Recombinant heparan sulfate for use in tissue engineering applications

Research output: Contribution to journalJournal articleResearchpeer-review

  • J. Whitelock
  • J.L. Ma
  • N. Davies
  • N. Nielsen
  • Chuang, Christine
  • M. Rees
  • R.V. Iozzo
  • S. Knox
  • M. Lord
Background: Heparan sulfate (HS) is an important component of many extracellular matrices that interacts with mitogens and morphogens to guide and control tissue and organ development. These interactions are controlled by its structure, which varies when produced by different cell types and different species. The major aim of the studies reported here was to isolate and characterize the HS expressed on the N-terminal domain of human perlecan when it is expressed in human cells. Results: The recombinant proteoglycan was expressed in greatest quantities when the cells were grown as monolayers in the presence of Medium 199. It was purified as a proteoglycan with a molecular weight between 75 and 150 kDa, which was decorated with HS, chondroitin sulfate (CS) and keratan sulfate (KS) in a similar way to the full-length perlecan from the same cells. Compositional analysis of the glycosaminoglycan (GAG) chains suggested that it contained the same amount of CS and HS, suggesting that one of the attachment sites may not be glycosylated. The HS chains were responsible for the binding of fibroblast growth factor 2 (FGF2), while the specific roles of the CS and KS remain unclear. Conclusion: Expressing the N-terminal domain of the proteoglycan perlecan results in a hybrid truncated molecule that binds to growth factors via it's HS and may prove useful to add to scaffolds to encourage cells to respond to growth signals, such as those produced by the FGFs. © 2008 Society of Chemical Industry.
Original languageEnglish
JournalJournal of Chemical Technology and Biotechnology
Volume83
Issue number4
Pages (from-to)496-504
Number of pages9
ISSN0268-2575
DOIs
Publication statusPublished - 2008

Bibliographical note

Cited By :7

Export Date: 24 June 2016

References: Bishop, J.R., Schuksz, M., Esko, J.D., Heparan sulphate proteoglycans fine-tune mammalian physiology (2007) Nature, 446, pp. 1030-1037; Whitelock, J.M., Iozzo, R.V., Heparan sulfate: A complex polymer charged with biological activity (2005) Chem Rev, 105, pp. 2745-2764; Knox, S.M., Whitelock, J.M., Perlecan: How does one molecule do so many things? (2006) Cell Mol Life Sci, 63, pp. 2435-2445; Winzen, U., Cole, G.J., Halfter, W., Agrin is a chimeric proteoglycan with the attachment sites for heparan sulfate/chondroitin sulfate located in two multiple serine-glycine clusters (2003) J Biol Chem, 278, pp. 30106-30114; Dong, S., Cole, G.J., Halfter, W., Expression of collagen XVIII and localization of its glycosaminoglycan attachment sites (2003) J Biol Chem, 278, pp. 1700-1707; Dolan, M., Horchar, T., Rigatti, B., Hassell, J.R., Identification of sites in domain I of perlecan that regulate heparan sulfate synthesis (1997) J Biol Chem, 272, pp. 4316-4322; Costell, M., Mann, K., Yamada, Y., Timpl, R., Characterization of recombinant perlecan domain I and its substitution by glycosaminoglycans and oligosaccharides (1997) Eur J Biochem, 243, pp. 115-121; Kokenyesi, R., Silbert, J.E., Formation of heparan sulfate or chondroitin/ dermatan sulfate on recombinant domain I of mouse perlecan expressed in Chinese hamster ovary cells (1995) Biochem Biophys Res Commun, 211, pp. 262-267; Friedrich, M.V., Gohring, W., Morgelin, M., Brancaccio, A., David, G., Timpl, R., Structural basis of glycosaminoglycan modification and of heterotypic interactions of perlecan domain V (1999) J Mol Biol, 294, pp. 259-270; Brown, J.C., Sasaki, T., Gohring, W., Yamada, Y., Timpl, R., The C-terminal domain V of perlecan promotes betal integrin-mediated cell adhesion, binds heparin, nidogen and fibulin-2 and can be modified by glycosaminoglycans (1997) Eur J Biochem, 250, pp. 39-46; Graham, L.D., Whitelock, J.M., Underwood, P.A., Expression of human perlecan domain I as a recombinant heparan sulfate proteoglycan with 20-kDa glycosaminoglycan chains (1999) Biochem Biophys Res Commun, 256, pp. 542-548; Whitelock, J., Purification of perlecan from endothelial cells (2001) Methods Mol Biol, 171, pp. 27-34; Whitelock, J.M., Murdoch, A.D., Iozzo, R.V., Underwood, P.A., The degradation of human endothelial cell-derived perlecan and release of bound basic fibroblast growth factor by stromelysin, collagenase, plasmin, and heparanases (1996) J Biol Chem, 271, pp. 10079-10086; Lehrman, A.A.A., Gao, N., Alternative and sources of reagents and supplies of fluorophore-assisted carbohydrate electrophoresis (FACE) (2003) Glycobiology, 13, pp. 1G-3G; Calabro, A., Benavides, M., Tammi, M., Hascall, V.C., Midura, R.J., Microanalysis of enzyme digests of hyaluronan and chondroitin/dermatan sulfate by fluorophore-assisted carbohydrate electrophoresis (FACE) (2000) Glycobiology, 10, pp. 273-281; Klock, J.C., Starr, C.M., Polyacrylamide gel electrophoresis of fluorophore-labeled carbohydrates from glycoproteins (1998) Methods Mol Biol, 76, pp. 115-129; Whitelock, J.M., Graham, L.D., Melrose, J., Murdoch, A.D., Iozzo, R.V., Underwood, P.A., Human perlecan immunopurified from different endothelial cell sources has different adhesive properties for vascular cells (1999) Matrix Biol, 18, pp. 163-178; Gerena-Lopez, Y., Nolan, J., Wang, L., Gaigalas, A., Schwartz, A., Fernandez-Repollet, E., Quantification of EGFP expression on Molt-4 T cells using calibration standards (2004) Cytometry A, 60, pp. 21-28; Harper, B.K., Mabon, S.A., Leffel, S.M., Halthill, M.D., Richards, H.A., Moyer, K.A., Green fluorescent protein as a marker for expression of a second gene in transgenic plants (1999) Nat Biotechnol, 17, pp. 1125-1129; Richards, H.A., Halfhill, M.D., Millwood, R.J., Stewart Jr, C.N., Quantitative GFP fluorescence as an indicator of recombinant protein synthesis in transgenic plants (2003) Plant Cell Rep, 22, pp. 117-121; Remans, T., Schenk, P.M., Manners, J.M., Grof, C.P.L., Elliott, A.R., A protocol for the fluorometric quantification of mGFP5-ER and sGFP(S65T) in transgenic plants (1999) Plant Mol Biol Rep, 17, pp. 385-395; Kokenyesi, R., Bernfield, M., Core protein structure and sequence determine the site and presence of heparan sulfate and chondroitin sulfate on syndecan-1 (1994) J Biol Chem, 269, pp. 12304-12309; Chen, R.L., Lander, A.D., Mechanisms underlying preferential assembly of heparan sulfate on glypican-1 (2001) J Biol Chem, 276, pp. 7507-7517; Sugahara, K., Kitagawa, H., Heparin and heparan sulfate biosynthesis (2002) IUBMB Life, 54, pp. 163-175; Zhang, L., David, G., Esko, J.D., Repetitive Ser-Gly sequences enhance heparan sulfate assembly in proteoglycans (1995) J Biol Chem, 270, pp. 27127-27135; Wang, H., Julenius, K., Hryhorenko, J., Hagen, F.K., Systematic analysis of proteoglycan modification sites in Caenorhabditis elegans by scanning mutagenesis (2007) J Biol Chem, 282, pp. 14586-14597; Smith, S.M., West, L.A., Govindraj, P., Zhang, X., Ornitz, D.M., Hassell, J.R., Heparan and chondroitin sulfate on growth plate perlecan mediate binding and delivery of FGF-2 to FGF receptors (2007) Matrix Biol, 26, pp. 175-184; Mongiat, M., Sweeney, S.M., San Antonio, J.D., Fu, J., Iozzo, R.V., Endorepellin, a novel inhibitor of angiogenesis derived from the C terminus of perlecan (2003) J Biol Chem, 278, pp. 4238-4249; Knox, S., Merry, C., Stringer, S., Melrose, J., Whitelock, J., Not all perlecans are created equal: Interactions with fibroblast growth factor (FGF) 2 and FGF receptors (2002) J Biol Chem, 277, pp. 14657-14665; Knox, S., Melrose, J., Whitelock, J., Electrophoretic, biosensor, and bioactivity analyses of perlecans of different cellular origins (2001) Proteomics, 1, pp. 1534-1541; Ornitz, D.M., Yayon, A., Flanagan, J.G., Svahn, C.M., Levi, E., Leder, P., Heparin is required for cell-free binding of basic fibroblast growth factor to a soluble receptor and for mitogenesis in whole cells (1992) Mol Cell Biol, 12, pp. 240-247; Springer, B.A., Pantoliano, M.W., Barbera, F.A., Gunyuzlu, P.L., Thompson, L.D., Herblin, W.F., Identification and concerted function of two receptor binding surfaces on basic fibroblast growth factor required for mitogenesis (1994) J Biol Chem, 269, pp. 26879-26884; Robinson CJ, Harmer NJ, Goodger SJ, Blundell TL and Gallagher JT, Cooperative dimerization of fibroblast growth factor 1 (FGF1) upon a single heparin saccharide may drive the formation of 2:2:1 FGF1.FGFR2c.heparin ternary complexes. J Biol Chem 280:42274-42282 (2005)Ibrahimi, O.A., Zhang, F., Hrstka, S.C., Mohammadi, M., Linhardt, R.J., Kinetic model for FGF, FGFR, and proteoglycan signal transduction complex assembly (2004) Biochemistry, 43, pp. 4724-4730; Jastrebova, N., Vanwildemeersch, M., Rapraeger, A.C., Gimenez-Gallego, G., Lindahl, U., Spillmann, D., Heparan sulfate-related oligosaccharides in ternary complex formation with fibroblast growth factors 1 and 2 and their receptors (2006) J Biol Chem, 281, pp. 26884-26892; French, M.M., Gomes Jr, R.R., Timpl, R., Hook, M., Czymmek, K., Farach-Carson, M.C., Chondrogenic activity of the heparan sulfate proteoglycan perlecan maps to the N-terminal domain I (2002) J Bone Miner Res, 17, pp. 48-55; Yang, W.D., Gomes Jr, R.R., Alicknavitch, M., Farach-Carson, M.C., Carson, D.D., Perlecan domain I promotes fibroblast growth factor 2 delivery in collagen I fibril scaffolds (2005) Tissue Eng, 11, pp. 76-89; Yang, W., Gomes, R.R., Brown, A.J., Burdett, A.R., Alicknavitch, M., Farach-Carson, M.C., Chondrogenic differentiation on perlecan domain I, collagen II, and bone morphogenetic protein-2-based matrices (2006) Tissue Eng, 12, pp. 2009-2024; Casper, C.L., Yang, W., Farach-Carson, M.C., Rabolt, J.F., Coating electrospun collagen and gelatin fibers with perlecan domain I for increased growth factor binding (2007) Biomacromolecules, 8, pp. 1116-1123

    Research areas

  • Growth factor, Heparan sulfate, Recombinant, Tissue engineering

ID: 162758223