular Pathology and ImmunologyUniversity of Porto-IPATIMUPPortoPortugal
  3. 3.Faculty of MedicineUniversity of PortoPortoPortugal
  4. 4.Institute of Biomedical Sciences of Abel Salazar (ICBAS)PortoPortugal

Personalised recommendations