Skip to main content
NCBI home page
The EMBO Journal logoLink to The EMBO Journal
. 1997 Jun 2;16(11):3035–3043. doi: 10.1093/emboj/16.11.3035

Dimer model for the microfibrillar protein fibulin-2 and identification of the connecting disulfide bridge.

T Sasaki 1, K Mann 1, H Wiedemann 1, W Göhring 1, A Lustig 1, J Engel 1, M L Chu 1, R Timpl 1
PMCID: PMC1169922  PMID: 9214621

Abstract

Fibulin-2 is a novel extracellular matrix protein frequently found in close association with microfibrils containing either fibronectin or fibrillin. The entire protein and its predicted domains were obtained as recombinant products and examined by ultracentrifugation and electron microscopy. This demonstrated a disulfide-linked homodimer of 175 kDa subunits. Partial reduction to monomers identified specifically an odd Cys574 residue responsible for dimer formation in one of three anaphylatoxin-like modules that constitute the central globular domain I (13 kDa) of fibulin-2. Furthermore, a Cys574-Ser mutation abolished disulfide connection but not non-covalent dimerization of fibulin-2. The C-terminal region (85 kDa) was shown to represent a 35-nm-long rod consisting of 11 calcium-binding EGF-like modules (domain II) and a small terminal globe (domain III). The unique N-terminal domain N (55 kDa) was also rod-shaped (approximately 38 nm) and rich in galactosamine indicating extensive O-glycosylation. A dimer model is proposed indicating mainly a rod-like shape of 80 nm length based on an anti-parallel association of two subunits through their domains I. This model also implies alignment of domains II and N between different subunits. This was demonstrated by surface plasmon resonance assay which showed a distinct interaction between domains N and II with a Kd of approximately 0.7 microM.

Full Text

The Full Text of this article is available as a PDF (691.5 KB).

Selected References

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

  1. Balbona K., Tran H., Godyna S., Ingham K. C., Strickland D. K., Argraves W. S. Fibulin binds to itself and to the carboxyl-terminal heparin-binding region of fibronectin. J Biol Chem. 1992 Oct 5;267(28):20120–20125. [PubMed] [Google Scholar]
  2. 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]
  3. Fässler R., Sasaki T., Timpl R., Chu M. L., Werner S. Differential regulation of fibulin, tenascin-C, and nidogen expression during wound healing of normal and glucocorticoid-treated mice. Exp Cell Res. 1996 Jan 10;222(1):111–116. doi: 10.1006/excr.1996.0014. [DOI] [PubMed] [Google Scholar]
  4. Gooley A. A., Classon B. J., Marschalek R., Williams K. L. Glycosylation sites identified by detection of glycosylated amino acids released from Edman degradation: the identification of Xaa-Pro-Xaa-Xaa as a motif for Thr-O-glycosylation. Biochem Biophys Res Commun. 1991 Aug 15;178(3):1194–1201. doi: 10.1016/0006-291x(91)91019-9. [DOI] [PubMed] [Google Scholar]
  5. Huber R., Scholze H., Pâques E. P., Deisenhofer J. Crystal structure analysis and molecular model of human C3a anaphylatoxin. Hoppe Seylers Z Physiol Chem. 1980 Sep;361(9):1389–1399. doi: 10.1515/bchm2.1980.361.2.1389. [DOI] [PubMed] [Google Scholar]
  6. Kohfeldt E., Göhring W., Mayer U., Zweckstetter M., Holak T. A., Chu M. L., Timpl R. Conversion of the Kunitz-type module of collagen VI into a highly active trypsin inhibitor by site-directed mutagenesis. Eur J Biochem. 1996 Jun 1;238(2):333–340. doi: 10.1111/j.1432-1033.1996.0333z.x. [DOI] [PubMed] [Google Scholar]
  7. Mann K., Mechling D. E., Bächinger H. P., Eckerskorn C., Gaill F., Timpl R. Glycosylated threonine but not 4-hydroxyproline dominates the triple helix stabilizing positions in the sequence of a hydrothermal vent worm cuticle collagen. J Mol Biol. 1996 Aug 16;261(2):255–266. doi: 10.1006/jmbi.1996.0457. [DOI] [PubMed] [Google Scholar]
  8. Mayer U., Aumailley M., Mann K., Timpl R., Engel J. Calcium-dependent binding of basement membrane protein BM-40 (osteonectin, SPARC) to basement membrane collagen type IV. Eur J Biochem. 1991 May 23;198(1):141–150. doi: 10.1111/j.1432-1033.1991.tb15996.x. [DOI] [PubMed] [Google Scholar]
  9. Mayer U., Nischt R., Pöschl E., Mann K., Fukuda K., Gerl M., Yamada Y., Timpl R. A single EGF-like motif of laminin is responsible for high affinity nidogen binding. EMBO J. 1993 May;12(5):1879–1885. doi: 10.1002/j.1460-2075.1993.tb05836.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Mayer U., Pöschl E., Gerecke D. R., Wagman D. W., Burgeson R. E., Timpl R. Low nidogen affinity of laminin-5 can be attributed to two serine residues in EGF-like motif gamma 2III4. FEBS Lett. 1995 May 29;365(2-3):129–132. doi: 10.1016/0014-5793(95)00438-f. [DOI] [PubMed] [Google Scholar]
  11. Miosge N., Götz W., Sasaki T., Chu M. L., Timpl R., Herken R. The extracellular matrix proteins fibulin-1 and fibulin-2 in the early human embryo. Histochem J. 1996 Feb;28(2):109–116. doi: 10.1007/BF02331415. [DOI] [PubMed] [Google Scholar]
  12. Pan T. C., Sasaki T., Zhang R. Z., Fässler R., Timpl R., Chu M. L. Structure and expression of fibulin-2, a novel extracellular matrix protein with multiple EGF-like repeats and consensus motifs for calcium binding. J Cell Biol. 1993 Dec;123(5):1269–1277. doi: 10.1083/jcb.123.5.1269. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Pfaff M., Sasaki T., Tangemann K., Chu M. L., Timpl R. Integrin-binding and cell-adhesion studies of fibulins reveal a particular affinity for alpha IIb beta 3. Exp Cell Res. 1995 Jul;219(1):87–92. doi: 10.1006/excr.1995.1208. [DOI] [PubMed] [Google Scholar]
  14. Reinhardt D. P., Keene D. R., Corson G. M., Pöschl E., Bächinger H. P., Gambee J. E., Sakai L. Y. Fibrillin-1: organization in microfibrils and structural properties. J Mol Biol. 1996 Apr 26;258(1):104–116. doi: 10.1006/jmbi.1996.0237. [DOI] [PubMed] [Google Scholar]
  15. Reinhardt D. P., Sasaki T., Dzamba B. J., Keene D. R., Chu M. L., Göhring W., Timpl R., Sakai L. Y. Fibrillin-1 and fibulin-2 interact and are colocalized in some tissues. J Biol Chem. 1996 Aug 9;271(32):19489–19496. doi: 10.1074/jbc.271.32.19489. [DOI] [PubMed] [Google Scholar]
  16. Sasaki T., Göhring W., Pan T. C., Chu M. L., Timpl R. Binding of mouse and human fibulin-2 to extracellular matrix ligands. J Mol Biol. 1995 Dec 15;254(5):892–899. doi: 10.1006/jmbi.1995.0664. [DOI] [PubMed] [Google Scholar]
  17. Sasaki T., Kostka G., Göhring W., Wiedemann H., Mann K., Chu M. L., Timpl R. Structural characterization of two variants of fibulin-1 that differ in nidogen affinity. J Mol Biol. 1995 Jan 20;245(3):241–250. doi: 10.1006/jmbi.1994.0020. [DOI] [PubMed] [Google Scholar]
  18. Sasaki T., Mann K., Murphy G., Chu M. L., Timpl R. Different susceptibilities of fibulin-1 and fibulin-2 to cleavage by matrix metalloproteinases and other tissue proteases. Eur J Biochem. 1996 Sep 1;240(2):427–434. doi: 10.1111/j.1432-1033.1996.0427h.x. [DOI] [PubMed] [Google Scholar]
  19. Sasaki T., Wiedemann H., Matzner M., Chu M. L., Timpl R. Expression of fibulin-2 by fibroblasts and deposition with fibronectin into a fibrillar matrix. J Cell Sci. 1996 Dec;109(Pt 12):2895–2904. doi: 10.1242/jcs.109.12.2895. [DOI] [PubMed] [Google Scholar]
  20. Wilson I. B., Gavel Y., von Heijne G. Amino acid distributions around O-linked glycosylation sites. Biochem J. 1991 Apr 15;275(Pt 2):529–534. doi: 10.1042/bj2750529. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Zhang H. Y., Chu M. L., Pan T. C., Sasaki T., Timpl R., Ekblom P. Extracellular matrix protein fibulin-2 is expressed in the embryonic endocardial cushion tissue and is a prominent component of valves in adult heart. Dev Biol. 1995 Jan;167(1):18–26. doi: 10.1006/dbio.1995.1003. [DOI] [PubMed] [Google Scholar]
  22. Zhang H. Y., Timpl R., Sasaki T., Chu M. L., Ekblom P. Fibulin-1 and fibulin-2 expression during organogenesis in the developing mouse embryo. Dev Dyn. 1996 Mar;205(3):348–364. doi: 10.1002/(SICI)1097-0177(199603)205:3<348::AID-AJA13>3.0.CO;2-0. [DOI] [PubMed] [Google Scholar]

Articles from The EMBO Journal are provided here courtesy of Nature Publishing Group

RESOURCES