MUTATIONAL ANALYSIS OF N-LINKED GLYCOSYLATION OF ESTERASE-6 IN DROSOPHILA-MELANOGASTER

The primary sequence of the esterase 6 (EST6) enzyme of Drosophila mel anogaster contains four potential N-linked glycosylation sites, at res idues 21, 399, 435, and 485. Here we determine the extent to which EST 6 is glycosylated and how the glycosylation affects the biochemistry a nd physiology of the enzyme. We have abolished each of the four potent ial glycosylation sites by replacing the required Asn residues with Gl n by in vitro mutagenesis. Five mutant genes were made, four containin g mutations of each site individually and the fifth site containing al l four mutations. Germline transformation was used to introduce the mu tant genes into a strain of D. melanogaster null for EST6. Electrophor etic 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 si te is nor utilized on all EST6 molecules, resulting in two molecular f orms of the enzyme. Digestion with specific endoglycosidases showed th at the glycan attached at the second site is of the high-mannose type while the other three sites carry more complex oligosaccharides. The t hermostability 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 doe s not reduce thermostability. Quantitative comparisons of EST6 activit ies showed that abolition of glycosylation does not affect the secreti on 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 p ersist 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.