Skip to main content
SpringerLink
Log in

Sialylation of vitronectin regulates stress fiber formation and cell spreading of dermal fibroblasts via a heparin-binding site

  • Original Article
  • Published:
Glycoconjugate Journal Aims and scope Submit manuscript

Abstract

Vitronectin (VN) plays an important role in tissue regeneration. We previously reported that VN from partial hepatectomized (PH) rats results in a decrease of sialylation of VN and de-sialylation of VN decreases the cell spreading of hepatic stellate cells. In this study, we analyzed the mechanism how sialylation of VN regulates the properties of mouse primary cultured dermal fibroblasts (MDF) and a dermal fibroblast cell line, Swiss 3T3 cells. At first, we confirmed that VN from PH rats or de-sialylated VN also decreased cell spreading in MDF and Swiss 3T3 cells. The de-sialylation suppressed stress fiber formation in Swiss 3T3 cells. Next, we analyzed the effect of the de-sialylation of VN on stress fiber formation in Swiss 3T3 cells. RGD peptide, an inhibitor for a cell binding site of VN, did not affect the cell attachment of Swiss 3T3 cells on untreated VN but significantly decreased it on de-sialylated VN, suggesting that the de-sialylation attenuates the binding activity of an RGD-independent binding site in VN. To analyze a candidate RGD-independent binding site, an inhibition experiment of stress fiber formation for a heparin binding site was performed. The addition of heparin and treatment of cells with heparinase decreased stress fiber formation in Swiss 3T3 cells. Furthermore, de-sialylation increased the binding activity of VN to heparin, as detected by surface plasmon resonance (SPR). These results demonstrate that sialylation of VN glycans regulates stress fiber formation and cell spreading of dermal fibroblast cells via a heparin binding site.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Similar content being viewed by others

Abbreviations

Con A:

Concanavalin A

ECM:

Extracellular matrix

FAK:

Focal adhesion kinase

HRP:

Horseradish peroxidase

MDF:

Mouse dermal fibroblast

NO:

Non-operated

PAI-1:

Plasminogen activator inhibitor-1

PH:

Partially hepatectomized

PNGase F:

Peptide-N4-(N-acetyl-β-D-glucosaminyl) asparagine amidase from Fravobacterium meningosepticum

SO:

Sham-operated

SNA:

Sambucus nigra agglutinin

SPR:

Surface plasmon resonance

VN:

Vitronectin

References

  1. Preissner K.T., Reuning U.: Vitronectin in vascular context: facets of a multitalented matricellular protein. Semin Thromb Hemost. 37(4), 408–424 (2011)

    Article  CAS  PubMed  Google Scholar 

  2. Leavesley D.I., Kashyap A.S., Croll T., Sivaramakrishnan M., Shokoohmand A., Hollier B.G., Upton Z.: Vitronectin–master controller or micromanager? IUBMB Life. 65(10), 807–818 (2013)

    CAS  PubMed  Google Scholar 

  3. Preissner K.T.: Structure and biological role of vitronectin. Annu Rev Cell Biol. 7, 275–310 (1991)

    Article  CAS  PubMed  Google Scholar 

  4. Schvartz I., Seger D., Shaltiel S.: Vitronectin Int J Biochem Cell Biol. 31(5), 539–544 (1999)

    Article  CAS  PubMed  Google Scholar 

  5. Fay W.P., Parker A.C., Ansari M.N., Zheng X., Ginsburg D.: Vitronectin inhibits the thrombotic response to arterial injury in mice. Blood. 93(6), 1825–1830 (1999)

    CAS  PubMed  Google Scholar 

  6. Wang A.G., Yen M.Y., Hsu W.M., Fann M.J.: Induction of vitronectin and integrin alphav in the retina after optic nerve injury. Mol Vis. 12, 76–84 (2006)

    PubMed  Google Scholar 

  7. Li R., Luo M., Ren M., Chen N., Xia J., Deng X., Zeng M., Yan K., Luo T., Wu J.: Vitronectin regulation of vascular endothelial growth factor-mediated angiogenesis. J Vasc Res. 51(2), 110–117 (2014)

    Article  CAS  PubMed  Google Scholar 

  8. Seiffert D.: Evidence that conformational changes upon the transition of the native to the modified form of vitronectin are not limited to the heparin binding domain. FEBS Lett. 368(1), 155–159 (1995)

    Article  CAS  PubMed  Google Scholar 

  9. Seiffert D., Loskutoff D.J.: Type 1 plasminogen activator inhibitor induces multimerization of plasma vitronectin. A suggested mechanism for the generation of the tissue form of vitronectin in vivo. J Biol Chem. 271(47), 29644–29651 (1996)

    Article  CAS  PubMed  Google Scholar 

  10. Izumi M., Yamada K.M., Hayashi M.: Vitronectin exists in two structurally and functionally distinct forms in human plasma. Biochim Biophys Acta. 990(2), 101–108 (1989)

    Article  CAS  PubMed  Google Scholar 

  11. Stockmann A., Hess S., Declerck P., Timpl R., Preissner K.T.: Multimeric vitronectin. Identification and characterization of conformation-dependent self-association of the adhesive protein. J Biol Chem. 268(30), 22874–22882 (1993)

    CAS  PubMed  Google Scholar 

  12. Preissner K.T., Muller-Berghaus G.: Neutralization and binding of heparin by S protein/vitronectin in the inhibition of factor Xa by antithrombin III. Involvement of an inducible heparin-binding domain of S protein/vitronectin. J. Biol. Chem. 262(25), 12247–12253 (1987)

  13. Seiffert D.: The glycosaminoglycan binding site governs ligand binding to the somatomedin B domain of vitronectin. J. Biol. Chem. 272(15), 9971–9978 (1997)

  14. Ogawa H., Sano K., Sobukawa N., Asanuma-Date K.: “Matrix Restructuring During Liver Regeneration is Regulated by Glycosylation of the Matrix Glycoprotein Vitronectin”. In: Baptista P. (ed.) “Liver regeneration, pp. 79–98. InTech Publishers,, Open access (2012)

    Google Scholar 

  15. Dufourcq P., Couffinhal T., Alzieu P., Daret D., Moreau C., Duplaa C., Bonnet J.: Vitronectin is up-regulated after vascular injury and vitronectin blockade prevents neointima formation. Cardiovasc Res. 53(4), 952–962 (2002)

    Article  CAS  PubMed  Google Scholar 

  16. Sano K., Miyamoto Y., Kawasaki N., Hashii N., Itoh S., Murase M., Date K., Yokoyama M., Sato C., Kitajima K., Ogawa H.: Survival signals of hepatic stellate cells in liver regeneration are regulated by glycosylation changes in rat vitronectin, especially decreased sialylation. J Biol Chem. 285(23), 17301–17309 (2010)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Uchibori-Iwaki H., Yoneda A., Oda-Tamai S., Kato S., Akamatsu N., Otsuka M., Murase K., Kojima K., Suzuki R., Maeya Y., Tanabe M., Ogawa H.: The changes in glycosylation after partial hepatectomy enhance collagen binding of vitronectin in plasma. Glycobiology. 10(9), 865–874 (2000)

    Article  CAS  PubMed  Google Scholar 

  18. Yasukawa Z., Sato C., Sano K., Ogawa H., Kitajima K.: Identification of disialic acid-containing glycoproteins in mouse serum: a novel modification of immunoglobulin light chains, vitronectin, and plasminogen. Glycobiology. 16(7), 651–665 (2006)

    Article  CAS  PubMed  Google Scholar 

  19. Yatohgo T., Izumi M., Kashiwagi H., Hayashi M.: Novel purification of vitronectin from human plasma by heparin affinity chromatography. Cell Struct Funct. 13(4), 281–292 (1988)

    Article  CAS  PubMed  Google Scholar 

  20. Ueda H., Kojima K., Saitoh T., Ogawa H.: Interaction of a lectin from Psathyrella velutina mushroom with N-acetylneuraminic acid. FEBS Lett. 448(1), 75–80 (1999)

    Article  CAS  PubMed  Google Scholar 

  21. Yoneda A., Ogawa H., Kojima K., Matsumoto I.: Characterization of the ligand binding activities of vitronectin: interaction of vitronectin with lipids and identification of the binding domains for various ligands using recombinant domains. Biochemistry. 37(18), 6351–6360 (1998)

    Article  CAS  PubMed  Google Scholar 

  22. Fischer S.M., Viaje A., Mills G.D., Slaga T.J.: Explant methods for epidermal cell culture. Methods Cell Biol. 21A, 207–227 (1980)

    Article  CAS  PubMed  Google Scholar 

  23. Takekawa H., Ina C., Sato R., Toma K., Ogawa H.: Novel carbohydrate-binding activity of pancreatic trypsins to N-linked glycans of glycoproteins. J Biol Chem. 281(13), 8528–8538 (2006)

    Article  CAS  PubMed  Google Scholar 

  24. Osmond R.I., Kett W.C., Skett S.E., Coombe D.R.: Protein-heparin interactions measured by BIAcore 2000 are affected by the method of heparin immobilization. Anal Biochem. 310(2), 199–207 (2002)

    Article  CAS  PubMed  Google Scholar 

  25. Nakagawa K., Nakamura K., Haishima Y., Yamagami M., Saito K., Sakagami H., Ogawa H.: Pseudoproteoglycan (pseudoPG) probes that simulate PG macromolecular structure for screening and isolation of PG-binding proteins. Glycoconj J. 26(8), 1007–1017 (2009)

    Article  CAS  PubMed  Google Scholar 

  26. Yoneda A., Ogawa H., Matsumoto I., Ishizuka I., Hase S., Seno N.: Structures of the N-linked oligosaccharides on porcine plasma vitronectin. Eur J Biochem. 218(3), 797–806 (1993)

    Article  CAS  PubMed  Google Scholar 

  27. Jurjus R.A., Liu Y., Pal-Ghosh S., Tadvalkar G., Stepp M.A.: Primary dermal fibroblasts derived from sdc-1 deficient mice migrate faster and have altered alphav integrin function. Wound Repair Regen. 16(5), 649–660 (2008)

    Article  PubMed  PubMed Central  Google Scholar 

  28. Bass M.D., Williamson R.C., Nunan R.D., Humphries J.D., Byron A., Morgan M.R., Martin P., Humphries M.J.: A syndecan-4 hair trigger initiates wound healing through caveolin- and RhoG-regulated integrin endocytosis. Dev Cell. 21(4), 681–693 (2011)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Granes F., Garcia R., Casaroli-Marano R.P., Castel S., Rocamora N., Reina M., Urena J.M., Vilaro S.: Syndecan-2 induces filopodia by active cdc42Hs. Exp Cell Res. 248(2), 439–456 (1999)

    Article  CAS  PubMed  Google Scholar 

  30. Gailit J., Clark R.A.: Studies in vitro on the role of alpha v and beta 1 integrins in the adhesion of human dermal fibroblasts to provisional matrix proteins fibronectin, vitronectin, and fibrinogen. J Investig Dermatol. 106(1), 102–108 (1996)

    Article  CAS  PubMed  Google Scholar 

  31. Reynolds L.E., Conti F.J., Lucas M., Grose R., Robinson S., Stone M., Saunders G., Dickson C., Hynes R.O., Lacy-Hulbert A., Hodivala-Dilke K.: Accelerated re-epithelialization in beta3-integrin-deficient- mice is associated with enhanced TGF-beta1 signaling. Nat Med. 11(2), 167–174 (2005)

    Article  CAS  PubMed  Google Scholar 

  32. Sano K., Asanuma-Date K., Arisaka F., Hattori S., Ogawa H.: Changes in glycosylation of vitronectin modulate multimerization and collagen binding during liver regeneration. Glycobiology. 17(7), 784–794 (2007)

    Article  CAS  PubMed  Google Scholar 

  33. Naski M.C., Lawrence D.A., Mosher D.F., Podor T.J., Ginsburg D.: Kinetics of inactivation of alpha-thrombin by plasminogen activator inhibitor-1. Comparison of the effects of native and urea-treated forms of vitronectin. J. Biol. Chem. 268(17), 12367–12372 (1993)

  34. Tschopp J., Masson D., Schafer S., Peitsch M., Preissner K.T.: The heparin binding domain of S-protein/vitronectin binds to complement components C7, C8, and C9 and perforin from cytolytic T-cells and inhibits their lytic activities. Biochemistry. 27(11), 4103–4109 (1988)

    Article  CAS  PubMed  Google Scholar 

  35. Wilkins-Port C.E., Sanderson R.D., Tominna-Sebald E., McKeown-Longo P.J.: Vitronectin's basic domain is a syndecan ligand which functions in trans to regulate vitronectin turnover. Cell Commun Adhes. 10(2), 85–103 (2003)

    CAS  PubMed  Google Scholar 

  36. Wilkins-Port C.E., McKeown-Longo P.J.: Heparan sulfate proteoglycans function in the binding and degradation of vitronectin by fibroblast monolayers. Biochem Cell Biol. 74(6), 887–897 (1996)

    Article  CAS  PubMed  Google Scholar 

  37. Echtermeyer F., Baciu P.C., Saoncella S., Ge Y., Goetinck P.F.: Syndecan-4 core protein is sufficient for the assembly of focal adhesions and actin stress fibers. J Cell Sci. 112(Pt 20), 3433–3441 (1999)

    CAS  PubMed  Google Scholar 

  38. Dovas A., Yoneda A., Couchman J.R.: PKCbeta-dependent activation of RhoA by syndecan-4 during focal adhesion formation. J Cell Sci. 119(Pt 13), 2837–2846 (2006)

    Article  CAS  PubMed  Google Scholar 

  39. Couchman J.R., Woods A.: Syndecan-4 and integrins: combinatorial signaling in cell adhesion. J Cell Sci. 112(Pt 20), 3415–3420 (1999)

    CAS  PubMed  Google Scholar 

  40. Cardin A.D., Weintraub H.J.: Molecular modeling of protein-glycosaminoglycan interactions. Arteriosclerosis. 9(1), 21–32 (1989)

    Article  CAS  PubMed  Google Scholar 

  41. Lane D.A., Flynn A.M., Pejler G., Lindahl U., Choay J., Preissner K.: Structural requirements for the neutralization of heparin-like saccharides by complement S protein/vitronectin. J. Biol. Chem. 262(34), 16343–16348 (1987)

  42. Liang O.D., Rosenblatt S., Chhatwal G.S., Preissner K.T.: Identification of novel heparin-binding domains of vitronectin. FEBS Lett. 407(2), 169–172 (1997)

    Article  CAS  PubMed  Google Scholar 

  43. Gibson A.D., Lamerdin J.A., Zhuang P., Baburaj K., Serpersu E.H., Peterson C.B.: Orientation of heparin-binding sites in native vitronectin. Analyses of ligand binding to the primary glycosaminoglycan-binding site indicate that putative secondary sites are not functional. J. Biol. Chem. 274(10), 6432–6442 (1999)

  44. Yoneda A., Kojima K., Matsumoto I., Yamamoto K., Ogawa H.: Porcine vitronectin, the most compact form of single-chain vitronectin: the smallest molecular mass among vitronectins was ascribed to deletion and substitution of base pairs, and proteolytic trimming of the peptide. J Biochem. 120(5), 954–960 (1996)

    Article  CAS  PubMed  Google Scholar 

  45. Chillakuri C.R., Jones C., Mardon H.J.: Heparin binding domain in vitronectin is required for oligomerization and thus enhances integrin mediated cell adhesion and spreading. FEBS Lett. 584(15), 3287–3291 (2010)

    Article  CAS  PubMed  Google Scholar 

  46. Clark R.A., Tonnesen M.G., Gailit J., Cheresh D.A.: Transient functional expression of alphaVbeta 3 on vascular cells during wound repair. Am J Pathol. 148(5), 1407–1421 (1996)

    CAS  PubMed  PubMed Central  Google Scholar 

  47. Weckroth M., Vaheri A., Virolainen S., Saarialho-Kere U., Jahkola T., Siren V.: Epithelial tissue-type plasminogen activator expression, unlike that of urokinase, its receptor, and plasminogen activator inhibitor-1, is increased in chronic venous ulcers. Br J Dermatol. 151(6), 1189–1196 (2004)

    Article  CAS  PubMed  Google Scholar 

  48. Olczyk P., Komosinska-Vassev K., Winsz-Szczotka K., Kozma E.M., Wisowski G., Stojko J., Klimek K., Olczyk K.: Propolis modulates vitronectin, laminin, and heparan sulfate/heparin expression during experimental burn healing. J Zhejiang Univ Sci B. 13(11), 932–941 (2012)

    Article  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgments

We thank Dr. Atsuko Yoneda (Tokyo University of Pharmacy and Life Sciences, Tokyo, JAPAN) for valuable suggestions and critical discussion.

Author information

Authors and Affiliations

  1. The Graduate School of Humanities and Sciences and The Glycoscience Institute, Ochanomizu University, Tokyo, 112-8610, Japan

    Yasunori Miyamoto, Mio Tanabe, Kimie Date, Kanoko Sakuda, Kotone Sano & Haruko Ogawa

Authors
  1. Yasunori Miyamoto
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Mio Tanabe
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Kimie Date
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Kanoko Sakuda
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Kotone Sano
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Haruko Ogawa
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Yasunori Miyamoto.

Electronic supplementary material

Fig. S1

(PDF 115 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Miyamoto, Y., Tanabe, M., Date, K. et al. Sialylation of vitronectin regulates stress fiber formation and cell spreading of dermal fibroblasts via a heparin-binding site. Glycoconj J 33, 227–236 (2016). https://doi.org/10.1007/s10719-016-9660-8

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10719-016-9660-8

Keywords

Search

Navigation