Purification and characterization of the plasma membrane glycosidases of Drosophila melanogaster spermatozoa.
ABSTRACT 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.
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ABSTRACT: Sperm storage organs are common and broadly distributed among animal taxa. However, little is known about how these organs function at the molecular level. Additionally, there is a paucity of knowledge about the evolution of genes expressed in these organs. This investigation is an evolutionary expressed sequence tag (EST) study of genes expressed in the seminal receptacle, one of the sperm storage organs in Drosophila. The incidence of positive selection is higher for the seminal receptacle genes than Drosophila reproductive genes as a whole, but lower than genes associated with the spermatheca, a second type of Drosophila sperm storage organ. By identifying overrepresented classes of proteins and classes for which sperm storage function is suggested by the nature of the proteins, candidate genes were discovered. These candidates belong to protein classes such as muscle contraction, odorant binding and odorant receptor, protease inhibitor and immunity.Journal of Evolutionary Biology 07/2010; 23(7):1386-98. DOI:10.1111/j.1420-9101.2010.01998.x · 3.48 Impact Factor
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ABSTRACT: Insect β-N-acetyl-d-hexosaminidases are of particular interest due to their multiple physiological roles in many life processes. Chitinolytic β-N-acetyl-d-hexosaminidases, which function only in chitin degradation in insects, have long been regarded as species-specific target potentials in developing environmental friendly pesticides. Here the chitinolytic β-N-acetyl-d-hexosaminidase from the insect Ostrinia furnacalis was cloned and expressed in the yeast strain, Pichia pastoris, to meet the demands of biochemical studies and drug development. Enzymatic assay as well as Western blot confirmed that the high-level expression could be achieved after the induction of methanol for 120h. Through the sequential combination of ammonium sulfate precipitation, metal chelating chromatography as well as anion exchange chromatography, 7.7mg of the recombinant OfHex1 with high purity was obtained from 1 liter of culture supernatant. The recombinant OfHex1, characterized as a homodimer with molecular weight of 130kDa, exhibited the same enzymatic activities as its native form, which could efficiently degrade the chitooligosaccharide substrate (GlcNAc)2 and release 4-methylumbelliferone (4MU) from substrates, 4MU-β-GlcNAc and 4MU-β-GalNAc. This work provides a low-costing and high-efficient purification procedure for the preparation of insect β-N-acetyl-d-hexosaminidases.Protein Expression and Purification 11/2009; 68(1):99-103. DOI:10.1016/j.pep.2009.06.004 · 1.51 Impact Factor
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ABSTRACT: Sperm surface beta-N-acetylhexosaminidases are among the molecules mediating early gamete interactions in invertebrates and vertebrates, including man. The plasma membrane of Drosophila spermatozoa contains two beta-N-acetylhexosaminidases, DmHEXA and DmHEXB, which are required for egg fertilization. Here, we demonstrate that three putative Drosophila melanogaster genes predicted to code for beta-N-acetylhexosaminidases, Hexo1, Hexo2, and fdl, are all expressed in the male germ line. fdl codes for a homolog of the alpha-subunit of the mammalian lysosomal beta-N-acetylhexosaminidase Hex A. Hexo1 and Hexo2 encode two homologs of the beta-subunit of all known beta-N-acetylhexosaminidases, which we have named beta(1) and beta(2), respectively. Immunoblot analysis of sperm proteins indicated that the gene products associate in different heterodimeric combinations forming DmHEXA, with an alphabeta(2) structure, and DmHEXB, with a beta(1)beta(2) structure. Immunofluorescence demonstrated that all the gene products localized to the sperm plasma membrane. Although none of the genes was testis-specific, fdl was highly and preferentially expressed in the testis, whereas Hexo1 and Hexo2 showed broader tissue expression. Enzyme assays carried out on testis and on a variety of somatic tissues corroborated the results of gene expression analysis. These findings for the first time show the in vivo expression in insects of genes encoding beta-N-acetylhexosaminidases, the only molecules so far identified as involved in sperm/egg recognition in this class, whereas in mammals, the organisms where these enzymes have been best studied, only two types of polypeptide chains forming dimeric functional beta-N-acetylhexosaminidases are present in Drosophila three different gene products are available that might generate numerous dimeric isoforms.Glycobiology 10/2006; 16(9):786-800. DOI:10.1093/glycob/cwl007 · 3.75 Impact Factor