1887

Abstract

Highly purified spore coats of each contained about 5 × 10 protein molecules as determined by amino acid composition analysis. By two-dimensional gel electrophoresis the coats were found to contain nine major-abundance and numerous minor protein species, most of which were highly enriched relative to the adjacent interspore matrix. Protein was nearly quantitatively eluted by denaturants and 2-mercaptoethanol, showing that it was not irreversibly cross-linked. Because a reducing agent is required together with denaturants to elute most proteins if their free thiol groups have been prealkylated, it was concluded that the spore coat proteins are disulfide cross-linked into the matrix. One major coat protein, SP75, was partially sequenced and found to be encoded by the previously identified DP87 gene; this finding was supported by additional physical, genetic, biochemical and microscopic evidence. The five major proteins for which genes have been cloned were associated with the outer layer of the coat. In coats missing one or more of four of these proteins as a result of gene disruption, there were physical changes but, with one exception, the other major coat proteins appeared to be incorporated normally. Sequence analysis showed that these five outer layer coat proteins are homologous and consist of alternating sequence motifs related to epithelial mucin repeats, basic proline repeats found in salivary acidic proline-rich proteins, the NH-terminal subdomain of epidermal growth factor modules and other cysteine repeats. Based on these and other observations, outer layer coat proteins are predicted to organize indeterminately to form a cell surface microenvironment supportive of cellulose morphogenesis during spore coat formation.

Loading

Article metrics loading...

/content/journal/micro/10.1099/13500872-142-8-2227
1996-08-01
2024-11-06
Loading full text...

Full text loading...

/deliver/fulltext/micro/142/8/mic-142-8-2227.html?itemId=/content/journal/micro/10.1099/13500872-142-8-2227&mimeType=html&fmt=ahah

References

  1. Akalehiywot T., Siu C.-H. 1983; Phosphorylation of spore coat proteins of Dictjostelium discoideum . Can J Biochem Cell Biol 61:996–1001
    [Google Scholar]
  2. Ann D.K., Gadbois D., Carlson D.M. 1987; Structure, organization, and regulation of a hamster proline-rich protein gene. J Biol Chem 262:3958–3963
    [Google Scholar]
  3. Aparicio J.G., Erdos G.W., West C.M. 1990; The spore coat is altered in modB glycosylation mutants of Dictjostelium discoideum . J Cell Biochem 42:255–266
    [Google Scholar]
  4. Bhargava A.K., Woitach J.T., Davidson E.A., Bhavanandan V.P. 1990; Cloning and cDNA sequence of a bovine submaxillary gland mucin-like protein containing two distinct domains. Proc Natl Acad Sci USA 876798–6802
    [Google Scholar]
  5. Ceccarelli A., McRobbie S.J., Jermyn K.A., Duffy K., Early A., Williams J.G. 1987; Structural and functional characterization of a Dictjostelium gene encoding a DIF inducible, prestalk-enriched mRNA sequence. Nucleic Acids Res 15:7463–7476
    [Google Scholar]
  6. Cotter D.A. 1981; Spore activation. In The Fungal Spore: Morphogenetic Controls pp. 385–411 Edited by Turian G., Flohl H.R. New York: Academic Press;
    [Google Scholar]
  7. Creighton T.E., Zapun A., Darby N.J. 1995; Mechanisms and catalysts of disulphide bond formation in proteins. Trends Biotechnol 13:18–23
    [Google Scholar]
  8. Devine P.L., McKenzie I.F.C. 1992; Mucins: structure, function, and associations with malignancy. Bioessays 14:619–625
    [Google Scholar]
  9. Devine K.M., Morrissey J.H., Loomis W.F. 1982; Differential synthesis of spore coat proteins in prespore and prestalk cells of Dictyostelium . Proc Natl Acad Sci USA 797361–7365
    [Google Scholar]
  10. Devine K., Bergmann J., Loomis W. F. 1983; Spore coat proteins of Dictjostelium discoideum are packaged in prespore vesicles. Dev Biol 99:437–446
    [Google Scholar]
  11. Dupont E., El Aoumari A., Briand J.P., Fromaget C., Gros D. 1989; Cross-linking of cardiac gap junction connexons by thiol/disulfide exchanges. J Membr Biol 108:247–252
    [Google Scholar]
  12. Engel L. 1989; EGF-like domains in extracellular matrix proteins: localized signals for growth and differentiation?. FEBS Lett 251:1–7
    [Google Scholar]
  13. Erdos G.W., Whitaker D. 1983; Failure to detect immuno cytochemically reactive endogenous lectin on the cell surface of Dictjostelium discoideum . J Cell Biol 97:993–1000
    [Google Scholar]
  14. Erdos G.W., West C.M. 1989; Formation and organization of the spore coat of Dictjostelium discoideum . Exp Mycol 13:169–182
    [Google Scholar]
  15. Fosnaugh K.L., Loomis W.F. 1989a; Sequence of the Dictjostelium discoideum spore coat gene SP96. Nucleic Acids Res 17:9489
    [Google Scholar]
  16. Fosnaugh K.L., Loomis W.F. 1989b; Spore coat genes SP60 and SP70 of Dictjostelium discoideum . Mol Cell Biol 9:5215–5218
    [Google Scholar]
  17. Fosnaugh K.L., Fuller D., Loomis W.F. 1995; Structural roles of the spore coat proteins in Dictjostelium discoideum . Dev Biol 166:823–825
    [Google Scholar]
  18. Freeze H.H. 1996; Dictjostelium discoideum glycoproteins: using a model system for organismic glycobiology. In New Comprehensive Biochemistrj Ch.20. Edited by Neuberger A., Van Deenen L.L.M. Cambridge: Elsevier; in press
    [Google Scholar]
  19. Gerken T.A., Butenhof K.J., Shogren R. 1989; Effects of glycosylation on the conformation and dynamics of O -linked glycoproteins: carbon-13 NMR studies of ovine submaxillary mucin. Biochemistry 28:5536–5543
    [Google Scholar]
  20. Gonzalez-Yanes B., Mandell R.B., Girard M., Henry S., Aparicio O., Gritzali M., Brown R.D., Erdos G.W., West C.M. 1989; The spore coat of a fucosylation mutant in Dictjostelium discoideum . Dev Biol 133:576–587
    [Google Scholar]
  21. Gooley A.A., Marschchalek R., Williams K.L. 1992; Size polymorphisms due to changes in the number of O -glycosylated tandem repeats in the Dictjostelium discoideum glycoprotein PsA. Genetics 130:749–756
    [Google Scholar]
  22. Grauschopf U., Winther J.R., Korber P., Zander T., Dallinger P., Bardwell J.C.A. 1995; Why is DsbA such an oxidizing disulfide catalyst?. Cell 83:947–955
    [Google Scholar]
  23. Graves B.J., Crowther R.L., Chandran C., Rumberger J.M., Li S., Huang K.-S., Presky D.H., Familletti P.C., Wolitzky B.A., Burns D.K. 1994; Insight into E-selectin/ligand interaction from the crystal structure and mutagenesis of the lec/EGF domains. Nature 367:532–538
    [Google Scholar]
  24. Gum J.R., Hicks J.W., Toribara N., Rothe E.-M., Lagace R.E., Kim Y.S. 1992; The human MUC2 intestinal mucin has cyteine rich subdomains located both upstream and downstream of its central repetitive region. J Biol Chem 267:21375–21383
    [Google Scholar]
  25. Haberstroh L., Firtel R.A. 1990; A spatial gradient of expression of a cAMP-regulated prespore cell-type-specific gene in Dictyostelium . Genes Dev 4:596–612
    [Google Scholar]
  26. Hansson G.C., Baeckstrom D., Caristedt I., Klinga-Levan K. 1994; Molecular cloning of a cDNA coding for a region of an apoprotein from the ‘insoluble’ mucin complex of rat small intestine. Biochem Biophjs Res Commun 198:181–190
    [Google Scholar]
  27. Harris R.J., Spellman M.W. 1993; O -linked fucose and other post-translational modifications unique to EGF modules. Glycobiology 3:219–224
    [Google Scholar]
  28. von Heijne G. 1985; Signal sequences: the limits of variation. J Mol Biol 184:99–105
    [Google Scholar]
  29. Hildebrandt M., Humbel B.M., Nellen W. 1991; The Dictjostelium discoideum EB4 gene product and a truncated mutant form of the protein are localized in prespore vesicles but absent from mature spores. Dev Biol 144:212–214
    [Google Scholar]
  30. Hilkens J., Ligtenberg M.J.L., Vos H.L., Litvinov S.V. 1992; Cell membrane-associated mucins and their adhesion-modulating properties. Trends Biochem Sci 17:359–363
    [Google Scholar]
  31. Hoffmann W., Hauser F. 1993; The P-domain or trefoil motif: a role in renewal and pathology of mucous epithelia?. Trends Biochem Sci 18:239–243
    [Google Scholar]
  32. John S.A., Revel J.-P. 1991; Connexon integrity is maintained by non-covalent bonds: intramolecular disulfide bonds link the extracellular domains in rat connexin-43. Biochem Biophjs Res Commun 178:1312–1318
    [Google Scholar]
  33. Kieffer B., Driscoll P.C., Campbell I.D., Willis A.C., van der Merwe P.A., Davis S.J. 1994; Three-dimensional solution structure of the extracellular region of the complement regulatory protein CD59 a new cell-surface protein domain related to snake venom neurotoxins. Biochemistry 33:4471–4482
    [Google Scholar]
  34. Kozarov E., van der Wel H., Field M., Gritzali M., Brown R.D., West C.M. 1995; Characterization of FP21 a cytosolic glycoprotein from Dictjostelium . J Biol Chem 270:3022–3030
    [Google Scholar]
  35. Kuspa A., Loomis W.F. 1994; REMI-RFLP mapping in the Dictjostelium genome. Genetics 138:665–674
    [Google Scholar]
  36. Kuspa A., Loomis W.F. 1996; Ordered yeast artificial chromosome clones representing the Dictjostelium discoideum genome . Proc Natl Acad Sci USA 935562–5566
    [Google Scholar]
  37. Lenhard J.M., Siegel A., Free S.J. 1989; Developing Dictyostelium cells contain the lycosomal enzyme α-mannosidase in a secretory granule. J Cell Biol 109:2761–2769
    [Google Scholar]
  38. Lydan M.A., Cotter D.A. 1994; Spore swelling in Dictyostelium is a dynamic process mediated bv calmodulin. FEMS Microbiol Lett 115:137–142
    [Google Scholar]
  39. Mayadas T.N., Wagner D.D. 1992; Vicinal cysteines in the prosequence play a role in von Willebrand factor multimer assembly. Proc Natl Acad Sci USA 893531–3535
    [Google Scholar]
  40. Meerzaman D., Charles P., Daskal E., Polymeropoulos M.H., Martin B.M., Rose M.C. 1994; Cloning and analysis of cDNA encoding a major airway glycoprotein, human tracheobronchial mucin (MUC5). J Biol Chem 169:12932–12939
    [Google Scholar]
  41. Nakao H., Yamamoto A., Takeuchi I., Tasaka M. 1994; Dictyostelium prespore-specific gene (DP87) encodes a sorus matrix protein. J Cell Sci 107:397–403
    [Google Scholar]
  42. Orlowski M., Loomis W.F. 1979; Plasma membrane proteins of Dictyostelium : the spore coat proteins. Dev Biol 71:297–307
    [Google Scholar]
  43. Ozaki T., Nakao H., Orii H., Morio T., Takeuchi I., Tasaka M. 1993; Developmental regulation of transcription of a novel prespore-specific gene (DP87) in Dictyostelium discoideum . Development 117:1299–1308
    [Google Scholar]
  44. Peters R. 1986; Fluorescence microphotolysis to measure nucleocytoplasmic transport and intracellular mobility. Biocbim Biophys Acta 864:305–359
    [Google Scholar]
  45. Ploug M., Jensen A.L., Barkholt V. 1989; Determination of amino acid compositions and NH2-terminal sequences of peptides electroblotted onto PVDF membranes from tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis: application to peptide mapping of human complement component C3. Anal Biochem 181:33–39
    [Google Scholar]
  46. Powell-Coffman J.A., Firtel R.A. 1994; Characterization of a novel Dictyostelium discoideum prespore-specific gene, PspB, reveals conserved regulatory sequences. Development 120:1601–1611
    [Google Scholar]
  47. Probst J.C., Gertzen E.-M., Hoffmann W. 1990; An integumentary mucin (FIM-B.l) from Nenopus laevis homologous with von Willebrand factor. Biochemistry 29:6240–6244
    [Google Scholar]
  48. Riley G.R., West C.M., Henderson E.J. 1993; Cell differentiation in Dictyostelium discoideum controls assembly of proteinlinked glycans. Glycobiology 3:165–177
    [Google Scholar]
  49. Seshadri J., Dimond R., Cotter D. 1986; The characterization and secretion pattern of the lycosomal trehalases of Dictyostelium discoideum . Exp Mycol 10:131–143
    [Google Scholar]
  50. Stenflo J. 1991; Structure-function relationships of epidermal growth factor modules in vitamin K-dependent clotting factors. Blood 78:1637–1651
    [Google Scholar]
  51. Stone K.L., Williams K.R. 1993; Enzymatic digestion of proteins and HPLC peptide isolation. In A Practical Guide to Protein and Peptide Purification for Microsequencing, 2nd edn.. pp. 43–69 Edited by Matsudaira P. San Diego: Academic Press;
    [Google Scholar]
  52. Tasaka M., Hasegawa M., Ozaki T., Iwabuchi M., Takeuchi I. 1990; Isolation and characterization of spore coat protein (SP96) gene of Dictyostelium discoideum . Cell Differ Dev 31:1–9
    [Google Scholar]
  53. Taylor-Papadimitriou, Epenetos A. A. 1994; Exploiting altered glycosylation patterns in cancer, progress and challenges in diagnosis and therapy. Trends Biotechnol 12:227–233
    [Google Scholar]
  54. Verma M., Davidson E.A. 1993; Molecular cloning and sequencing of a canine tracheo-bronchial mucin cDNA containing a cysteine-rich domain. Proc Natl Acad Sci USA 907144–7148
    [Google Scholar]
  55. Verma M., Davidson E.A. 1994; Mucin genes: structure, expression and regulation. Glycoconj J 11:172–179
    [Google Scholar]
  56. Wagner G., Wyss D.F. 1994; Cell surface adhesion receptors. Curr Opin Struct Biol 4:841–851
    [Google Scholar]
  57. Watson N., Williams K.L., Alexander S. 1993; A developmentally regulated glycoprotein complex from Dictyostelium discoideum . J Biol Chem 268:22634–22641
    [Google Scholar]
  58. Watson N., McGuire V., Alexander S. 1994; The PsB glycoprotein is secreted as a preassembled precursor of the spore coat in Dictyostelium discoideum . J Cell Sci 107:2567–2579
    [Google Scholar]
  59. West C.M., Erdos G.W. 1988; The expression of glycoproteins in the extracellular matrix of the cellular slime mold Dictyostelium discoideum . Cell Differ 23:1–16
    [Google Scholar]
  60. West C.M., Erdos G.W. 1990; Formation of the Dictyostelium spore coat. Dev Genet 11:492–506
    [Google Scholar]
  61. West C.M., Erdos G.W. 1992; Incorporation of protein into spore coats is not cell-autonomous in Dictyostelium . J Cell Biol 116:1291–1300
    [Google Scholar]
  62. West C.M., Loomis W.F. 1985; Absence of a carbohydrate modification does not affect the level or subcellular localization of three membrane glycoproteins in modB mutants of Dictyostelium discoideum . J Biol Chem 260:13803–13809
    [Google Scholar]
  63. Wilkinson D.G., Hames B.D. 1983; Characterisation of the spore coat proteins of Dictyostelium discoideum . Eur J Biochem 129:637–643
    [Google Scholar]
  64. Wray W., Boulikas T., Wray V.P., Hancock R. 1981; Silver staining of proteins in polyacrylamide gels. Anal Biochem 118:197–203
    [Google Scholar]
  65. Yoder B.K., Mao J., Erdos G.W., West C.M., Blumberg D.D. 1994; Identification of a new spore coat protein gene in the cellular slime mold Dictyostelium discoideum . Dev Biol 163:49–65
    [Google Scholar]
  66. Yurchenko P.D., Cheng Y.-S., Colognato H. 1992; Laminin forms an independent network in basement membranes. J Cell Biol 117:1119–1133
    [Google Scholar]
  67. Zhou-Chou T., Wilkins M.R., Vardy P.H., Gooley A.A., Williams K.L. 1995; Glycoprotein complexes interacting with cellulose in the ‘cell print’ zones of the Dictyostelium discoideum extracellular matrix. Dev Biol 168:332–341
    [Google Scholar]
/content/journal/micro/10.1099/13500872-142-8-2227
Loading
/content/journal/micro/10.1099/13500872-142-8-2227
Loading

Data & Media loading...

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error