Purification and characterization of plasma membrane glycosidases of Drosophila melanogaster spermatozoa
Previous studies from our laboratory have demonstrated the presence of two integral proteins with glycosidase activity in the plasma membrane of Drosophila melanogaster spermatozoa and we have suggested that these enzymes might have a role in sperm-egg binding. In this study the glycosidases have been purified and characterized. We have evidenced the presence of three distinct enzymes, two beta-N-acetylhexosaminidase isoforms, named HEX 1 and HEX 2, and an alpha-mannosidase. The molecular size of the native enzymes estimated by gel filtration was 158 kDa for beta-hexosaminidases and 317 kDa for alpha-mannosidase. SDS-PAGE showed that HEX 1 and HEX 2 are dimers formed by subunits with different molecular sizes, whereas alpha-mannosidase consists of three subunits with different molecular weights. All the enzymes are terminally glycosylated. Characterization of the purified enzymes included their 4-methylumbelliferyl-substrate preferences, kinetic properties, inhibitor constants and thermal stability. On the basis of substrate specificity, kinetics and the results of inhibition studies, beta-hexosaminidases appear to differ from each other. HEX 1 and HEX 2 are similar to mammalian isoenzyme A and isoenzyme B, respectively. These findings represent the first report on the characterization of sperm proteins that are potentially involved in interactions with the egg in Insects.
Available from: Jari Intra
- "No labeling was observed in the controls. The localization over the acrosome and the tail is similar to that reported for other glycosidases of D. melanogaster sperm plasma membrane, including two b-N-acetylhexosaminidase isoforms that are involved in egg fertilization (Perotti et al., 2001; Cattaneo et al., 2002, 2006). A comment is necessary in relation to the presence on the surface of the tail of a-L- Table 1 Purification of plasma membrane a-L-fucosidase from D. ananassae spermatozoa. "
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ABSTRACT: Sperm-oocyte interaction during fertilization is multiphasic, with multicomponent events, taking place between egg's glycoproteins and sperm surface receptors. Protein-carbohydrate complementarities in gamete recognition have observed in cases throughout the whole evolutionary scale. Sperm-associated α-L-fucosidases have been identified in various organisms. Their wide distribution and known properties reflect the hypothesis that fucose and α-L-fucosidases have fundamental function(s) during gamete interactions. An α-L-fucosidase has been detected as transmembrane protein on the surface of spermatozoa of eleven species across the genus Drosophila. Immunofluorescence labelling showed that the protein is localized in the sperm plasma membrane over the acrosome and the tail, in D. melanogaster. In the present study, efforts were made to analyse with solid phase assays the oligosaccharide recognition ability of fruit fly sperm α-L-fucosidase with defined carbohydrate chains that can functionally mimic egg glycoconjugates. Our results showed that α-L-fucosidase bound to fucose residue and in particular it prefers N-glycans carrying core α1,6-linked fucose and core α1,3-linked fucose in N-glycans carrying only a terminal mannose residue. The ability of sperm α-L-fucosidase to bind to the micropylar chorion and to the vitelline envelope was examined in in vitro assays in presence of α-L-fucosidase, either alone or in combination with molecules containing fucose residues. No binding was detected when α-L-fucosidase was pre-incubated with fucoidan, a polymer of α-L-fucose and the monosaccharide fucose. Furthermore, egg labeling with anti-horseradish peroxidase, that recognized only core α1,3-linked fucose, correlates with α-L-fucosidase micropylar binding. Collectively, these data support the hypothesis of the potential role of this glycosidase in sperm-egg interactions in Drosophila.
Copyright © 2015. Published by Elsevier Ltd.
Available from: Richard A Cardullo
- "Drosophila sperm possess fucosidase, mannosidase and b-N-hexosaminidase that have been proposed to recognize complementary fucose, mannose and b-N-acetylglucosamine residues on the surface surrounding the micropyle of the egg chorion (Intra et al., 2006, 2011; Perotti, 2001). The presence of multiple glycosidases suggests that multiple molecules may be involved in a complex sperm-egg recognition event (Perotti et al., 2001; Intra et al., 2006; Cattaneo et al., 2002). Similar to Drosophila, Ceratitis capitata sperm possess surface associated fucosidase, mannosidase and b-N-acetylglucosaminidase (Intra et al., 2011), although the presence of cognate sugars on the egg chorion was not determined. "
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ABSTRACT: For successful fertilization to occur, molecules on the surface of male and female gametes must recognize each other in a complementary manner. In some organisms, sperm possess a glycosidase on the plasma membrane overlying the head while eggs have glycoproteins that are recognized by those glycosidases resulting in sperm-egg recognition. In this study, two glycosidases, mannosidase and β-N-acetylglucosaminidase, were identified and biochemically characterized in Aquarius remigis sperm. The mannosidase had a Km of 2.36 ± 0.19 mM, a Vmax of 27.49 ± 0.88 pmol/min and a Hill coefficient of 0.94 ± 0.18 at its optimal pH of 7.0. The mannosidase was extracted most efficiently with CHAPSO but was also efficiently extracted with sodium chloride. Mannosidase activity was effectively inhibited by swainsonine, but not by kifunesine, and was significantly reduced in the presence of Mn(2+) and Mg(2+), but not Zn(2+). N-acetylglucosaminidase had a Km of .093 ± 0.01 mM, a Vmax of 153.80 ± 2.97 pmol/min and a Hill coefficient of 0.96 ± 0.63 at its optimal pH of 7.0. N-acetylglucosaminidase was extracted most efficiently with potassium iodide but was also efficiently extracted with Triton X-100 and Zn(2+), but not Ca(2+), Co(2+), Mn(2+) or Mg(2+), significantly inhibited its activity. Taken together, these results indicate that the A. remigis sperm surface contains at least two glycosidases that may recognize complementary glycoconjugates on the surface of water strider eggs.
Copyright © 2015 Elsevier Ltd. All rights reserved.
Available from: Tian Liu
- "DmFDL (GenBank ID: AAL55992) . OfHex3 belonged to Group III, which also included BmHex3 (GenBank ID: BAF52531) , DmHexo2 (GenBank ID: AAF46406)  and TcNag2 (GenBank ID: ABQ95983) . "
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ABSTRACT: Insects require molting fluids to shed the old cuticle during molting. β-N-acetyl-D-hexosaminidase, known as Hex1, together with various chitinases, is responsible for degrading the chitin component of the old cuticle. This study showed that another β-N-acetyl-D-hexosaminidase, termed OfHex3, interacted with Hex1 and functioned in the molting fluid, although the homolog of OfHex3 was known as a sperm-plasma enzyme functioning in egg-sperm recognition. OfHex3 is an enzyme cloned from the insect Asian corn borer, Ostrinia furnacalis, which is one of the most destructive pests of maize. The enzymatic activity analysis indicated that OfHex3 was able to degrade chitooligosaccharides, but at a lower rate than that of OfHex1. Because OfHex3 did not have substrate inhibition, we deduced that the presence of OfHex3 might help OfHex1 relieve substrate inhibition during chitin degradation during molting. The expression patterns of OfHex3 during O. furnacalis development were studied by real-time PCR as well as western blot. The results showed that both gene transcription and protein translation levels of OfHex3 were up-regulated during larval-larval molting. The tissue-specific expression pattern analysis indicated that OfHex3 was mostly localized in the fat body and testis. All these data further supported that Hex3 was involved in molting as well as in fertilization. This study may help to understand the complexity of cuticle degradation during insect molting, and may provide a possible target for pest control.
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