Skip to content
1887

Abstract

The baculovirus enzyme ecdysteroid UDP-glucosyltransferase (EGT) disrupts the hormonal balance of the insect host by catalysing the conjugation of ecdysteroids, the moulting hormones, with the sugar moiety from UDP-glucose or UDP-galactose. In this study, EGT has been overproduced using a recombinant Autographa californica nucleopolyhedrovirus and an antiserum has been raised against the purified protein. This antiserum was used to visualize the kinetics of expression of EGT by wild-type AcMNPVL-1 and by the overproducing recombinant virus. The inclusion of tunicamycin during these time-course experiments suggested that EGT is glycosylated. This was confirmed by Endo F treatment, which showed that glycosylation increased the apparent subunit molecular mass by approximately 11 kDa. These sugars do not appear to be required for enzyme activity. EGT activity invariantly elutes from gel-filtration columns as a single peak corresponding to a 260 kDa (+/-50 kDa) protein. This suggests that the enzyme is an oligomer of three to five subunits, since the subunit molecular mass is 56 kDa.

Loading

Article metrics loading...

/content/journal/jgv/10.1099/0022-1317-80-2-485
1999-02-01
2025-04-26
Loading full text...

Full text loading...

References

  1. Bruni S., Chang T. M. S. 1995; Kinetic analysis of UDP-glucuronosyltransferase in bilirubin conjugation by encapsulated hepato-cytes for transplantation into Gunn rats. Artificial Organs 19:449–457
    [Google Scholar]
  2. Burchell B., Coughtrie M. 1989; UDP-glucuronosyltransferases. Pharmacological Therapy 43:261–289
    [Google Scholar]
  3. Chowdhury J. R., Chowdhury N. R., Falany C. N., Tephly T. R., Arias I. M. 1986; Isolation and characterization of multiple forms of rat liver UDP-glucuronate glucuronosyltransferase. Biochemical Journal 233:827–837
    [Google Scholar]
  4. Coudron T. A., Law J. A., Koeppe J. K. 1981; Insect hormones. Trends in Biochemical Sciences 6:248–251
    [Google Scholar]
  5. Dong M., Owens I. S., Sheen Y. Y. 1997; Cloning and expression of human liver UDP-glucuronosyltransferase cDNA, UDPGTh2. Archives of Pharmacological Research 20:459–464
    [Google Scholar]
  6. Erps L. T., Ritter J. K., Hersh J. H., Blossom D., Martin N. C., Owen I. S. 1994; Identification of two single base substitutions in the UGT1 gene locus which abolish bilirubin uridine diphosphate glucuronosyl-transferase activity in vitro . Journal of Clinical Investigation 93:564–570
    [Google Scholar]
  7. Evans O. P., O’Reilly D. R. 1998; Purification and kinetic analysis of a baculovirus ecdysteroid UDP-glucosyltransferase. Biochemical Journal 330:1265–1270
    [Google Scholar]
  8. Green M. D., Tephly T. R. 1989; N-glycosylation of purified rat and rabbit hepatic UDP-glucuronosyltransferases. Archives of Biochemistry and Biophysics 273:72–78
    [Google Scholar]
  9. Harlow E., Lane D. P. 1988 Antibodies: A Laboratory Manual Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  10. Laemmli U. K. 1970; Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685
    [Google Scholar]
  11. Lee H. H., Miller L. K. 1978; Isolation of genotypic variants of Autographa californica nuclear polyhedrosis virus. Journal of Virology 27:754–767
    [Google Scholar]
  12. Mackenzie P. 1990; Expression of chimeric cDNAs in cell culture defines a region of UDP-glucuronosyltransferase involved in substrate selection. Journal of Biological Chemistry 265:3432–3435
    [Google Scholar]
  13. Meech R., Mackenzie P. 1997; Structure and function of uridine diphosphate glucuronosyltransferases. Clinical and Experimental Pharmacology and Physiology 24:907–915
    [Google Scholar]
  14. O’Reilly D. R. 1995; Baculovirus-encoded ecdysteroid UDP-glucosyl-transferases. Insect Biochemistry and Molecular Biology 25:541–550
    [Google Scholar]
  15. O’Reilly D.R., Miller L.K. 1989; A baculovirus blocks insect molting by producing ecdysteroid UDP-glucosyltransferase. Science 245:1110–1112
    [Google Scholar]
  16. O’Reilly D. R., Miller L. K. 1990; Regulation of expression of a baculovirus ecdysteroid UDP-glucosyltransferase gene. Journal of Virology 64:1321–1328
    [Google Scholar]
  17. O’Reilly D. R., Miller L. K. 1991; Improvement of a baculovirus pesticide by deletion of the egt gene. Bio/Technology 9:1086–1089
    [Google Scholar]
  18. O’Reilly D. R., Howarth O.W., Rees H.H., Miller L. K. 1991; Structure of the ecdysone glucoside formed by a baculovirus ecdysteroid UDP-glucosyltransferase. Insect Biochemistry 21:795–801
    [Google Scholar]
  19. O’Reilly D. R., Brown M. R., Miller L. K. 1992a; Alteration of ecdysteroid metabolism due to baculovirus infection of the fall armyworm Spodoptera frugiperda: host ecdysteroids are conjugated with galactose. Insect Biochemistry and Molecular Biology 22:313–320
    [Google Scholar]
  20. O’Reilly D. R., Miller L. K., Luckow V. A. 1992b Baculovirus Expression Vectors: A Laboratory Manual New York: W. H. Freeman;
    [Google Scholar]
  21. O’Reilly D. R., Hails R. S., Kelly T. J. 1998; The impact of host developmental status on baculovirus replication. Journal of Invertebrate Pathology 72:269–275
    [Google Scholar]
  22. Park E. J., Burand J. P., Yin C.-H. 1993; The effect of baculovirus infection on ecdysteroid titer in gypsy moth larvae (Lymantria dispar). Journal of Insect Physiology 39:791–796
    [Google Scholar]
  23. Peters W. H. M., Jansen P. L. M., Nauta H. 1984; The molecular weights of UDP-glucuronyltransferase determined with radiation-inactivation analysis – a molecular model of bilirubin UDP-glucuronyl-transferase. Journal of Biological Chemistry 259:1701–1705
    [Google Scholar]
  24. Peters W. H. M., Jansen P. L. M., Cuypers H. T. M., de Abreu R. A., Nauta H. 1986; Deconjugation of glucuronides catalysed by UDP-glucuronyltransferase. Biochimica et Biophysica Acta 873:252–259
    [Google Scholar]
  25. Puig J. F., Tephly T. R. 1986; Isolation and purification of rat liver morphine UDP-glucuronosyltransferase. Molecular Pharmacology 30:558–565
    [Google Scholar]
  26. Sambrook J., Fritsch E. F., Maniatis T. 1989 Molecular Cloning: A Laboratory Manual Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  27. Shepherd S. R. P., Baird S. J., Hallinan T., Burchell B. 1989; An investigation of the transverse topology of bilirubin UDP-glucuronosyl-transferase in rat hepatic endoplasmic reticulum. Biochemical Journal 259:617–620
    [Google Scholar]
  28. Vaughn J. L., Goodwin R. H., Tompkins G. J., McCawley P. 1977; The establishment of two cell lines from the insect Spodoptera frugiperda (Lepidoptera: Noctuidae). In Vitro 13:213–217
    [Google Scholar]
  29. Wang X., Ooi B. G., Miller L. K. 1991; Baculovirus vectors for multiple gene expression and for occluded virus production. Gene 100:131–137
    [Google Scholar]
/content/journal/jgv/10.1099/0022-1317-80-2-485
Loading
/content/journal/jgv/10.1099/0022-1317-80-2-485
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