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. 1992 Dec;11(12):4281–4290. doi: 10.1002/j.1460-2075.1992.tb05527.x

Structure of recombinant N-terminal globule of type VI collagen alpha 3 chain and its binding to heparin and hyaluronan.

U Specks 1, U Mayer 1, R Nischt 1, T Spissinger 1, K Mann 1, R Timpl 1, J Engel 1, M L Chu 1
PMCID: PMC557001  PMID: 1425570

Abstract

A large portion of the N-terminal globule of human collagen VI was prepared from the culture medium of stably transfected human embryonic kidney cell clones. The recombinant product corresponds to sequence positions 1-1586 of the alpha 3 (VI) chain that consists of eight homologous approximately 200 residue motifs (N9 to N2) being similar to the A domain motif of von Willebrand factor. By ultracentrifugation fragment N9-N2 showed a molecular mass of 180 kDa and an asymmetric shape. Elongated structures that consist of eight small globes (diameter approximately 5 nm) were demonstrated by electron microscopy. The data indicate that each A domain motif represents a separate folding unit which are connected to each other by short protease-sensitive peptide segments. Circular dichroism studies demonstrated about 38% alpha helix, 14% beta sheets and 17% beta turns. Fragment N9-N2 showed binding to heparin which could be abolished by moderate salt concentrations. Heparin binding was assigned to domains N9, N6 and N3 which were obtained after partial proteolysis. Domains N7, N5 and N4 lacked affinity for heparin. In addition, N9-N2 showed strong binding to hyaluronan that required exposure to 6 M urea for full dissociation. Ligand binding studies indicated some affinity of N9-N2 for the triple helical region of collagen VI suggesting a role of the N-terminal globule in the self-assembly of microfibrils. No or only little binding was, however, observed to fibril-forming collagens I and III, several basement membrane proteins and other extracellular proteins. Fragment N9-N2 was also an inactive substrate for cell adhesion.

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Selected References

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

  1. Aumailley M., Mann K., von der Mark H., Timpl R. Cell attachment properties of collagen type VI and Arg-Gly-Asp dependent binding to its alpha 2(VI) and alpha 3(VI) chains. Exp Cell Res. 1989 Apr;181(2):463–474. doi: 10.1016/0014-4827(89)90103-1. [DOI] [PubMed] [Google Scholar]
  2. Aumailley M., Wiedemann H., Mann K., Timpl R. Binding of nidogen and the laminin-nidogen complex to basement membrane collagen type IV. Eur J Biochem. 1989 Sep 1;184(1):241–248. doi: 10.1111/j.1432-1033.1989.tb15013.x. [DOI] [PubMed] [Google Scholar]
  3. Ayad S., Marriott A., Morgan K., Grant M. E. Bovine cartilage types VI and IX collagens. Characterization of their forms in vivo. Biochem J. 1989 Sep 15;262(3):753–761. doi: 10.1042/bj2620753. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. BITTER T., MUIR H. M. A modified uronic acid carbazole reaction. Anal Biochem. 1962 Oct;4:330–334. doi: 10.1016/0003-2697(62)90095-7. [DOI] [PubMed] [Google Scholar]
  5. Bonaldo P., Colombatti A. The carboxyl terminus of the chicken alpha 3 chain of collagen VI is a unique mosaic structure with glycoprotein Ib-like, fibronectin type III, and Kunitz modules. J Biol Chem. 1989 Dec 5;264(34):20235–20239. [PubMed] [Google Scholar]
  6. Bonaldo P., Russo V., Bucciotti F., Bressan G. M., Colombatti A. Alpha 1 chain of chick type VI collagen. The complete cDNA sequence reveals a hybrid molecule made of one short collagen and three von Willebrand factor type A-like domains. J Biol Chem. 1989 Apr 5;264(10):5575–5580. [PubMed] [Google Scholar]
  7. Bonaldo P., Russo V., Bucciotti F., Doliana R., Colombatti A. Structural and functional features of the alpha 3 chain indicate a bridging role for chicken collagen VI in connective tissues. Biochemistry. 1990 Feb 6;29(5):1245–1254. doi: 10.1021/bi00457a021. [DOI] [PubMed] [Google Scholar]
  8. Bruns R. R., Press W., Engvall E., Timpl R., Gross J. Type VI collagen in extracellular, 100-nm periodic filaments and fibrils: identification by immunoelectron microscopy. J Cell Biol. 1986 Aug;103(2):393–404. doi: 10.1083/jcb.103.2.393. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Chu M. L., Conway D., Pan T. C., Baldwin C., Mann K., Deutzmann R., Timpl R. Amino acid sequence of the triple-helical domain of human collagen type VI. J Biol Chem. 1988 Dec 15;263(35):18601–18606. [PubMed] [Google Scholar]
  10. Chu M. L., Mann K., Deutzmann R., Pribula-Conway D., Hsu-Chen C. C., Bernard M. P., Timpl R. Characterization of three constituent chains of collagen type VI by peptide sequences and cDNA clones. Eur J Biochem. 1987 Oct 15;168(2):309–317. doi: 10.1111/j.1432-1033.1987.tb13422.x. [DOI] [PubMed] [Google Scholar]
  11. Chu M. L., Pan T. C., Conway D., Kuo H. J., Glanville R. W., Timpl R., Mann K., Deutzmann R. Sequence analysis of alpha 1(VI) and alpha 2(VI) chains of human type VI collagen reveals internal triplication of globular domains similar to the A domains of von Willebrand factor and two alpha 2(VI) chain variants that differ in the carboxy terminus. EMBO J. 1989 Jul;8(7):1939–1946. doi: 10.1002/j.1460-2075.1989.tb03598.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Chu M. L., Zhang R. Z., Pan T. C., Stokes D., Conway D., Kuo H. J., Glanville R., Mayer U., Mann K., Deutzmann R. Mosaic structure of globular domains in the human type VI collagen alpha 3 chain: similarity to von Willebrand factor, fibronectin, actin, salivary proteins and aprotinin type protease inhibitors. EMBO J. 1990 Feb;9(2):385–393. doi: 10.1002/j.1460-2075.1990.tb08122.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Colombatti A., Ainger K., Colizzi F. Type VI collagen: high yields of a molecule with multiple forms of alpha 3 chain from avian and human tissues. Matrix. 1989 Jun;9(3):177–185. doi: 10.1016/s0934-8832(89)80048-4. [DOI] [PubMed] [Google Scholar]
  14. Colombatti A., Bonaldo P., Ainger K., Bressan G. M., Volpin D. Biosynthesis of chick type VI collagen. I. Intracellular assembly and molecular structure. J Biol Chem. 1987 Oct 25;262(30):14454–14460. [PubMed] [Google Scholar]
  15. Colombatti A., Bonaldo P. The superfamily of proteins with von Willebrand factor type A-like domains: one theme common to components of extracellular matrix, hemostasis, cellular adhesion, and defense mechanisms. Blood. 1991 Jun 1;77(11):2305–2315. [PubMed] [Google Scholar]
  16. Devereux J., Haeberli P., Smithies O. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 1):387–395. doi: 10.1093/nar/12.1part1.387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Doliana R., Bonaldo P., Colombatti A. Multiple forms of chicken alpha 3(VI) collagen chain generated by alternative splicing in type A repeated domains. J Cell Biol. 1990 Nov;111(5 Pt 1):2197–2205. doi: 10.1083/jcb.111.5.2197. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Eaton D. L., Wood W. I., Eaton D., Hass P. E., Hollingshead P., Wion K., Mather J., Lawn R. M., Vehar G. A., Gorman C. Construction and characterization of an active factor VIII variant lacking the central one-third of the molecule. Biochemistry. 1986 Dec 30;25(26):8343–8347. doi: 10.1021/bi00374a001. [DOI] [PubMed] [Google Scholar]
  19. Engel J., Furthmayr H. Electron microscopy and other physical methods for the characterization of extracellular matrix components: laminin, fibronectin, collagen IV, collagen VI, and proteoglycans. Methods Enzymol. 1987;145:3–78. doi: 10.1016/0076-6879(87)45003-9. [DOI] [PubMed] [Google Scholar]
  20. Engvall E., Hessle H., Klier G. Molecular assembly, secretion, and matrix deposition of type VI collagen. J Cell Biol. 1986 Mar;102(3):703–710. doi: 10.1083/jcb.102.3.703. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Fox J. W., Mayer U., Nischt R., Aumailley M., Reinhardt D., Wiedemann H., Mann K., Timpl R., Krieg T., Engel J. Recombinant nidogen consists of three globular domains and mediates binding of laminin to collagen type IV. EMBO J. 1991 Nov;10(11):3137–3146. doi: 10.1002/j.1460-2075.1991.tb04875.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Furthmayr H., Wiedemann H., Timpl R., Odermatt E., Engel J. Electron-microscopical approach to a structural model of intima collagen. Biochem J. 1983 May 1;211(2):303–311. doi: 10.1042/bj2110303. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Hennessey J. P., Jr, Johnson W. C., Jr Information content in the circular dichroism of proteins. Biochemistry. 1981 Mar 3;20(5):1085–1094. doi: 10.1021/bi00508a007. [DOI] [PubMed] [Google Scholar]
  24. Keene D. R., Engvall E., Glanville R. W. Ultrastructure of type VI collagen in human skin and cartilage suggests an anchoring function for this filamentous network. J Cell Biol. 1988 Nov;107(5):1995–2006. doi: 10.1083/jcb.107.5.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Kielty C. M., Boot-Handford R. P., Ayad S., Shuttleworth C. A., Grant M. E. Molecular composition of type VI collagen. Evidence for chain heterogeneity in mammalian tissues and cultured cells. Biochem J. 1990 Dec 15;272(3):787–795. doi: 10.1042/bj2720787. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Koller E., Winterhalter K. H., Trueb B. The globular domains of type VI collagen are related to the collagen-binding domains of cartilage matrix protein and von Willebrand factor. EMBO J. 1989 Apr;8(4):1073–1077. doi: 10.1002/j.1460-2075.1989.tb03475.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Komoriya A., Green L. J., Mervic M., Yamada S. S., Yamada K. M., Humphries M. J. The minimal essential sequence for a major cell type-specific adhesion site (CS1) within the alternatively spliced type III connecting segment domain of fibronectin is leucine-aspartic acid-valine. J Biol Chem. 1991 Aug 15;266(23):15075–15079. [PubMed] [Google Scholar]
  28. Maurer P., Mayer U., Bruch M., Jenö P., Mann K., Landwehr R., Engel J., Timpl R. High-affinity and low-affinity calcium binding and stability of the multidomain extracellular 40-kDa basement membrane glycoprotein (BM-40/SPARC/osteonectin). Eur J Biochem. 1992 Apr 1;205(1):233–240. doi: 10.1111/j.1432-1033.1992.tb16773.x. [DOI] [PubMed] [Google Scholar]
  29. McDevitt C. A., Marcelino J., Tucker L. Interaction of intact type VI collagen with hyaluronan. FEBS Lett. 1991 Dec 9;294(3):167–170. doi: 10.1016/0014-5793(91)80660-u. [DOI] [PubMed] [Google Scholar]
  30. Miller E. J., Rhodes R. K. Preparation and characterization of the different types of collagen. Methods Enzymol. 1982;82(Pt A):33–64. doi: 10.1016/0076-6879(82)82059-4. [DOI] [PubMed] [Google Scholar]
  31. Mohri H., Yoshioka A., Zimmerman T. S., Ruggeri Z. M. Isolation of the von Willebrand factor domain interacting with platelet glycoprotein Ib, heparin, and collagen and characterization of its three distinct functional sites. J Biol Chem. 1989 Oct 15;264(29):17361–17367. [PubMed] [Google Scholar]
  32. Mould A. P., Wheldon L. A., Komoriya A., Wayner E. A., Yamada K. M., Humphries M. J. Affinity chromatographic isolation of the melanoma adhesion receptor for the IIICS region of fibronectin and its identification as the integrin alpha 4 beta 1. J Biol Chem. 1990 Mar 5;265(7):4020–4024. [PubMed] [Google Scholar]
  33. Nischt R., Pottgiesser J., Krieg T., Mayer U., Aumailley M., Timpl R. Recombinant expression and properties of the human calcium-binding extracellular matrix protein BM-40. Eur J Biochem. 1991 Sep 1;200(2):529–536. doi: 10.1111/j.1432-1033.1991.tb16214.x. [DOI] [PubMed] [Google Scholar]
  34. Odermatt E., Risteli J., van Delden V., Timpl R. Structural diversity and domain composition of a unique collagenous fragment (intima collagen) obtained from human placenta. Biochem J. 1983 May 1;211(2):295–302. doi: 10.1042/bj2110295. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Okada Y., Naka K., Minamoto T., Ueda Y., Oda Y., Nakanishi I., Timpl R. localization of type VI collagen in the lining cell layer of normal and rheumatoid synovium. Lab Invest. 1990 Nov;63(5):647–656. [PubMed] [Google Scholar]
  36. Paulsson M., Aumailley M., Deutzmann R., Timpl R., Beck K., Engel J. Laminin-nidogen complex. Extraction with chelating agents and structural characterization. Eur J Biochem. 1987 Jul 1;166(1):11–19. doi: 10.1111/j.1432-1033.1987.tb13476.x. [DOI] [PubMed] [Google Scholar]
  37. Paulsson M., Yurchenco P. D., Ruben G. C., Engel J., Timpl R. Structure of low density heparan sulfate proteoglycan isolated from a mouse tumor basement membrane. J Mol Biol. 1987 Sep 20;197(2):297–313. doi: 10.1016/0022-2836(87)90125-2. [DOI] [PubMed] [Google Scholar]
  38. Pejler G., Bäckström G., Lindahl U., Paulsson M., Dziadek M., Fujiwara S., Timpl R. Structure and affinity for antithrombin of heparan sulfate chains derived from basement membrane proteoglycans. J Biol Chem. 1987 Apr 15;262(11):5036–5043. [PubMed] [Google Scholar]
  39. Pejler G., David G. Basement-membrane heparan sulphate with high affinity for antithrombin synthesized by normal and transformed mouse mammary epithelial cells. Biochem J. 1987 Nov 15;248(1):69–77. doi: 10.1042/bj2480069. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Ronzière M. C., Ricard-Blum S., Tiollier J., Hartmann D. J., Garrone R., Herbage D. Comparative analysis of collagens solubilized from human foetal, and normal and osteoarthritic adult articular cartilage, with emphasis on type VI collagen. Biochim Biophys Acta. 1990 Apr 19;1038(2):222–230. doi: 10.1016/0167-4838(90)90209-x. [DOI] [PubMed] [Google Scholar]
  41. Sixma J. J., Schiphorst M. E., Verweij C. L., Pannekoek H. Effect of deletion of the A1 domain of von Willebrand factor on its binding to heparin, collagen and platelets in the presence of ristocetin. Eur J Biochem. 1991 Mar 14;196(2):369–375. doi: 10.1111/j.1432-1033.1991.tb15826.x. [DOI] [PubMed] [Google Scholar]
  42. Sonnenberg A., Linders C. J., Modderman P. W., Damsky C. H., Aumailley M., Timpl R. Integrin recognition of different cell-binding fragments of laminin (P1, E3, E8) and evidence that alpha 6 beta 1 but not alpha 6 beta 4 functions as a major receptor for fragment E8. J Cell Biol. 1990 Jun;110(6):2145–2155. doi: 10.1083/jcb.110.6.2145. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Stokes D. G., Saitta B., Timpl R., Chu M. L. Human alpha 3(VI) collagen gene. Characterization of exons coding for the amino-terminal globular domain and alternative splicing in normal and tumor cells. J Biol Chem. 1991 May 5;266(13):8626–8633. [PubMed] [Google Scholar]
  44. Tengblad A. Affinity chromatography on immobilized hyaluronate and its application to the isolation of hyaluronate binding properties from cartilage. Biochim Biophys Acta. 1979 Jun 19;578(2):281–289. doi: 10.1016/0005-2795(79)90158-2. [DOI] [PubMed] [Google Scholar]
  45. Timpl R. Antibodies to collagens and procollagens. Methods Enzymol. 1982;82(Pt A):472–498. doi: 10.1016/0076-6879(82)82079-x. [DOI] [PubMed] [Google Scholar]
  46. Timpl R., Glanville R. W., Nowack H., Wiedemann H., Fietzek P. P., Kühn K. Isolation, chemical and electron microscopical characterization of neutral-salt-soluble type III collagen and procollagen from fetal bovine skin. Hoppe Seylers Z Physiol Chem. 1975 Nov;356(11):1783–1792. doi: 10.1515/bchm2.1975.356.2.1783. [DOI] [PubMed] [Google Scholar]
  47. Titani K., Kumar S., Takio K., Ericsson L. H., Wade R. D., Ashida K., Walsh K. A., Chopek M. W., Sadler J. E., Fujikawa K. Amino acid sequence of human von Willebrand factor. Biochemistry. 1986 Jun 3;25(11):3171–3184. doi: 10.1021/bi00359a015. [DOI] [PubMed] [Google Scholar]
  48. Titani K., Walsh K. A. Human von Willebrand factor: the molecular glue of platelet plugs. Trends Biochem Sci. 1988 Mar;13(3):94–97. doi: 10.1016/0968-0004(88)90048-5. [DOI] [PubMed] [Google Scholar]
  49. Trüeb B., Winterhalter K. H. Type VI collagen is composed of a 200 kd subunit and two 140 kd subunits. EMBO J. 1986 Nov;5(11):2815–2819. doi: 10.1002/j.1460-2075.1986.tb04573.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Wayner E. A., Carter W. G. Identification of multiple cell adhesion receptors for collagen and fibronectin in human fibrosarcoma cells possessing unique alpha and common beta subunits. J Cell Biol. 1987 Oct;105(4):1873–1884. doi: 10.1083/jcb.105.4.1873. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Wu J. J., Eyre D. R., Slayter H. S. Type VI collagen of the intervertebral disc. Biochemical and electron-microscopic characterization of the native protein. Biochem J. 1987 Dec 1;248(2):373–381. doi: 10.1042/bj2480373. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Yatohgo T., Izumi M., Kashiwagi H., Hayashi M. Novel purification of vitronectin from human plasma by heparin affinity chromatography. Cell Struct Funct. 1988 Aug;13(4):281–292. doi: 10.1247/csf.13.281. [DOI] [PubMed] [Google Scholar]
  53. Yurchenco P. D., Furthmayr H. Self-assembly of basement membrane collagen. Biochemistry. 1984 Apr 10;23(8):1839–1850. doi: 10.1021/bi00303a040. [DOI] [PubMed] [Google Scholar]
  54. van der Rest M., Garrone R. Collagen family of proteins. FASEB J. 1991 Oct;5(13):2814–2823. [PubMed] [Google Scholar]
  55. von der Mark H., Aumailley M., Wick G., Fleischmajer R., Timpl R. Immunochemistry, genuine size and tissue localization of collagen VI. Eur J Biochem. 1984 Aug 1;142(3):493–502. doi: 10.1111/j.1432-1033.1984.tb08313.x. [DOI] [PubMed] [Google Scholar]

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