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. 1995 Apr;15(4):2294–2303. doi: 10.1128/mcb.15.4.2294

Proteolytic processing yields two secreted forms of sonic hedgehog.

D A Bumcrot 1, R Takada 1, A P McMahon 1
PMCID: PMC230457  PMID: 7891723

Abstract

Sonic hedgehog (Shh) is expressed in tissues with known signalling capacities, such as the notochord, the floor plate of the central nervous system, and the zone of polarizing activity in the limb. Several lines of evidence indicate that Shh is involved in floor plate induction, somite patterning, and regulation of anterior-posterior polarity in the vertebrate limb. In this report, we investigate the biochemical behavior of Shh in a variety of expression systems and embryonic tissues. Expression of mouse Shh in Xenopus oocytes, COS cells, and baculovirus-infected insect cells demonstrates that in addition to signal peptide cleavage and N-linked glycosylation, chicken and mouse Shh proteins undergo additional proteolytic processing to yield two peptides with molecular masses of approximately 19 kDa (amino terminus) and 27 kDa (carboxy terminus), both of which are secreted. In transfected COS cells, we show that the 19-kDa peptide does not accumulate significantly in the medium unless heparin or suramin is added, suggesting that this peptide associates with the cell surface or extracellular matrix. This retention appears to depend on sequences in the carboxy-terminal part of the peptide. Finally, detection of the 19-kDa product in a variety of mouse and chicken embryonic tissues demonstrates that the proteolytic processing observed in cell culture is a normal aspect of Shh processing in embryonic development. These results raise the possibility that amino- and carboxyl-terminal regions of Shh may have distinct functions in regulating cell-cell interactions in the vertebrate embryo.

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Selected References

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  1. Basler K., Struhl G. Compartment boundaries and the control of Drosophila limb pattern by hedgehog protein. Nature. 1994 Mar 17;368(6468):208–214. doi: 10.1038/368208a0. [DOI] [PubMed] [Google Scholar]
  2. Bradley R. S., Brown A. M. The proto-oncogene int-1 encodes a secreted protein associated with the extracellular matrix. EMBO J. 1990 May;9(5):1569–1575. doi: 10.1002/j.1460-2075.1990.tb08276.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Buffinger N., Stockdale F. E. Myogenic specification in somites: induction by axial structures. Development. 1994 Jun;120(6):1443–1452. doi: 10.1242/dev.120.6.1443. [DOI] [PubMed] [Google Scholar]
  4. Chang D. T., López A., von Kessler D. P., Chiang C., Simandl B. K., Zhao R., Seldin M. F., Fallon J. F., Beachy P. A. Products, genetic linkage and limb patterning activity of a murine hedgehog gene. Development. 1994 Nov;120(11):3339–3353. doi: 10.1242/dev.120.11.3339. [DOI] [PubMed] [Google Scholar]
  5. Echelard Y., Epstein D. J., St-Jacques B., Shen L., Mohler J., McMahon J. A., McMahon A. P. Sonic hedgehog, a member of a family of putative signaling molecules, is implicated in the regulation of CNS polarity. Cell. 1993 Dec 31;75(7):1417–1430. doi: 10.1016/0092-8674(93)90627-3. [DOI] [PubMed] [Google Scholar]
  6. Evan G. I., Lewis G. K., Ramsay G., Bishop J. M. Isolation of monoclonal antibodies specific for human c-myc proto-oncogene product. Mol Cell Biol. 1985 Dec;5(12):3610–3616. doi: 10.1128/mcb.5.12.3610. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Fan C. M., Tessier-Lavigne M. Patterning of mammalian somites by surface ectoderm and notochord: evidence for sclerotome induction by a hedgehog homolog. Cell. 1994 Dec 30;79(7):1175–1186. doi: 10.1016/0092-8674(94)90009-4. [DOI] [PubMed] [Google Scholar]
  8. Heemskerk J., DiNardo S. Drosophila hedgehog acts as a morphogen in cellular patterning. Cell. 1994 Feb 11;76(3):449–460. doi: 10.1016/0092-8674(94)90110-4. [DOI] [PubMed] [Google Scholar]
  9. Ingham P. W., Hidalgo A. Regulation of wingless transcription in the Drosophila embryo. Development. 1993 Jan;117(1):283–291. doi: 10.1242/dev.117.1.283. [DOI] [PubMed] [Google Scholar]
  10. Ingham P. W. Pattern formation. Hedgehog points the way. Curr Biol. 1994 Apr 1;4(4):347–350. doi: 10.1016/s0960-9822(00)00076-2. [DOI] [PubMed] [Google Scholar]
  11. Johnson R. L., Laufer E., Riddle R. D., Tabin C. Ectopic expression of Sonic hedgehog alters dorsal-ventral patterning of somites. Cell. 1994 Dec 30;79(7):1165–1173. doi: 10.1016/0092-8674(94)90008-6. [DOI] [PubMed] [Google Scholar]
  12. Kornfeld R., Kornfeld S. Assembly of asparagine-linked oligosaccharides. Annu Rev Biochem. 1985;54:631–664. doi: 10.1146/annurev.bi.54.070185.003215. [DOI] [PubMed] [Google Scholar]
  13. Krauss S., Concordet J. P., Ingham P. W. A functionally conserved homolog of the Drosophila segment polarity gene hh is expressed in tissues with polarizing activity in zebrafish embryos. Cell. 1993 Dec 31;75(7):1431–1444. doi: 10.1016/0092-8674(93)90628-4. [DOI] [PubMed] [Google Scholar]
  14. Laufer E. D. Vertebrate development: factoring in the limb. Curr Biol. 1993 May 1;3(5):306–308. doi: 10.1016/0960-9822(93)90187-s. [DOI] [PubMed] [Google Scholar]
  15. Laufer E., Nelson C. E., Johnson R. L., Morgan B. A., Tabin C. Sonic hedgehog and Fgf-4 act through a signaling cascade and feedback loop to integrate growth and patterning of the developing limb bud. Cell. 1994 Dec 16;79(6):993–1003. doi: 10.1016/0092-8674(94)90030-2. [DOI] [PubMed] [Google Scholar]
  16. Lee J. J., Ekker S. C., von Kessler D. P., Porter J. A., Sun B. I., Beachy P. A. Autoproteolysis in hedgehog protein biogenesis. Science. 1994 Dec 2;266(5190):1528–1537. doi: 10.1126/science.7985023. [DOI] [PubMed] [Google Scholar]
  17. Lee J. J., von Kessler D. P., Parks S., Beachy P. A. Secretion and localized transcription suggest a role in positional signaling for products of the segmentation gene hedgehog. Cell. 1992 Oct 2;71(1):33–50. doi: 10.1016/0092-8674(92)90264-d. [DOI] [PubMed] [Google Scholar]
  18. Marshall R. D. Glycoproteins. Annu Rev Biochem. 1972;41:673–702. doi: 10.1146/annurev.bi.41.070172.003325. [DOI] [PubMed] [Google Scholar]
  19. Middaugh C. R., Mach H., Burke C. J., Volkin D. B., Dabora J. M., Tsai P. K., Bruner M. W., Ryan J. A., Marfia K. E. Nature of the interaction of growth factors with suramin. Biochemistry. 1992 Sep 22;31(37):9016–9024. doi: 10.1021/bi00152a044. [DOI] [PubMed] [Google Scholar]
  20. Mohler J., Vani K. Molecular organization and embryonic expression of the hedgehog gene involved in cell-cell communication in segmental patterning of Drosophila. Development. 1992 Aug;115(4):957–971. doi: 10.1242/dev.115.4.957. [DOI] [PubMed] [Google Scholar]
  21. Munro S., Pelham H. R. A C-terminal signal prevents secretion of luminal ER proteins. Cell. 1987 Mar 13;48(5):899–907. doi: 10.1016/0092-8674(87)90086-9. [DOI] [PubMed] [Google Scholar]
  22. Nüsslein-Volhard C., Wieschaus E. Mutations affecting segment number and polarity in Drosophila. Nature. 1980 Oct 30;287(5785):795–801. doi: 10.1038/287795a0. [DOI] [PubMed] [Google Scholar]
  23. Perrimon N. The genetic basis of patterned baldness in Drosophila. Cell. 1994 Mar 11;76(5):781–784. doi: 10.1016/0092-8674(94)90351-4. [DOI] [PubMed] [Google Scholar]
  24. Placzek M., Jessell T. M., Dodd J. Induction of floor plate differentiation by contact-dependent, homeogenetic signals. Development. 1993 Jan;117(1):205–218. doi: 10.1242/dev.117.1.205. [DOI] [PubMed] [Google Scholar]
  25. Placzek M., Tessier-Lavigne M., Yamada T., Jessell T., Dodd J. Mesodermal control of neural cell identity: floor plate induction by the notochord. Science. 1990 Nov 16;250(4983):985–988. doi: 10.1126/science.2237443. [DOI] [PubMed] [Google Scholar]
  26. Placzek M., Yamada T., Tessier-Lavigne M., Jessell T., Dodd J. Control of dorsoventral pattern in vertebrate neural development: induction and polarizing properties of the floor plate. Dev Suppl. 1991;Suppl 2:105–122. [PubMed] [Google Scholar]
  27. Riddle R. D., Johnson R. L., Laufer E., Tabin C. Sonic hedgehog mediates the polarizing activity of the ZPA. Cell. 1993 Dec 31;75(7):1401–1416. doi: 10.1016/0092-8674(93)90626-2. [DOI] [PubMed] [Google Scholar]
  28. Roelink H., Augsburger A., Heemskerk J., Korzh V., Norlin S., Ruiz i Altaba A., Tanabe Y., Placzek M., Edlund T., Jessell T. M. Floor plate and motor neuron induction by vhh-1, a vertebrate homolog of hedgehog expressed by the notochord. Cell. 1994 Feb 25;76(4):761–775. doi: 10.1016/0092-8674(94)90514-2. [DOI] [PubMed] [Google Scholar]
  29. Rong P. M., Teillet M. A., Ziller C., Le Douarin N. M. The neural tube/notochord complex is necessary for vertebral but not limb and body wall striated muscle differentiation. Development. 1992 Jul;115(3):657–672. doi: 10.1242/dev.115.3.657. [DOI] [PubMed] [Google Scholar]
  30. Ruiz i Altaba A., Jessell T. M. Midline cells and the organization of the vertebrate neuraxis. Curr Opin Genet Dev. 1993 Aug;3(4):633–640. doi: 10.1016/0959-437x(93)90100-4. [DOI] [PubMed] [Google Scholar]
  31. Smith J. C. Dorso-ventral patterning in the neural tube. Curr Biol. 1993 Sep 1;3(9):582–585. doi: 10.1016/0960-9822(93)90003-7. [DOI] [PubMed] [Google Scholar]
  32. Smith J. C. Hedgehog, the floor plate, and the zone of polarizing activity. Cell. 1994 Jan 28;76(2):193–196. doi: 10.1016/0092-8674(94)90325-5. [DOI] [PubMed] [Google Scholar]
  33. Smith J. L., Schoenwolf G. C. Notochordal induction of cell wedging in the chick neural plate and its role in neural tube formation. J Exp Zool. 1989 Apr;250(1):49–62. doi: 10.1002/jez.1402500107. [DOI] [PubMed] [Google Scholar]
  34. Smolich B. D., McMahon J. A., McMahon A. P., Papkoff J. Wnt family proteins are secreted and associated with the cell surface. Mol Biol Cell. 1993 Dec;4(12):1267–1275. doi: 10.1091/mbc.4.12.1267. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Tabata T., Eaton S., Kornberg T. B. The Drosophila hedgehog gene is expressed specifically in posterior compartment cells and is a target of engrailed regulation. Genes Dev. 1992 Dec;6(12B):2635–2645. doi: 10.1101/gad.6.12b.2635. [DOI] [PubMed] [Google Scholar]
  36. Tabata T., Kornberg T. B. Hedgehog is a signaling protein with a key role in patterning Drosophila imaginal discs. Cell. 1994 Jan 14;76(1):89–102. doi: 10.1016/0092-8674(94)90175-9. [DOI] [PubMed] [Google Scholar]
  37. Tabin C. J. Retinoids, homeoboxes, and growth factors: toward molecular models for limb development. Cell. 1991 Jul 26;66(2):199–217. doi: 10.1016/0092-8674(91)90612-3. [DOI] [PubMed] [Google Scholar]
  38. Tarentino A. L., Trimble R. B., Plummer T. H., Jr Enzymatic approaches for studying the structure, synthesis, and processing of glycoproteins. Methods Cell Biol. 1989;32:111–139. doi: 10.1016/s0091-679x(08)61169-3. [DOI] [PubMed] [Google Scholar]
  39. Tashiro S., Michiue T., Higashijima S., Zenno S., Ishimaru S., Takahashi F., Orihara M., Kojima T., Saigo K. Structure and expression of hedgehog, a Drosophila segment-polarity gene required for cell-cell communication. Gene. 1993 Feb 28;124(2):183–189. doi: 10.1016/0378-1119(93)90392-g. [DOI] [PubMed] [Google Scholar]
  40. Taylor A. M., Nakano Y., Mohler J., Ingham P. W. Contrasting distributions of patched and hedgehog proteins in the Drosophila embryo. Mech Dev. 1993 Jul;42(1-2):89–96. doi: 10.1016/0925-4773(93)90101-3. [DOI] [PubMed] [Google Scholar]
  41. Tickle C. The number of polarizing region cells required to specify additional digits in the developing chick wing. Nature. 1981 Jan 22;289(5795):295–298. doi: 10.1038/289295a0. [DOI] [PubMed] [Google Scholar]
  42. Wolpert L. Positional information and the spatial pattern of cellular differentiation. J Theor Biol. 1969 Oct;25(1):1–47. doi: 10.1016/s0022-5193(69)80016-0. [DOI] [PubMed] [Google Scholar]
  43. Yamada T., Pfaff S. L., Edlund T., Jessell T. M. Control of cell pattern in the neural tube: motor neuron induction by diffusible factors from notochord and floor plate. Cell. 1993 May 21;73(4):673–686. doi: 10.1016/0092-8674(93)90248-o. [DOI] [PubMed] [Google Scholar]
  44. Yamada T., Placzek M., Tanaka H., Dodd J., Jessell T. M. Control of cell pattern in the developing nervous system: polarizing activity of the floor plate and notochord. Cell. 1991 Feb 8;64(3):635–647. doi: 10.1016/0092-8674(91)90247-v. [DOI] [PubMed] [Google Scholar]

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