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. 1994 Jul 2;126(2):563–574. doi: 10.1083/jcb.126.2.563

67-kD elastin-binding protein is a protective "companion" of extracellular insoluble elastin and intracellular tropoelastin

PMCID: PMC2200028  PMID: 8034752

Abstract

The 67-kD elastin-binding protein (EBP) mediates cell adhesion to elastin and elastin fiber assembly, and it is similar, if not identical, to the 67-kD enzymatically inactive, alternatively spliced beta-galactosidase. The latter contains an elastin binding domain (S- GAL) homologous both to the aorta EBP and to NH2-terminal sequences of serine proteinases (Hinek, A., M. Rabinovitch, F. W. Keeley, and J. Callahan. 1993. J. Clin. Invest. 91:1198-1205). We now confirm the functional importance of this homology by showing that elastolytic activity of a representative serine elastase, porcine pancreatic elastase, was prevented by an antibody (anti-S-GAL) and by competing with purified EBP or S-GAL peptide. Immunohistochemistry of adult aorta indicates that the EBP exists as a permanent component of mature elastic fibers. This observation, together with the in vitro studies, suggests that the EBP could protect insoluble elastin from extracellular proteolysis and contribute to the extraordinary stability of this protein. Double immunolabeling of fetal lamb aorta with anti-S- GAL and antitropoelastin antibodies demonstrated, under light and electron microscopy, intracellular colocalization of the proteins in smooth muscle cells (SMC). Incubation of SMC with galactosugars to dissociate tropoelastin from EBP caused intracellular aggregation of tropoelastin. A tropoelastin/EBP complex was extracted from SMC lysates by coimmunoprecipitation and cross-linking, and its functional significance was addressed by showing that its dissociation by galactosugars caused degradation of tropoelastin by endogenous serine proteinase(s). This suggests that the EBP may also serve as a "companion" to intracellular tropoelastin, protecting this highly hydrophobic protein from self-aggregation and proteolytic degradation.

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Selected References

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  1. Agard D. A. To fold or not to fold.... Science. 1993 Jun 25;260(5116):1903–1904. doi: 10.1126/science.8100365. [DOI] [PubMed] [Google Scholar]
  2. Banda M. J., Werb Z. Mouse macrophage elastase. Purification and characterization as a metalloproteinase. Biochem J. 1981 Feb 1;193(2):589–605. doi: 10.1042/bj1930589. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Degen E., Williams D. B. Participation of a novel 88-kD protein in the biogenesis of murine class I histocompatibility molecules. J Cell Biol. 1991 Mar;112(6):1099–1115. doi: 10.1083/jcb.112.6.1099. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Doolittle R. F., Feng D. F. Reconstructing the evolution of vertebrate blood coagulation from a consideration of the amino acid sequences of clotting proteins. Cold Spring Harb Symp Quant Biol. 1987;52:869–874. doi: 10.1101/sqb.1987.052.01.095. [DOI] [PubMed] [Google Scholar]
  5. Doolittle R. F. Similar amino acid sequences revisited. Trends Biochem Sci. 1989 Jul;14(7):244–245. doi: 10.1016/0968-0004(89)90055-8. [DOI] [PubMed] [Google Scholar]
  6. Gibson M. A., Kumaratilake J. S., Cleary E. G. The protein components of the 12-nanometer microfibrils of elastic and nonelastic tissues. J Biol Chem. 1989 Mar 15;264(8):4590–4598. [PubMed] [Google Scholar]
  7. Hinek A., Mecham R. P., Keeley F., Rabinovitch M. Impaired elastin fiber assembly related to reduced 67-kD elastin-binding protein in fetal lamb ductus arteriosus and in cultured aortic smooth muscle cells treated with chondroitin sulfate. J Clin Invest. 1991 Dec;88(6):2083–2094. doi: 10.1172/JCI115538. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Hinek A., Rabinovitch M., Keeley F., Okamura-Oho Y., Callahan J. The 67-kD elastin/laminin-binding protein is related to an enzymatically inactive, alternatively spliced form of beta-galactosidase. J Clin Invest. 1993 Mar;91(3):1198–1205. doi: 10.1172/JCI116280. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Hinek A., Rabinovitch M. The ductus arteriosus migratory smooth muscle cell phenotype processes tropoelastin to a 52-kDa product associated with impaired assembly of elastic laminae. J Biol Chem. 1993 Jan 15;268(2):1405–1413. [PubMed] [Google Scholar]
  10. Hinek A., Wrenn D. S., Mecham R. P., Barondes S. H. The elastin receptor: a galactoside-binding protein. Science. 1988 Mar 25;239(4847):1539–1541. doi: 10.1126/science.2832941. [DOI] [PubMed] [Google Scholar]
  11. Hornebeck W., Derouette J. C., Robert L. Isolation, purification and properties of aortic elastase. FEBS Lett. 1975 Oct 15;58(1):66–70. doi: 10.1016/0014-5793(75)80227-4. [DOI] [PubMed] [Google Scholar]
  12. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  13. Landry S. J., Jordan R., McMacken R., Gierasch L. M. Different conformations for the same polypeptide bound to chaperones DnaK and GroEL. Nature. 1992 Jan 30;355(6359):455–457. doi: 10.1038/355455a0. [DOI] [PubMed] [Google Scholar]
  14. Langer T., Lu C., Echols H., Flanagan J., Hayer M. K., Hartl F. U. Successive action of DnaK, DnaJ and GroEL along the pathway of chaperone-mediated protein folding. Nature. 1992 Apr 23;356(6371):683–689. doi: 10.1038/356683a0. [DOI] [PubMed] [Google Scholar]
  15. Leake D. S., Hornebeck W., Bréchemier D., Robert L., Peters T. J. Properties and subcellular localization of elastase-like activities of arterial smooth muscle cells in culture. Biochim Biophys Acta. 1983 Nov 22;761(1):41–47. doi: 10.1016/0304-4165(83)90360-4. [DOI] [PubMed] [Google Scholar]
  16. McKenzie H. A., White F. H., Jr Lysozyme and alpha-lactalbumin: structure, function, and interrelationships. Adv Protein Chem. 1991;41:173–315. doi: 10.1016/s0065-3233(08)60198-9. [DOI] [PubMed] [Google Scholar]
  17. Mecham R. P., Hinek A., Cleary E. G., Kucich U., Lee S. J., Rosenbloom J. Development of immunoreagents to ciliary zonules that react with protein components of elastic fiber microfibrils and with elastin-producing cells. Biochem Biophys Res Commun. 1988 Mar 15;151(2):822–826. doi: 10.1016/s0006-291x(88)80355-3. [DOI] [PubMed] [Google Scholar]
  18. Mecham R. P., Hinek A., Entwistle R., Wrenn D. S., Griffin G. L., Senior R. M. Elastin binds to a multifunctional 67-kilodalton peripheral membrane protein. Biochemistry. 1989 May 2;28(9):3716–3722. doi: 10.1021/bi00435a014. [DOI] [PubMed] [Google Scholar]
  19. Mecham R. P., Hinek A., Griffin G. L., Senior R. M., Liotta L. A. The elastin receptor shows structural and functional similarities to the 67-kDa tumor cell laminin receptor. J Biol Chem. 1989 Oct 5;264(28):16652–16657. [PubMed] [Google Scholar]
  20. Mecham R. P., Whitehouse L., Hay M., Hinek A., Sheetz M. P. Ligand affinity of the 67-kD elastin/laminin binding protein is modulated by the protein's lectin domain: visualization of elastin/laminin-receptor complexes with gold-tagged ligands. J Cell Biol. 1991 Apr;113(1):187–194. doi: 10.1083/jcb.113.1.187. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Morreau H., Galjart N. J., Gillemans N., Willemsen R., van der Horst G. T., d'Azzo A. Alternative splicing of beta-galactosidase mRNA generates the classic lysosomal enzyme and a beta-galactosidase-related protein. J Biol Chem. 1989 Dec 5;264(34):20655–20663. [PubMed] [Google Scholar]
  22. Nakai A., Satoh M., Hirayoshi K., Nagata K. Involvement of the stress protein HSP47 in procollagen processing in the endoplasmic reticulum. J Cell Biol. 1992 May;117(4):903–914. doi: 10.1083/jcb.117.4.903. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Prosser I. W., Whitehouse L. A., Parks W. C., Stahle-Bäckdahl M., Hinek A., Park P. W., Mecham R. P. Polyclonal antibodies to tropoelastin and the specific detection and measurement of tropoelastin in vitro. Connect Tissue Res. 1991;25(3-4):265–279. doi: 10.3109/03008209109029162. [DOI] [PubMed] [Google Scholar]
  24. Rosenbloom J., Weinbaum G., Abrams W., Ornsten-Goldstein N., Indik Z., Kucich U. Newly determined carboxy terminal sequences in tropoelastin: immunologic identification in insoluble elastin. Coll Relat Res. 1986 Dec;6(5):423–433. doi: 10.1016/s0174-173x(86)80018-8. [DOI] [PubMed] [Google Scholar]
  25. Senior R. M., Griffin G. L., Mecham R. P., Wrenn D. S., Prasad K. U., Urry D. W. Val-Gly-Val-Ala-Pro-Gly, a repeating peptide in elastin, is chemotactic for fibroblasts and monocytes. J Cell Biol. 1984 Sep;99(3):870–874. doi: 10.1083/jcb.99.3.870. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Shotton D. M., Hartley B. S. Amino-acid sequence of porcine pancreatic elastase and its homologies with other serine proteinases. Nature. 1970 Feb 28;225(5235):802–806. doi: 10.1038/225802a0. [DOI] [PubMed] [Google Scholar]
  27. Takahashi S., Seifter S., Yang F. C. A new radioactive assay for enzymes with elastolytic activity using reduced tritiated elastin. The effect of sodium dodecyl sulfate on elastolysis. Biochim Biophys Acta. 1973 Nov 15;327(1):138–145. doi: 10.1016/0005-2744(73)90111-3. [DOI] [PubMed] [Google Scholar]
  28. Urry D. W., Starcher B., Partridge S. M. Coacervation of solubilized elastin effects a notable conformational change. Nature. 1969 May 24;222(5195):795–796. doi: 10.1038/222795a0. [DOI] [PubMed] [Google Scholar]
  29. Volpin D., Urry D. W., Cox B. A., Gotte L. Optical diffraction of tropoelastin and alpha-elastin coacervates. Biochim Biophys Acta. 1976 Jul 19;439(1):253–258. doi: 10.1016/0005-2795(76)90181-1. [DOI] [PubMed] [Google Scholar]
  30. Wrenn D. S., Griffin G. L., Senior R. M., Mecham R. P. Characterization of biologically active domains on elastin: identification of a monoclonal antibody to a cell recognition site. Biochemistry. 1986 Sep 9;25(18):5172–5176. doi: 10.1021/bi00366a028. [DOI] [PubMed] [Google Scholar]
  31. Wrenn D. S., Mecham R. P. Immunology of elastin. Methods Enzymol. 1987;144:246–259. doi: 10.1016/0076-6879(87)44182-7. [DOI] [PubMed] [Google Scholar]
  32. Zhu L., Dagher E., Johnson D. J., Bedell-Hogan D., Keeley F. W., Kagan H. M., Rabinovitch M. A developmentally regulated program restricting insolubilization of elastin and formation of laminae in the fetal lamb ductus arteriosus. Lab Invest. 1993 Mar;68(3):321–331. [PubMed] [Google Scholar]

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