Conservation of glycosidases of the sperm plasma membrane among Drosophila species (original) (raw)
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Glycosidases are present on the surface ofDrosophila melanogaster spermatozoa
Molecular Reproduction and Development, 1997
We investigated the presence of enzymes on the surface of Drosophila melanogaster spermatozoa that might bind to the carbohydrate residues of the egg shell. Spectrophotometric and fluorimetric studies were used on whole spermatozoa to assay galactosyltransferase and glycosidase activities. No galactosyltransferase is present on the sperm surface, whereas two glycosidases, b-N-acetylglucosaminidase (GlcNAc8ase) and a-mannosidase (Man8ase), have been evidenced. They have an optimal pH of 6-6.5 and 4, respectively. The same glycosidases were detected as soluble forms probably secreted by the seminal vesicle epithelium. We suggest that these enzymes might be involved in the recognition of a-mannose and b-N-acetylglucosamine residues present on the egg shell at the site of sperm entry. Mol. Reprod.
Glycosidases in the plasma membrane of Ceratitis capitata spermatozoa
Insect Biochemistry and Molecular Biology, 2011
Fruit flies in the family Tephritidae are rated among the world's most destructive agricultural pests. The Mediterranean fruit fly Ceratitis capitata is emerging as a model organism to study the fertilization in Insects. Three integral proteins with glycosidase activity are present in the plasma membrane of spermatozoa. The glycosidases have been purified and characterized. We have demonstrated the presence of three enzymes, a b-N-acetylhexosaminidase, an a-mannosidase and an a-L-fucosidase. The molecular mass of the native enzymes estimated by gel filtration was 160 kDa for b-N-acetylhexosaminidase, 310 kDa for a-mannosidase and 140 kDa for a-L-fucosidase. SDS-PAGE showed that b-N-acetylhexosaminidase is a dimer of a single protein of 73 kDa, a-mannosidase consists of six subunits with different molecular weights and a-L-fucosidase is a dimer made up by two different monomers. Characterization of the purified enzymes included glycosylation pattern, pI, optimal pH, substrate preference, kinetic properties and thermal stability. Soluble forms similar to the sperm associated glycosidases are present. Polyclonal antibodies raised against synthetic peptides designed from the predicted products of the Drosophila melanogaster genes encoding b-N-acetylhexosaminidase and a-L-fucosidase were used.
Expression study of an α-l-fucosidase gene in the Drosophilidae family
Gene, 2008
The plasma membrane of Drosophila (Sophophora) melanogaster spermatozoa contains an α-L-fucosidase that might be involved in fertilization by interacting with α-L-fucose residues on the micropyle of the eggshell. D. (S.) melanogaster has a single gene called CG6128 or Fuca encoding for a putative α-L-fucosidase. Two transcripts have been annotated, RA of 3514 bp, and RB of 1673 bp. While both transcripts encode an α-L-fucosidase, RA contains an upstream open reading frame, translated into a polypeptide containing a predicted BTB/POZ domain. We demonstrate that Fuca is expressed in male and female germ lines. RT-PCR analysis indicated a broader tissue expression. Homologous genes are expressed in the same tissues in several drosophilid flies belonging to the genera Drosophila and Scaptodrosophila. However, the long transcript is restricted to species belonging to the subgenus Sophophora. The presence of two transcripts in species of the subgenus Sophophora and only one in species belonging to the subgenus Drosophila might be related to the phylogenetic relationships of these subgenera. Immunofluorescence demonstrated that the gene product, localized to the sperm plasma membrane, is absent from Scaptodrosophila lebanonensis spermatozoa. These findings support the hypothesis that the enzyme is involved in the molecular events of primary gamete interactions that are conserved among drosophilids belonging to Drosophila genus.
Glycobiology, 2006
Sperm surface b-N-acetylhexosaminidases are among the molecules mediating early gamete interactions in invertebrates and vertebrates, including man. The plasma membrane of Drosophila spermatozoa contains two b-N-acetylhexosaminidases, DmHEXA and DmHEXB, which are required for egg fertilization. Here, we demonstrate that three putative Drosophila melanogaster genes predicted to code for b-N-acetylhexosaminidases, Hexo1, Hexo2, and fdl, are all expressed in the male germ line. fdl codes for a homolog of the a-subunit of the mammalian lysosomal b-N-acetylhexosaminidase Hex A. Hexo1 and Hexo2 encode two homologs of the b-subunit of all known b-N-acetylhexosaminidases, which we have named b 1 and b 2 , respectively. Immunoblot analysis of sperm proteins indicated that the gene products associate in different heterodimeric combinations forming DmHEXA, with an ab 2 structure, and DmHEXB, with a b 1 b 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 b-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 b-N-acetylhexosaminidases are present in Drosophila three different gene products are available that might generate numerous dimeric isoforms.
BMC Genomics, 2011
Background: The evolutionary diversification of gene families through gene creation (and loss) is a dynamic process believed to be critical to the evolution of functional novelty. Previous identification of a closely related family of eight annotated metalloprotease genes of the M17 Merops family in the Drosophila sperm proteome (termed, Sperm-LeucylAminoPeptidases, S-LAPs 1-8) led us to hypothesize that this gene family may have experienced such a diversification during insect evolution. Results: To assess putative functional activities of S-LAPs, we (i) demonstrated that all S-LAPs are specifically expressed in the testis, (ii) confirmed their presence in sperm by two-dimensional gel electrophoresis and mass spectrometry, (iii) determined that they represent a major portion of the total protein in sperm and (iv) identified aminopeptidase enzymatic activity in sperm extracts using LAP-specific substrates. Functionally significant divergence at the canonical M17 active site indicates that the largest phylogenetic group of S-LAPs lost catalytic activity and likely acquired novel, as yet undetermined, functions in sperm prior to the expansion of the gene family. Conclusions: Comparative genomic and phylogenetic analyses revealed the dramatic expansion of the S-LAP gene family during Drosophila evolution and copy number heterogeneity in the genomes of related insects. This finding, in conjunction with the loss of catalytic activity and potential neofunctionalization amongst some family members, extends empirical support for pervasive "revolving door" turnover in the evolution of reproductive gene family composition and function.
Journal of Experimental Zoology, 1995
The important roles played by glycoconjugates in the recognition of gametes and in fertilization are well documented. In the present study, the nature and distribution of carbohydrate residues of the plasma membrane of spermatozoa of Drosophila melanogaster were characterized by use of FITC-conjugated lectins as probes. The plasma membrane bound agglutinins from Concanavalia ensiformis (Con A) and Pisum sativum (PSA), native and succinylated agglutinins from wheat germ (WGA and s-WGA), the A4 isoform of agglutinin-I from Griffonia simplicifolia (GSA-I A4), and, to a lesser extent, the lectins from Dolichus biflorus (DBA), Lotus tetragonolobus (LTA), and Glycine maximus (SBA). Each lectin gave a specific pattern of binding. The extent of binding of Con A, WGA, s-WGA, and GSA-I A4 over the acrosomal region was greater than over nonacrosomal regions, indicating the concentration of α-mannose/α-glucose, β-N-acetylglucosamine, and α-N-acetylgalactosamine residues in this area of the plasma membrane. The surface of the sperm failed to react with lectins from Ricinus communis (RCA-I), Limulus polyphemus (LPA), and Limax flavus (LFA) and with the B4 isoform of agglutinin-I from Griffonia simplicifolia-I (GSA-I B4). The plasma membrane over the nucleus did not react with any of the lectins tested.Quantitative analysis of binding of Con A, s-WGA, and GSA-I A4 to spermatozoa showed that only Con A bound consistently to the sperm surface, showing high affinity for the acrosomal area of the plasma membrane. The other lectins tested bound only to limited and variably sized fractions of the total population of sperm. Therefore, only residues of α-mannose/α-glucose are a constitutive component of the plasma membrane, and they are characteristic of the acrosomal area. Con A can be used as a marker of the acrosome portion of sperm from Drosophila for visualization and assessment of acrosome status; labelling with FITC-conjugated Con A provides a simple and reliable method for visualization of the acrosome of Drosophila sperm that is otherwise detectable only by ultrastructural examination. © 1995 Wiley-Liss, Inc.
Expression of genes encoding hexosaminidases in the testis of D. melanogaster
Sperm surface b-N-acetylhexosaminidases are among the molecules mediating early gamete interactions in invertebrates and vertebrates, including man. The plasma membrane of Drosophila spermatozoa contains two b-N-acetylhexosaminidases, DmHEXA and DmHEXB, which are required for egg fertilization. Here, we demonstrate that three putative Drosophila melanogaster genes predicted to code for b-N-acetylhexosaminidases, Hexo1, Hexo2, and fdl, are all expressed in the male germ line. fdl codes for a homolog of the a-subunit of the mammalian lysosomal b-N-acetylhexosaminidase Hex A. Hexo1 and Hexo2 encode two homologs of the b-subunit of all known b-N-acetylhexosaminidases, which we have named b 1 and b 2 , respectively. Immunoblot analysis of sperm proteins indicated that the gene products associate in different heterodimeric combinations forming DmHEXA, with an ab 2 structure, and DmHEXB, with a b 1 b 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 b-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 b-N-acetylhexosaminidases are present in Drosophila three different gene products are available that might generate numerous dimeric isoforms.
Biochemical and Biophysical Research Communications, 1997
subsequent steps leading to successful fertilization. The male reproductive system of the mollusc bivalve There is also evidence that other glycosidases such as Unio elongatulus contains two distinct forms of a-Lb-N-acetylglucosaminidase (6-9) and a-D-mannosidase fucosidase, one present in the gonad fluid and a second (10) play a similar role in fertilization. In all cases, the one associated with the sperm plasma membrane. Both glycosidase on the sperm has been found to match the activities were purified to homogeneity. The soluble functional sugar residues of the egg coat glycoproteins. seminal plasma enzyme had an oligomeric MW of 56 In a previous study, we showed that the ligand for kDa as determined by MALDI-TOF mass spectrometry, sperm binding in the mollusc bivalve Unio elongatulus whereas the enzyme purified from sperm plasma memis a 220 kDa glycoprotein and that fucose residues of its branes had an MW of 68 kDa. Analyzed by lectin blotoligosaccharide chains play a key role in the interaction ting with ConA and PNA, the 68 kDa enzyme did not mechanism (11,12). We postulated that a-L-fucosidase bind either lectin, whereas the 56 kDa form bound could be a receptor molecule on the sperm. In this re-ConA only. Both fucosidases followed a Michaelis-Menport, we demonstrate that two forms of a-L-fucosidase, ten kinetics with the K m of the sperm-bound enzyme one soluble and the other bound to the sperm membeing 7.1 1 10 04 M and that of the seminal enzyme being brane, are present in the gonads of Unio elongatulus. 9.1 1 10 04 M. Both had a pH optimum of 5.0. ᭧ 1997 Academic Press
Evolutionary, structural and functional analysis of Drosophila melanogaster seminal fluid proteins
2004
1 The work in this chapter has been accepted in the journal Genetics and is in press; Mueller, JL, Ravi Ram, K., McGraw, LA, Bloch Qazi, MC, Siggia, ED, Clark, AG, Aquadro, CF, Wolfner, MF, Cross-species comparison of Drosophila male accessory gland protein ...