Skip to main content
NCBI home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1992 May 15;89(10):4693–4697. doi: 10.1073/pnas.89.10.4693

Primary structure and expression of a gamete lytic enzyme in Chlamydomonas reinhardtii: similarity of functional domains to matrix metalloproteases.

T Kinoshita 1, H Fukuzawa 1, T Shimada 1, T Saito 1, Y Matsuda 1
PMCID: PMC49149  PMID: 1584806

Abstract

A gamete lytic enzyme (GLE) of Chlamydomonas reinhardtii is a zinc metalloprotease and mediates digestion of the cell walls of the two mating-type gametes during mating as a necessary prelude to cell fusion. The nucleotide sequence analysis of a cDNA revealed that GLE is synthesized in a preproenzyme form, a 638-amino acid polypeptide (Mr, 69,824) with a 28-amino acid signal peptide, a 155-amino acid propolypeptide, and a 455-amino acid mature polypeptide (Mr, 49,633). A potential site for autocatalytic activation was contained within the propolypeptide and a zinc binding site found within the mature polypeptide; both sites were highly homologous to those in mammalian collagenase. A putative calcium binding site was present in the near C-terminal region of the mature GLE. Both propolypeptide and mature polypeptide had potential sites for asparagine-linked glycosylation, and the Arg-(Pro)3 and Arg-(Pro)2 motifs, which are known to exist in hydroxyproline-rich glycoproteins of the Chlamydomonas cell wall. Northern blot analysis revealed that steady-state levels of the 2.4-kilobase GLE mRNA increased during growth and mitotic cell division in the vegetative cell cycle and also increased markedly during gametogenesis under nitrogen-starved conditions.

Full text

PDF
4693

Images in this article

4695Image
on p.4695
4696Image
on p.4696

Selected References

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

  1. Adair W. S., Apt K. E. Cell wall regeneration in Chlamydomonas: accumulation of mRNAs encoding cell wall hydroxyproline-rich glycoproteins. Proc Natl Acad Sci U S A. 1990 Oct;87(19):7355–7359. doi: 10.1073/pnas.87.19.7355. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Babu Y. S., Sack J. S., Greenhough T. J., Bugg C. E., Means A. R., Cook W. J. Three-dimensional structure of calmodulin. Nature. 1985 May 2;315(6014):37–40. doi: 10.1038/315037a0. [DOI] [PubMed] [Google Scholar]
  3. Buchanan M. J., Imam S. H., Eskue W. A., Snell W. J. Activation of the cell wall degrading protease, lysin, during sexual signalling in Chlamydomonas: the enzyme is stored as an inactive, higher relative molecular mass precursor in the periplasm. J Cell Biol. 1989 Jan;108(1):199–207. doi: 10.1083/jcb.108.1.199. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chou P. Y., Fasman G. D. Empirical predictions of protein conformation. Annu Rev Biochem. 1978;47:251–276. doi: 10.1146/annurev.bi.47.070178.001343. [DOI] [PubMed] [Google Scholar]
  5. Church G. M., Gilbert W. Genomic sequencing. Proc Natl Acad Sci U S A. 1984 Apr;81(7):1991–1995. doi: 10.1073/pnas.81.7.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Claes H. Autolyse der Zellwand bei den Gemeten von Chlamydomonas reinhardii. Arch Mikrobiol. 1971;78(2):180–188. [PubMed] [Google Scholar]
  7. Collier I. E., Wilhelm S. M., Eisen A. Z., Marmer B. L., Grant G. A., Seltzer J. L., Kronberger A., He C. S., Bauer E. A., Goldberg G. I. H-ras oncogene-transformed human bronchial epithelial cells (TBE-1) secrete a single metalloprotease capable of degrading basement membrane collagen. J Biol Chem. 1988 May 15;263(14):6579–6587. [PubMed] [Google Scholar]
  8. Ertl H., Mengele R., Wenzl S., Engel J., Sumper M. The extracellular matrix of Volvox carteri: molecular structure of the cellular compartment. J Cell Biol. 1989 Dec;109(6 Pt 2):3493–3501. doi: 10.1083/jcb.109.6.3493. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Goldberg G. I., Wilhelm S. M., Kronberger A., Bauer E. A., Grant G. A., Eisen A. Z. Human fibroblast collagenase. Complete primary structure and homology to an oncogene transformation-induced rat protein. J Biol Chem. 1986 May 15;261(14):6600–6605. [PubMed] [Google Scholar]
  10. Goodenough U. W., Heuser J. E. The Chlamydomonas cell wall and its constituent glycoproteins analyzed by the quick-freeze, deep-etch technique. J Cell Biol. 1985 Oct;101(4):1550–1568. doi: 10.1083/jcb.101.4.1550. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hubbard S. C., Ivatt R. J. Synthesis and processing of asparagine-linked oligosaccharides. Annu Rev Biochem. 1981;50:555–583. doi: 10.1146/annurev.bi.50.070181.003011. [DOI] [PubMed] [Google Scholar]
  12. Imam S. H., Snell W. J. The Chlamydomonas cell wall degrading enzyme, lysin, acts on two substrates within the framework of the wall. J Cell Biol. 1988 Jun;106(6):2211–2221. doi: 10.1083/jcb.106.6.2211. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Jongeneel C. V., Bouvier J., Bairoch A. A unique signature identifies a family of zinc-dependent metallopeptidases. FEBS Lett. 1989 Jan 2;242(2):211–214. doi: 10.1016/0014-5793(89)80471-5. [DOI] [PubMed] [Google Scholar]
  14. Kozak M. Compilation and analysis of sequences upstream from the translational start site in eukaryotic mRNAs. Nucleic Acids Res. 1984 Jan 25;12(2):857–872. doi: 10.1093/nar/12.2.857. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Kretsinger R. H., Nockolds C. E. Carp muscle calcium-binding protein. II. Structure determination and general description. J Biol Chem. 1973 May 10;248(9):3313–3326. [PubMed] [Google Scholar]
  16. Lawler J., Hynes R. O. The structure of human thrombospondin, an adhesive glycoprotein with multiple calcium-binding sites and homologies with several different proteins. J Cell Biol. 1986 Nov;103(5):1635–1648. doi: 10.1083/jcb.103.5.1635. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Lepage T., Gache C. Early expression of a collagenase-like hatching enzyme gene in the sea urchin embryo. EMBO J. 1990 Sep;9(9):3003–3012. doi: 10.1002/j.1460-2075.1990.tb07493.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Matsuda Y., Saito T., Yamaguchi T., Kawase H. Cell wall lytic enzyme released by mating gametes of Chlamydomonas reinhardtii is a metalloprotease and digests the sodium perchlorate-insoluble component of cell wall. J Biol Chem. 1985 May 25;260(10):6373–6377. [PubMed] [Google Scholar]
  19. Matsuda Y., Saito T., Yamaguchi T., Koseki M., Hayashi K. Topography of cell wall lytic enzyme in Chlamydomonas reinhardtii: form and location of the stored enzyme in vegetative cell and gamete. J Cell Biol. 1987 Feb;104(2):321–329. doi: 10.1083/jcb.104.2.321. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Matthews B. W., Weaver L. H., Kester W. R. The conformation of thermolysin. J Biol Chem. 1974 Dec 25;249(24):8030–8044. [PubMed] [Google Scholar]
  21. Pasquale S. M., Goodenough U. W. Cyclic AMP functions as a primary sexual signal in gametes of Chlamydomonas reinhardtii. J Cell Biol. 1987 Nov;105(5):2279–2292. doi: 10.1083/jcb.105.5.2279. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Saito T., Matsuda Y. Isolation and characterization of Chlamydomonas temperature-sensitive mutants affecting gametic differentiation under nitrogen-starved conditions. Curr Genet. 1991 Feb;19(2):65–71. doi: 10.1007/BF00326284. [DOI] [PubMed] [Google Scholar]
  23. Sanchez-Lopez R., Nicholson R., Gesnel M. C., Matrisian L. M., Breathnach R. Structure-function relationships in the collagenase family member transin. J Biol Chem. 1988 Aug 25;263(24):11892–11899. [PubMed] [Google Scholar]
  24. Siersma P. W., Chiang K. S. Conservation and degradation of cytoplasmic and chloroplast ribosomes in Chlamydomonas reinhardtii. J Mol Biol. 1971 May 28;58(1):167–185. doi: 10.1016/0022-2836(71)90239-7. [DOI] [PubMed] [Google Scholar]
  25. Snell W. J., Eskue W. A., Buchanan M. J. Regulated secretion of a serine protease that activates an extracellular matrix-degrading metalloprotease during fertilization in Chlamydomonas. J Cell Biol. 1989 Oct;109(4 Pt 1):1689–1694. doi: 10.1083/jcb.109.4.1689. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Springman E. B., Angleton E. L., Birkedal-Hansen H., Van Wart H. E. Multiple modes of activation of latent human fibroblast collagenase: evidence for the role of a Cys73 active-site zinc complex in latency and a "cysteine switch" mechanism for activation. Proc Natl Acad Sci U S A. 1990 Jan;87(1):364–368. doi: 10.1073/pnas.87.1.364. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Varner J. E., Lin L. S. Plant cell wall architecture. Cell. 1989 Jan 27;56(2):231–239. doi: 10.1016/0092-8674(89)90896-9. [DOI] [PubMed] [Google Scholar]
  28. Wilhelm S. M., Collier I. E., Kronberger A., Eisen A. Z., Marmer B. L., Grant G. A., Bauer E. A., Goldberg G. I. Human skin fibroblast stromelysin: structure, glycosylation, substrate specificity, and differential expression in normal and tumorigenic cells. Proc Natl Acad Sci U S A. 1987 Oct;84(19):6725–6729. doi: 10.1073/pnas.84.19.6725. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Woessner J. P., Goodenough U. W. Molecular characterization of a zygote wall protein: an extensin-like molecule in Chlamydomonas reinhardtii. Plant Cell. 1989 Sep;1(9):901–911. doi: 10.1105/tpc.1.9.901. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. von Heijne G. Signal sequences. The limits of variation. J Mol Biol. 1985 Jul 5;184(1):99–105. doi: 10.1016/0022-2836(85)90046-4. [DOI] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES