Skip to main content
NCBI home page
Genetics logoLink to Genetics
. 1994 Apr;136(4):1355–1365. doi: 10.1093/genetics/136.4.1355

Mutational Analysis of the Drosophila Snake Protease: An Essential Role for Domains within the Proenzyme Polypeptide Chain

C Smith 1, H Giordano 1, R DeLotto 1
PMCID: PMC1205916  PMID: 8013912

Abstract

Two genes involved in the generation of dorsoventral asymmetry in the developing Drosophila melanogaster embryo, snake and easter, encode the zymogen form of serine proteases. Mutant alleles of snake were cloned and sequenced revealing two types of lesions: point mutations which alter the amino acid sequence (snk(073) and snk(rm4)) and point mutations which alter the splicing (snk(229) or snk(233)) of intron 1 of the mRNA from the normal 3' end of the intron to a cryptic site. snake mutant embryos derived from homozygous mothers can be fully rescued by injection of RNA transcripts of the wild-type snake cDNA. RNA phenotypic rescue and site-directed mutagenesis experiments indicate that snake requires the serine, histidine and aspartic acid of the catalytic triad for normal activity. Deletion experiments show that an acidic proenzyme domain is required for snake rescue activity to be uniformly distributed throughout the embryo. A second proenzyme domain, called the disulfide knot, appears to be essential for normal regulation of activity of the snake catalytic chain. Transcripts encoding only the proenzyme polypeptides of either snake or easter can dorsalize wild type embryos. We propose a model in which the proenzyme determinants of both the snake and easter enzymes mediate interaction between the serine proteases and other components of the dorsal-ventral patterning system.

Full Text

The Full Text of this article is available as a PDF (3.6 MB).

Selected References

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

  1. Anderson K. V., Nüsslein-Volhard C. Information for the dorsal--ventral pattern of the Drosophila embryo is stored as maternal mRNA. Nature. 1984 Sep 20;311(5983):223–227. doi: 10.1038/311223a0. [DOI] [PubMed] [Google Scholar]
  2. Aroian R. V., Levy A. D., Koga M., Ohshima Y., Kramer J. M., Sternberg P. W. Splicing in Caenorhabditis elegans does not require an AG at the 3' splice acceptor site. Mol Cell Biol. 1993 Jan;13(1):626–637. doi: 10.1128/mcb.13.1.626. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Chasan R., Anderson K. V. The role of easter, an apparent serine protease, in organizing the dorsal-ventral pattern of the Drosophila embryo. Cell. 1989 Feb 10;56(3):391–400. doi: 10.1016/0092-8674(89)90242-0. [DOI] [PubMed] [Google Scholar]
  4. Chasan R., Jin Y., Anderson K. V. Activation of the easter zymogen is regulated by five other genes to define dorsal-ventral polarity in the Drosophila embryo. Development. 1992 Jun;115(2):607–616. doi: 10.1242/dev.115.2.607. [DOI] [PubMed] [Google Scholar]
  5. Craik C. S., Largman C., Fletcher T., Roczniak S., Barr P. J., Fletterick R., Rutter W. J. Redesigning trypsin: alteration of substrate specificity. Science. 1985 Apr 19;228(4697):291–297. doi: 10.1126/science.3838593. [DOI] [PubMed] [Google Scholar]
  6. Davis C. A., Riddell D. C., Higgins M. J., Holden J. J., White B. N. A gene family in Drosophila melanogaster coding for trypsin-like enzymes. Nucleic Acids Res. 1985 Sep 25;13(18):6605–6619. doi: 10.1093/nar/13.18.6605. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. DeLotto R., Spierer P. A gene required for the specification of dorsal-ventral pattern in Drosophila appears to encode a serine protease. Nature. 1986 Oct 23;323(6090):688–692. doi: 10.1038/323688a0. [DOI] [PubMed] [Google Scholar]
  8. Erickson A. H., Blobel G. Cell-free translation of messenger RNA in a wheat germ system. Methods Enzymol. 1983;96:38–50. doi: 10.1016/s0076-6879(83)96007-x. [DOI] [PubMed] [Google Scholar]
  9. Furie B. C., Blumenstein M., Furie B. Metal binding sites of a gamma-carboxyglutamic acid-rich fragment of bovine prothrombin. J Biol Chem. 1979 Dec 25;254(24):12521–12530. [PubMed] [Google Scholar]
  10. Furie B., Bing D. H., Feldmann R. J., Robison D. J., Burnier J. P., Furie B. C. Computer-generated models of blood coagulation factor Xa, factor IXa, and thrombin based upon structural homology with other serine proteases. J Biol Chem. 1982 Apr 10;257(7):3875–3882. [PubMed] [Google Scholar]
  11. Furie B., Furie B. C. The molecular basis of blood coagulation. Cell. 1988 May 20;53(4):505–518. doi: 10.1016/0092-8674(88)90567-3. [DOI] [PubMed] [Google Scholar]
  12. Ghosh S., Gifford A. M., Riviere L. R., Tempst P., Nolan G. P., Baltimore D. Cloning of the p50 DNA binding subunit of NF-kappa B: homology to rel and dorsal. Cell. 1990 Sep 7;62(5):1019–1029. doi: 10.1016/0092-8674(90)90276-k. [DOI] [PubMed] [Google Scholar]
  13. Graf L., Craik C. S., Patthy A., Roczniak S., Fletterick R. J., Rutter W. J. Selective alteration of substrate specificity by replacement of aspartic acid-189 with lysine in the binding pocket of trypsin. Biochemistry. 1987 May 5;26(9):2616–2623. doi: 10.1021/bi00383a031. [DOI] [PubMed] [Google Scholar]
  14. Greer J. Model for haptoglobin heavy chain based upon structural homology. Proc Natl Acad Sci U S A. 1980 Jun;77(6):3393–3397. doi: 10.1073/pnas.77.6.3393. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hashimoto C., Hudson K. L., Anderson K. V. The Toll gene of Drosophila, required for dorsal-ventral embryonic polarity, appears to encode a transmembrane protein. Cell. 1988 Jan 29;52(2):269–279. doi: 10.1016/0092-8674(88)90516-8. [DOI] [PubMed] [Google Scholar]
  16. Jin Y. S., Anderson K. V. Dominant and recessive alleles of the Drosophila easter gene are point mutations at conserved sites in the serine protease catalytic domain. Cell. 1990 Mar 9;60(5):873–881. doi: 10.1016/0092-8674(90)90100-s. [DOI] [PubMed] [Google Scholar]
  17. Kunkel T. A., Roberts J. D., Zakour R. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol. 1987;154:367–382. doi: 10.1016/0076-6879(87)54085-x. [DOI] [PubMed] [Google Scholar]
  18. Mann K. G., Jenny R. J., Krishnaswamy S. Cofactor proteins in the assembly and expression of blood clotting enzyme complexes. Annu Rev Biochem. 1988;57:915–956. doi: 10.1146/annurev.bi.57.070188.004411. [DOI] [PubMed] [Google Scholar]
  19. Muta T., Hashimoto R., Miyata T., Nishimura H., Toh Y., Iwanaga S. Proclotting enzyme from horseshoe crab hemocytes. cDNA cloning, disulfide locations, and subcellular localization. J Biol Chem. 1990 Dec 25;265(36):22426–22433. [PubMed] [Google Scholar]
  20. Park C. H., Tulinsky A. Three-dimensional structure of the kringle sequence: structure of prothrombin fragment 1. Biochemistry. 1986 Jul 15;25(14):3977–3982. doi: 10.1021/bi00362a001. [DOI] [PubMed] [Google Scholar]
  21. Patthy L., Trexler M., Váli Z., Bányai L., Váradi A. Kringles: modules specialized for protein binding. Homology of the gelatin-binding region of fibronectin with the kringle structures of proteases. FEBS Lett. 1984 Jun 4;171(1):131–136. doi: 10.1016/0014-5793(84)80473-1. [DOI] [PubMed] [Google Scholar]
  22. Rogers J. Exon shuffling and intron insertion in serine protease genes. Nature. 1985 Jun 6;315(6019):458–459. doi: 10.1038/315458a0. [DOI] [PubMed] [Google Scholar]
  23. Roth S., Stein D., Nüsslein-Volhard C. A gradient of nuclear localization of the dorsal protein determines dorsoventral pattern in the Drosophila embryo. Cell. 1989 Dec 22;59(6):1189–1202. doi: 10.1016/0092-8674(89)90774-5. [DOI] [PubMed] [Google Scholar]
  24. Rushlow C. A., Han K., Manley J. L., Levine M. The graded distribution of the dorsal morphogen is initiated by selective nuclear transport in Drosophila. Cell. 1989 Dec 22;59(6):1165–1177. doi: 10.1016/0092-8674(89)90772-1. [DOI] [PubMed] [Google Scholar]
  25. Smith C. L., DeLotto R. A common domain within the proenzyme regions of the Drosophila snake and easter proteins and Tachypleus proclotting enzyme defines a new subfamily of serine proteases. Protein Sci. 1992 Sep;1(9):1225–1226. doi: 10.1002/pro.5560010915. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Sprang S., Standing T., Fletterick R. J., Stroud R. M., Finer-Moore J., Xuong N. H., Hamlin R., Rutter W. J., Craik C. S. The three-dimensional structure of Asn102 mutant of trypsin: role of Asp102 in serine protease catalysis. Science. 1987 Aug 21;237(4817):905–909. doi: 10.1126/science.3112942. [DOI] [PubMed] [Google Scholar]
  27. St Johnston D., Nüsslein-Volhard C. The origin of pattern and polarity in the Drosophila embryo. Cell. 1992 Jan 24;68(2):201–219. doi: 10.1016/0092-8674(92)90466-p. [DOI] [PubMed] [Google Scholar]
  28. Stein D. S., Stevens L. M. Establishment of dorsal-ventral and terminal pattern in the Drosophila embryo. Curr Opin Genet Dev. 1991 Aug;1(2):247–254. doi: 10.1016/s0959-437x(05)80078-4. [DOI] [PubMed] [Google Scholar]
  29. Stein D., Roth S., Vogelsang E., Nüsslein-Volhard C. The polarity of the dorsoventral axis in the Drosophila embryo is defined by an extracellular signal. Cell. 1991 May 31;65(5):725–735. doi: 10.1016/0092-8674(91)90381-8. [DOI] [PubMed] [Google Scholar]
  30. Steward R. Dorsal, an embryonic polarity gene in Drosophila, is homologous to the vertebrate proto-oncogene, c-rel. Science. 1987 Oct 30;238(4827):692–694. doi: 10.1126/science.3118464. [DOI] [PubMed] [Google Scholar]
  31. Steward R., Zusman S. B., Huang L. H., Schedl P. The dorsal protein is distributed in a gradient in early Drosophila embryos. Cell. 1988 Nov 4;55(3):487–495. doi: 10.1016/0092-8674(88)90035-9. [DOI] [PubMed] [Google Scholar]
  32. Tai J. Y., Liu T. Y. Studies on Limulus amoebocyte lysate. Isolation of pro-clotting enzyme. J Biol Chem. 1977 Apr 10;252(7):2178–2181. [PubMed] [Google Scholar]
  33. Tulinsky A. The structures of domains of blood proteins. Thromb Haemost. 1991 Jul 12;66(1):16–31. [PubMed] [Google Scholar]
  34. Yun Y., Davis R. L. Levels of RNA from a family of putative serine protease genes are reduced in Drosophila melanogaster dunce mutants and are regulated by cyclic AMP. Mol Cell Biol. 1989 Feb;9(2):692–700. doi: 10.1128/mcb.9.2.692. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. de Both N. J., Vermey M., van't Hull E., Klootwijk-van-Dijke E., van Griensven L. J., Mol J. N., Stoof T. J. A new erythroid cell line induced by Rauscher murine leukaemia virus. Nature. 1978 Apr 13;272(5654):626–628. doi: 10.1038/272626a0. [DOI] [PubMed] [Google Scholar]

Articles from Genetics are provided here courtesy of Oxford University Press

RESOURCES