Skip to main content
PMC home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1994 Jun 1;125(5):1179–1188. doi: 10.1083/jcb.125.5.1179

Mapping the heparin-binding sites on type I collagen monomers and fibrils

PMCID: PMC2120046  PMID: 8195298

Abstract

The glycosaminoglycan chains of cell surface heparan sulfate proteoglycans are believed to regulate cell adhesion, proliferation, and extracellular matrix assembly, through their interactions with heparin-binding proteins (for review see Ruoslahti, E. 1988. Annu. Rev. Cell Biol. 4:229-255; and Bernfield, M., R. Kokenyesi, M. Kato, M. T. Hinkes, J. Spring, R. L. Gallo, and E. J. Lose. 1992. Annu. Rev. Cell Biol. 8:365-393). Heparin-binding sites on many extracellular matrix proteins have been described; however, the heparin-binding site on type I collagen, a ubiquitous heparin-binding protein of the extracellular matrix, remains undescribed. Here we used heparin, a structural and functional analogue of heparan sulfate, as a probe to study the nature of the heparan sulfate proteoglycan-binding site on type I collagen. We used affinity coelectrophoresis to study the binding of heparin to various forms of type I collagen, and electron microscopy to visualize the site(s) of interaction of heparin with type I collagen monomers and fibrils. Using affinity coelectrophoresis it was found that heparin has similar affinities for both procollagen and collagen fibrils (Kd's approximately 60-80 nM), suggesting that functionally similar heparin- binding sites exist in type I collagen independent of its aggregation state. Complexes of heparin-albumin-gold particles and procollagen were visualized by rotary shadowing and electron microscopy, and a preferred site of heparin binding was observed near the NH2 terminus of procollagen. Native or reconstituted type I collagen fibrils showed one region of significant heparin-gold binding within each 67-nm period, present near the division between the overlap and gap zones, within the "a" bands region. According to an accepted model of collagen fibril structure, our data are consistent with the presence of a single preferred heparin-binding site near the NH2 terminus of the collagen monomer. Correlating these data with known type I collagen sequences, we suggest that the heparin-binding site in type I collagen may consist of a highly basic triple helical domain, including several amino acids known sometimes to function as disaccharide acceptor sites. We propose that the heparin-binding site of type I collagen may play a key role in cell adhesion and migration within connective tissues, or in the cell- directed assembly or restructuring of the collagenous extracellular matrix.

Full Text

The Full Text of this article is available as a PDF (2.1 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Beeler D., Rosenberg R., Jordan R. Fractionation of low molecular weight heparin species and their interaction with antithrombin. J Biol Chem. 1979 Apr 25;254(8):2902–2913. [PubMed] [Google Scholar]
  2. Bernard M. P., Myers J. C., Chu M. L., Ramirez F., Eikenberry E. F., Prockop D. J. Structure of a cDNA for the pro alpha 2 chain of human type I procollagen. Comparison with chick cDNA for pro alpha 2(I) identifies structurally conserved features of the protein and the gene. Biochemistry. 1983 Mar 1;22(5):1139–1145. doi: 10.1021/bi00274a023. [DOI] [PubMed] [Google Scholar]
  3. Bernfield M., Kokenyesi R., Kato M., Hinkes M. T., Spring J., Gallo R. L., Lose E. J. Biology of the syndecans: a family of transmembrane heparan sulfate proteoglycans. Annu Rev Cell Biol. 1992;8:365–393. doi: 10.1146/annurev.cb.08.110192.002053. [DOI] [PubMed] [Google Scholar]
  4. Brennan M. J., Oldberg A., Hayman E. G., Ruoslahti E. Effect of a proteoglycan produced by rat tumor cells on their adhesion to fibronectin-collagen substrata. Cancer Res. 1983 Sep;43(9):4302–4307. [PubMed] [Google Scholar]
  5. Brodsky B., Eikenberry E. F. Characterization of fibrous forms of collagen. Methods Enzymol. 1982;82(Pt A):127–174. doi: 10.1016/0076-6879(82)82062-4. [DOI] [PubMed] [Google Scholar]
  6. Butler W. T., Cunningham L. W. Evidence for the linkage of a disaccharide to hydroxylysine in tropocollagen. J Biol Chem. 1966 Sep 10;241(17):3882–3888. [PubMed] [Google Scholar]
  7. Cardin A. D., Weintraub H. J. Molecular modeling of protein-glycosaminoglycan interactions. Arteriosclerosis. 1989 Jan-Feb;9(1):21–32. doi: 10.1161/01.atv.9.1.21. [DOI] [PubMed] [Google Scholar]
  8. Chapman J. A. The staining pattern of collagen fibrils. I. An analysis of electron micrographs. Connect Tissue Res. 1974;2(2):137–150. doi: 10.3109/03008207409152099. [DOI] [PubMed] [Google Scholar]
  9. Doyle B. B., Hukins D. W., Hulmes D. J., Miller A., Woodhead-Galloway J. Collagen polymorphism: its origins in the amino acid sequence. J Mol Biol. 1975 Jan 5;91(1):79–99. doi: 10.1016/0022-2836(75)90373-3. [DOI] [PubMed] [Google Scholar]
  10. Geuze H. J., Slot J. W., van der Ley P. A., Scheffer R. C. Use of colloidal gold particles in double-labeling immunoelectron microscopy of ultrathin frozen tissue sections. J Cell Biol. 1981 Jun;89(3):653–665. doi: 10.1083/jcb.89.3.653. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hulmes D. J., Miller A., Parry D. A., Piez K. A., Woodhead-Galloway J. Analysis of the primary structure of collagen for the origins of molecular packing. J Mol Biol. 1973 Sep 5;79(1):137–148. doi: 10.1016/0022-2836(73)90275-1. [DOI] [PubMed] [Google Scholar]
  12. Keller K. M., Keller J. M., Kühn K. The C-terminus of type I collagen is a major binding site for heparin. Biochim Biophys Acta. 1986 Jun 3;882(1):1–5. doi: 10.1016/0304-4165(86)90047-4. [DOI] [PubMed] [Google Scholar]
  13. Koda J. E., Bernfield M. Heparan sulfate proteoglycans from mouse mammary epithelial cells. Basal extracellular proteoglycan binds specifically to native type I collagen fibrils. J Biol Chem. 1984 Oct 10;259(19):11763–11770. [PubMed] [Google Scholar]
  14. Koda J. E., Rapraeger A., Bernfield M. Heparan sulfate proteoglycans from mouse mammary epithelial cells. Cell surface proteoglycan as a receptor for interstitial collagens. J Biol Chem. 1985 Jul 5;260(13):8157–8162. [PubMed] [Google Scholar]
  15. Koliakos G. G., Kouzi-Koliakos K., Furcht L. T., Reger L. A., Tsilibary E. C. The binding of heparin to type IV collagen: domain specificity with identification of peptide sequences from the alpha 1(IV) and alpha 2(IV) which preferentially bind heparin. J Biol Chem. 1989 Feb 5;264(4):2313–2323. [PubMed] [Google Scholar]
  16. Kuivaniemi H., Tromp G., Chu M. L., Prockop D. J. Structure of a full-length cDNA clone for the prepro alpha 2(I) chain of human type I procollagen. Comparison with the chicken gene confirms unusual patterns of gene conservation. Biochem J. 1988 Jun 15;252(3):633–640. doi: 10.1042/bj2520633. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Laurent T. C., Tengblad A., Thunberg L., Hök M., Lindahl U. The molecular-weight-dependence of the anti-coagulant activity of heparin. Biochem J. 1978 Nov 1;175(2):691–701. doi: 10.1042/bj1750691. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. LeBaron R. G., Hök A., Esko J. D., Gay S., Hök M. Binding of heparan sulfate to type V collagen. A mechanism of cell-substrate adhesion. J Biol Chem. 1989 May 15;264(14):7950–7956. [PubMed] [Google Scholar]
  19. Lee M. K., Lander A. D. Analysis of affinity and structural selectivity in the binding of proteins to glycosaminoglycans: development of a sensitive electrophoretic approach. Proc Natl Acad Sci U S A. 1991 Apr 1;88(7):2768–2772. doi: 10.1073/pnas.88.7.2768. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Marcum J. A., Rosenberg R. D. Role of endothelial cell surface heparin-like polysaccharides. Ann N Y Acad Sci. 1989;556:81–94. doi: 10.1111/j.1749-6632.1989.tb22492.x. [DOI] [PubMed] [Google Scholar]
  21. Margossian S. S., Krueger J. W., Sellers J. R., Cuda G., Caulfield J. B., Norton P., Slayter H. S. Influence of the cardiac myosin hinge region on contractile activity. Proc Natl Acad Sci U S A. 1991 Jun 1;88(11):4941–4945. doi: 10.1073/pnas.88.11.4941. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Monson J. M., Friedman J., McCarthy B. J. DNA sequence analysis of a mouse pro alpha 1 (I) procollagen gene: evidence for a mouse B1 element within the gene. Mol Cell Biol. 1982 Nov;2(11):1362–1371. doi: 10.1128/mcb.2.11.1362. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Obrink B. A study of the interactions between monomeric tropocollagen and glycosaminoglycans. Eur J Biochem. 1973 Mar 1;33(2):387–400. doi: 10.1111/j.1432-1033.1973.tb02695.x. [DOI] [PubMed] [Google Scholar]
  24. Olsen B. R., Hoffmann H., Prockop D. J. Interchain disulfide bonds at the COOH-terminal end of procollagen synthesized by matrix-free cells from chick embryonic tendon and cartilage. Arch Biochem Biophys. 1976 Jul;175(1):341–350. doi: 10.1016/0003-9861(76)90516-6. [DOI] [PubMed] [Google Scholar]
  25. Piepkorn M. W., Chapman D. L. Glycosaminoglycans and the substrate attachment of murine myeloma, 3T3, and cutaneous fibrosarcoma cells. Lab Invest. 1985 Jul;53(1):22–29. [PubMed] [Google Scholar]
  26. Pignatelli M., Bodmer W. F. Genetics and biochemistry of collagen binding-triggered glandular differentiation in a human colon carcinoma cell line. Proc Natl Acad Sci U S A. 1988 Aug;85(15):5561–5565. doi: 10.1073/pnas.85.15.5561. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Rosenberg R. D., Jordan R. E., Favreau L. V., Lam L. H. Highly active heparin species with multiple binding sites for antithrombin. Biochem Biophys Res Commun. 1979 Feb 28;86(4):1319–1324. doi: 10.1016/0006-291x(79)90260-2. [DOI] [PubMed] [Google Scholar]
  28. Ruoslahti E., Engvall E. Complexing of fibronectin glycosaminoglycans and collagen. Biochim Biophys Acta. 1980 Aug 13;631(2):350–358. doi: 10.1016/0304-4165(80)90308-6. [DOI] [PubMed] [Google Scholar]
  29. Ruoslahti E., Pierschbacher M. D. New perspectives in cell adhesion: RGD and integrins. Science. 1987 Oct 23;238(4826):491–497. doi: 10.1126/science.2821619. [DOI] [PubMed] [Google Scholar]
  30. Ruoslahti E. Structure and biology of proteoglycans. Annu Rev Cell Biol. 1988;4:229–255. doi: 10.1146/annurev.cb.04.110188.001305. [DOI] [PubMed] [Google Scholar]
  31. San Antonio J. D., Lander A. D., Wright T. C., Karnovsky M. J. Heparin inhibits the attachment and growth of Balb/c-3T3 fibroblasts on collagen substrata. J Cell Physiol. 1992 Jan;150(1):8–16. doi: 10.1002/jcp.1041500103. [DOI] [PubMed] [Google Scholar]
  32. San Antonio J. D., Slover J., Lawler J., Karnovsky M. J., Lander A. D. Specificity in the interactions of extracellular matrix proteins with subpopulations of the glycosaminoglycan heparin. Biochemistry. 1993 May 11;32(18):4746–4755. doi: 10.1021/bi00069a008. [DOI] [PubMed] [Google Scholar]
  33. Sanderson R. D., Sneed T. B., Young L. A., Sullivan G. L., Lander A. D. Adhesion of B lymphoid (MPC-11) cells to type I collagen is mediated by integral membrane proteoglycan, syndecan. J Immunol. 1992 Jun 15;148(12):3902–3911. [PubMed] [Google Scholar]
  34. Scott J. E., Parry D. A. Control of collagen fibril diameters in tissues. Int J Biol Macromol. 1992 Oct;14(5):292–293. doi: 10.1016/s0141-8130(05)80043-1. [DOI] [PubMed] [Google Scholar]
  35. Scott J. E. Proteoglycan-fibrillar collagen interactions. Biochem J. 1988 Jun 1;252(2):313–323. doi: 10.1042/bj2520313. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Scott J. E. Proteoglycan: collagen interactions in connective tissues. Ultrastructural, biochemical, functional and evolutionary aspects. Int J Biol Macromol. 1991 Jun;13(3):157–161. doi: 10.1016/0141-8130(91)90041-r. [DOI] [PubMed] [Google Scholar]
  37. Slayter H. S. Electron microscopic studies of fibrinogen structure: historical perspectives and recent experiments. Ann N Y Acad Sci. 1983 Jun 27;408:131–145. doi: 10.1111/j.1749-6632.1983.tb23241.x. [DOI] [PubMed] [Google Scholar]
  38. Slayter H. S. High-resolution metal replication of macromolecules. Ultramicroscopy. 1976 Sep-Oct;1(4):341–357. doi: 10.1016/0304-3991(76)90050-4. [DOI] [PubMed] [Google Scholar]
  39. Slayter H. S. Secretion of thrombospondin from human blood platelets. Methods Enzymol. 1989;169:251–268. doi: 10.1016/0076-6879(89)69066-0. [DOI] [PubMed] [Google Scholar]
  40. Staatz W. D., Fok K. F., Zutter M. M., Adams S. P., Rodriguez B. A., Santoro S. A. Identification of a tetrapeptide recognition sequence for the alpha 2 beta 1 integrin in collagen. J Biol Chem. 1991 Apr 25;266(12):7363–7367. [PubMed] [Google Scholar]
  41. Staatz W. D., Walsh J. J., Pexton T., Santoro S. A. The alpha 2 beta 1 integrin cell surface collagen receptor binds to the alpha 1 (I)-CB3 peptide of collagen. J Biol Chem. 1990 Mar 25;265(9):4778–4781. [PubMed] [Google Scholar]
  42. Stamatoglou S. C., Keller J. M. Interactions of cellular glycosaminoglycans with plasma fibronectin and collagen. Biochim Biophys Acta. 1982 Oct 28;719(1):90–97. doi: 10.1016/0304-4165(82)90311-7. [DOI] [PubMed] [Google Scholar]
  43. Stefanini M., De Martino C., Zamboni L. Fixation of ejaculated spermatozoa for electron microscopy. Nature. 1967 Oct 14;216(5111):173–174. doi: 10.1038/216173a0. [DOI] [PubMed] [Google Scholar]
  44. Tokuyasu K. T., Dutton A. H., Singer S. J. Immunoelectron microscopic studies of desmin (skeletin) localization and intermediate filament organization in chicken cardiac muscle. J Cell Biol. 1983 Jun;96(6):1736–1742. doi: 10.1083/jcb.96.6.1736. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Tokuyasu K. T., Singer S. J. Improved procedures for immunoferritin labeling of ultrathin frozen sections. J Cell Biol. 1976 Dec;71(3):894–906. doi: 10.1083/jcb.71.3.894. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Tromp G., Kuivaniemi H., Stacey A., Shikata H., Baldwin C. T., Jaenisch R., Prockop D. J. Structure of a full-length cDNA clone for the prepro alpha 1(I) chain of human type I procollagen. Biochem J. 1988 Aug 1;253(3):919–922. doi: 10.1042/bj2530919. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Tsilibary E. C., Koliakos G. G., Charonis A. S., Vogel A. M., Reger L. A., Furcht L. T. Heparin type IV collagen interactions: equilibrium binding and inhibition of type IV collagen self-assembly. J Biol Chem. 1988 Dec 15;263(35):19112–19118. [PubMed] [Google Scholar]
  48. Tsilibary E. C., Reger L. A., Vogel A. M., Koliakos G. G., Anderson S. S., Charonis A. S., Alegre J. N., Furcht L. T. Identification of a multifunctional, cell-binding peptide sequence from the a1(NC1) of type IV collagen. J Cell Biol. 1990 Oct;111(4):1583–1591. doi: 10.1083/jcb.111.4.1583. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Vogel K. G., Paulsson M., Heinegård D. Specific inhibition of type I and type II collagen fibrillogenesis by the small proteoglycan of tendon. Biochem J. 1984 Nov 1;223(3):587–597. doi: 10.1042/bj2230587. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Watkins S. C., Raso V., Slayter H. S. Immunoelectron-microscopic studies of human platelet thrombospondin, von Willebrand factor, and fibrinogen redistribution during clot formation. Histochem J. 1990 Sep;22(9):507–518. doi: 10.1007/BF01007236. [DOI] [PubMed] [Google Scholar]
  51. Watkins S. C., Slayter H. S., Codington J. F. Intracellular pathway of a mucin-type membrane glycoprotein in mouse mammary tumor cells. Carbohydr Res. 1991 Jun 25;213:185–200. doi: 10.1016/s0008-6215(00)90608-6. [DOI] [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES