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Mutational analysis ofN-linked glycosylation of esterase 6 inDrosophila melanogaster

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Abstract

The primary sequence of the esterase 6 (EST6) enzyme ofDrosophila melanogaster contains four potential N-linked glycosylation sites, at residues 21, 399, 435, and 485. Here we determine the extent to which EST6 is glycosylated and how the glycosylation affects the biochemistry and physiology of the enzyme. We have abolished each of the four potential glycosylation sites by replacing the required Asn residues with Gln byin vitro mutagenesis. Five mutant genes were made, four containing mutations of each site individually and the fifth site containing all four mutations. Germline transformation was used to introduce the mutant genes into a strain ofD. melanogaster null for EST6. Electrophoretic and Western blot comparisons of the mutant strains and wild-type controls showed that each of the four potential N-linked glycosylation sites in the wild-type protein is glycosylated. However, the fourth site is not utilized on all EST6 molecules, resulting in two molecular forms of the enzyme. Digestion with specific endoglycosidases showed that the glycan attached at the second site is of the high-mannose type, while the other three sites carry more complex oligosaccharides. The thermostability of the enzyme is not affected by abolition of the first, third, or fourth glycosylation sites but is reduced by abolition of the second site. Anomalously, abolition of all four sites together does not reduce thermostability. Quantitative comparisons of EST6 activities showed that abolition of glycosylation does not affect the secretion of the enzyme into the male sperm ejaculatory duct, its transfer to the female vagina during mating, or its subsequent translocation into her hemolymph. However, the activity of the mutant enzymes does not persist in the female's hemolymph for as long as wild-type esterase 6. The latter effect may compromise the role of the transferred enzyme in stimulating egg-laying and delaying receptivity to remating.

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References

  • Anderson, D. R., and Grimes, W. J. (1982). Heterogeneity of asparagine-linked oligosaccharides of five glycosylation sites on immunoglobulin M heavy chain from mineral oil plasmacytoma 104E.J. Biol. Chem. 25714858.

    PubMed  CAS  Google Scholar 

  • Bergman, L. W., and Kuehl, W. M. (1978). Temporal relationship of translation and glycosylation of immunoglobulin heavy chain and light chains.Biochemistry 175174.

    Article  PubMed  CAS  Google Scholar 

  • Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein using the principle of protein dye binding.Anal. Biochem. 22248.

    Article  Google Scholar 

  • Catty, D., and Raykundalia, C. (1988). Gel immunodiffusion, immunoelectrophoresis and immunostaining methods. In Catty, D. (ed.),Antibodies: A Practical Approach, Vol. 1 IRL Press, Oxford, pp. 137–167.

    Google Scholar 

  • Cochrane, B. J., and Richmond, R. C. (1979). Studies of esterase 6 inDrosophila melanogaster. II. The genetics and frequency distribution of naturally occurring variants studied by electrophoretic and heat stability criteria.Genetics 93461.

    PubMed  CAS  Google Scholar 

  • Collet, C., Nielsen, K. M., Russell, R. J., Karl, M., Oakeshott, J. G., and Richmond, R. C. (1990). Molecular analysis of duplicated esterase genes inDrosophila melanogaster.Mol. Biol. Evol. 79.

    PubMed  CAS  Google Scholar 

  • Cooke, P. H., and Oakeshott, J. G. (1989). Amino acid polymorphism for esterase 6 inDrosophila melanogaster.Proc. Natl. Acad. Sci. USA 861426.

    PubMed  CAS  Google Scholar 

  • Dorner, A. J., Bole, D. G., and Kaufman, R. J. (1987). The relationship of heavy chain binding protein association with the secretion of glycoproteins.J. Cell. Biol. 1052665.

    Article  PubMed  CAS  Google Scholar 

  • Drickamer, K. (1991). Clearing up glycoprotein hormones.Cell 671029.

    Article  PubMed  CAS  Google Scholar 

  • Dubé, S., Fisher, J. W., and Powell, J. S. (1988). Glycosylation at specific sites of erythropoietin is essential for biosynthesis, secretion and biological function.J. Biol. Chem. 26317516.

    PubMed  Google Scholar 

  • Gavel, Y., and von Heijne, G. (1990). Sequence differences between glycosylated and nonglycosylated Asn-X-Ser/Thr acceptor sites: Implications for protein engineering.Prot. Eng. 3433.

    CAS  Google Scholar 

  • Gilbert, D. G., and Richmond, R. C. (1982). Esterase 6 inDrosophila melanogaster: Reproductive function of active and null males at low temperatures.Proc. Natl. Acad. Sci. USA 792962.

    PubMed  CAS  Google Scholar 

  • Gilbert, D. G., Richmond, R. C., and Sheehan, K. B. (1981a). Studies of esterase 6 inDrosophila melanogaster. VII. Remating times of females inseminated by males having active or null alleles.Behav. Genet. 11195.

    Article  PubMed  CAS  Google Scholar 

  • Gilbert, D. G., Richmond, R. C., and Sheehan, K. B. (1981b). Studies of esterase 6 inDrosophila melanogaster. V. Progeny production and sperm use in females inseminated by males having active or null alleles.Evolution 3521.

    Article  CAS  Google Scholar 

  • Glabe, C. G., Hanover, J. A., and Lennarz, W. J. (1980). Glycosylation of ovalbumin nascent chains.J. Biol. Chem. 2559236.

    PubMed  CAS  Google Scholar 

  • Gromko, M. H., Gilbert, D. F., and Richmond, R. C. (1984a). Sperm transfer and use in the multiple mating system ofDrosophila. In Smith, R. L. (eds.),Sperm Competition and the Evolution of Animal Mating Systems Academic Press, New York, pp. 371–426.

    Google Scholar 

  • Gromko, M. H., Newport, M. E. A., and Koritier, M. G. (1984b). Sperm dependence on female receptivity to remating inDrosophila melanogaster.Evolution 381273.

    Article  Google Scholar 

  • Hansen, L., Blue, Y., Barone, K., Collen, D., and Larsen, G. R. (1988). Functional effects of asparagine linked oligosaccharide on natural and variant human tissue-type plasminogen activator.J. Biol. Chem. 26315713.

    PubMed  CAS  Google Scholar 

  • Haselbeck, A., and Hösel, W. (1988). Studies on the effect of the incubation conditions, various detergents and protein concentration on the enzymatic activity of N-glycosidase F and Endo-glycosidase F.Topics in Biochemistry No 8, Boehringer Mannheim.

  • Healy, M. J., Dumancic, M. M., and Oakeshott, J. G. (1991). Biochemical and physiological studies of soluble esterases fromDrosophila melanogaster.Biochem. Genet. 29365.

    Article  PubMed  CAS  Google Scholar 

  • Jackson, R. L., and Hirs, C. H. W. (1970). The primary structure of porcine pancreatic ribonuclease.J. Biol. Chem. 245624.

    PubMed  CAS  Google Scholar 

  • Kornfeld, S. (1987). Trafficking of lysosomal enzymes.FASEB J. 1462.

    PubMed  CAS  Google Scholar 

  • Kroetz, D.-L., McBride, O. W., and Gonzalez, F. J. (1993). Glycosylation dependent activity of baculovirus-expressed human liver carboxylesterases: cDNA cloning and characterisation of two highly similar enzyme forms.Biochemistry 3211606.

    Article  PubMed  CAS  Google Scholar 

  • Kurosaka, A., Yano, A., Itoh, N., Kuroda, Y., Nakagawa, T., and Kawasaki, T. (1991). The structure of a neural specific carbohydrate epitope of horseradish peroxidase recognized by anti-horseradish peroxidase antiserum.J. Biol. Chem. 2664168.

    PubMed  CAS  Google Scholar 

  • Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4.Nature 227680.

    Article  PubMed  CAS  Google Scholar 

  • Mane, S. D., Tepper, C. S., and Richmond, R. C. (1983). Studies of esterase 6 inDrosophila melanogaster. XIII. Purification and characterisation of the two major isozymes.Biochem. Genet. 211019.

    Article  PubMed  CAS  Google Scholar 

  • Meikle, D. B., Sheehan, K. B., Phillis, D. M., and Richmond, R. C. (1990). Localisation and longevity of seminal fluid esterase 6 in mated femaleDrosophila melanogaster.J. Insect Physiol. 3693.

    Article  CAS  Google Scholar 

  • Mutero, A., and Fournier, D. (1992). Post-translational modifications ofDrosophila acetylcholline esterase.J. Biol. Chem. 2671695.

    PubMed  CAS  Google Scholar 

  • Myers, M. A., Healy, M. J., and Oakeshott, J. G. (1993). Effects of the residue adjacent to the reactive serine on the substrate interactions ofDrosophila esterase 6.Biochem. Genet. 31259.

    Article  PubMed  CAS  Google Scholar 

  • Oakeshott, J. G., Collet, C., Phillis, R. W., Nielsen, K. M., Russell, R. J., Chambers, G. K., Ross, V., and Richmond, R. C. (1987). Molecular cloning and characterisation of esterase-6, a serine hydrolase ofDrosophila.Proc. Natl. Acad. Sci. USA 843359.

    PubMed  CAS  Google Scholar 

  • Oakeshott, J. G., Boyce, T. M., Russell, R. J., and Healy, M. J. (1995). Molecular insights into the evolution of an enzyme; Esterase 6 inDrosophila.TREE 10103.

    Google Scholar 

  • Olden, K., Parent, J. B., and White, S. L. (1982). Carbohydrate moieties of glycoproteins. A reevaluation of their function.Biochim. Biophys. Acta 650209.

    PubMed  CAS  Google Scholar 

  • Paulson, J. C. (1989). Glycoproteins: What are the sugar chains for?TIBS 14272.

    PubMed  CAS  Google Scholar 

  • Penninca, D., Holmes, W. E., Kohr, W. J., Harkins, R. N., Vehar, G. A., Ward, C. A., Bennet, W. P., Yelverton, E., Seeburg, P. H., Heynekar, H. L., Goeddel, D. V., and Collen, D. (1983). Cloning and expression of human tissue-type plasminogen activator cDNA inE. coli. Nature 301214.

    Article  Google Scholar 

  • Price, C. P., and Neuman, D. J. (eds.) (1991).Principles and Practice of Immunoassay Stockton Press, New York.

    Google Scholar 

  • Richmond, R. C., and Senior, A. (1981). Esterase 6 ofDrosophila melanogaster: Kinetics of transfer to females, decay in females and male recovery.J. Insect Physiol. 27849.

    Article  CAS  Google Scholar 

  • Richmond, R. C., Nielsen, K. M., Brady, J. P., and Snella, E. M. (1990). Physiology, biochemistry and molecular biology of theEst6 locus inDrosophila melanogaster. In Barker, J. S. F., Starmer, W. T., and MacIntyre, R. J. (eds.),Ecological and Evolutionary Genetics of Drosophila Plenum, New York, pp. 273–292.

    Google Scholar 

  • Robertson, H. M., Preston, C. R., Phillis, R. W., Johnson-Sohlez, D. M., Benz, W. K., and Engels, W. R. (1988). A stable genomic source of P-element transposase inD. melanogaster.Genetics 118461.

    PubMed  CAS  Google Scholar 

  • Saad, M., Game, A. Y., Healy, M. J., and Oakeshott, J. G. (1994). Associations of esterase 6 allozyme and activity variation with reproductive fitness inDrosophila melanogaster.Genetica 9443.

    Article  PubMed  CAS  Google Scholar 

  • Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989).Molecular Cloning: A Laboratory Manual Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.

    Google Scholar 

  • Sanger, F., Nicklen, S., and Coulson, A. R. (1977). DNA sequencing with chain terminating inhibitors.Proc. Natl. Acad. Sci. USA 745463.

    PubMed  CAS  Google Scholar 

  • Scott, D. (1986). Inhibition of femaleDrosophila melanogaster remating by a seminal fluid protein (esterase 6).Evolution 401084.

    Article  CAS  Google Scholar 

  • Sheehan, K., Richmond, R. C., and Cochrane, B. J. (1979). Studies of esterase 6 inDrosophila melanogaster. III. The developmental pattern and tissue distribution.Insect Biochem. 9443.

    Article  CAS  Google Scholar 

  • Tarentino, A. L., Gomez, C. M., and Plummer, T. H., Jr. (1985). Deglycosylation of asparagine-linked glycans by peptide: N-glycosidase-F.Biochemistry 244665.

    Article  PubMed  CAS  Google Scholar 

  • Taylor, A. K., and Wall, R. (1988). Selective removal of α-heavy chain glycosylation sites causes immunoglobulin A degradation and reduced secretion.Mol. Cell Biol. 84197.

    PubMed  CAS  Google Scholar 

  • Taylor, J. W., Ott, J., and Eckstein, F. (1985).In vitro mutagenesis.Nucl. Acids Res. 138764.

    Google Scholar 

  • Velan, B., Kronman, C., Ordentlich, A., Flashner, Y., Leitner, M., Cohen, S., and Shafferman, A. (1993). N glycosylation of human acetylcholinesterase: Effects on activity, stability and biosynthesis.Biochem. J. 296649.

    PubMed  CAS  Google Scholar 

  • Wang, X., Sun, B., Yasuyama, K., and Salvaterra, P. M. (1994). Biochemical analysis of proteins recognized by anti-HRP antibodies inDrosophila melanogaster: Identification and characterization of neuron specific and male specific glycoproteins.Insect Biochem. Mol. Biol. 24233.

    Article  PubMed  CAS  Google Scholar 

  • White, M. W., Mane, S. D., and Richmond, R. C. (1988). Studies of esterase-6 inDrosophila melanogaster XVIII: Biochemical differences between slow and fast allozymes.Mol. Biol. Evol. 541.

    PubMed  CAS  Google Scholar 

  • Wray, W., Boulikas, T., Wray, V. P., and Hancock, R. (1981). Silver staining of proteins in polyacrylamide gels.Anal. Biochem. 118197.

    Article  PubMed  CAS  Google Scholar 

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  1. Mark A. Myers

    Present address: Centre for Molecular Biology and Medicine, Monash University, Wellington Road, 3068, Clayton, Victoria, Australia

Authors and Affiliations

  1. CSIRO Division of Entomology, GPO Box 1700, ACT 2601, Canberra, Australia

    Mark A. Myers, Marion J. Healy & John G. Oakeshott

  2. Botany Department, Australian National University, ACT 2601, Canberra, Australia

    Mark A. Myers

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Myers, M.A., Healy, M.J. & Oakeshott, J.G. Mutational analysis ofN-linked glycosylation of esterase 6 inDrosophila melanogaster . Biochem Genet 34, 201–218 (1996). https://doi.org/10.1007/BF02407020

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  • DOI: https://doi.org/10.1007/BF02407020

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