Glycosidases in the plasma membrane of Ceratitis capitata spermatozoa (original) (raw)
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Conservation of glycosidases of the sperm plasma membrane among Drosophila species
We have studied the presence of four sperm glycosidases, a-mannosidase, a-Lfucosidase and two b-hexosaminidase isoforms, in 11 species of the genus Drosophila spanning approximately an evolutionary 60 MY period, and in Scaptodrosophila lebanonensis, belonging to the ancestor genus Scaptodrosophila. These enzymes had been previously identified in Drosophila melanogaster as putative receptors for glycoconjugates of the egg surface. Alpha-mannosidase and b-hexosaminidases are intrinsic proteins of the sperm plasma membrane in species closely related to D. melanogaster as well as in the divergent species D. willistoni, D. hydei, D. virilis, and S. lebanonensis. Alpha-L-fucosidase is restricted to the species of the genus Drosophila. Alpha-mannosidase and b-hexosaminidases have been purified and characterized in all species. Their molecular masses and optimal pHs are similar in all the species, whereas interspecific differences in enzyme activities were detected. Crossspecies comparison of kinetic parameters indicated a relationship between enzyme efficiency and phylogenetic relatedness. Beta-hexosaminidases were the most efficient enzymes. Lectin cytochemistry suggested the presence of carbohydrate residues complementary to the glycosidases on the eggshell at the site of sperm entry in all species. Bioinformatic analysis of the coding sequences of b-hexosaminases and a-L-fucosidase and of their predicted products showed no evidence of positive selection of the genes coding for these enzymes and a high degree of sequence identity of the predicted polypeptides among the species of the genus Drosophila. Collectively, our findings indicate that the Drosophila sperm glycosidases are structurally and functionally conserved and strengthen the hypothesis of their involvement in the interactions with the egg surface. Mol. Reprod. Dev. ß
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.
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.
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.
Journal of Insect Physiology, 2011
The Mediterranean fruit fly Ceratitis capitata (Diptera: Tephritidae) is one of the most destructive agricultural pests, a polyphagus insect of relevant economic importance and is widespread in many regions around the world. It is the best-studied fruit fly pest at genetic and molecular level and much has been learned on its ecology and behaviour. An a-L-fucosidase has been recently hypothesized to be involved in sperm-egg interactions in Drosophila melanogaster and in other Drosophila species. Here, a complete cDNA encoding a putative a-L-fucosidase of the medfly was amplified using the reverse polymerase chain reaction (RT-PCR) with degenerate based on the conserved coding sequence information of several insect a-L-fucosidases, cloned and sequenced (GenBank accession no. FJ177429).
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
Epididymal α- l -fucosidase and its possible role in remodelling the surface of bull spermatozoa
Theriogenology, 2017
The mammalian epididymis provides an appropriate environment for sperm maturation. During the epididymal transit, spermatozoa undergo biochemical and morphological changes that lead to the acquisition of the fertilizing capacity. In this study we analysed the fucosylation status of membrane glycoproteins in the spermatozoa obtained from different regions of the bull epididymis. High amounts of fucose were detected on caput spermatozoa (R.F.I.= 1010 ± 20.35), mostly located in the post-acrosome zone. A significant decrease in the fucose levels was detected toward the cauda (R.F.I.= 540.5 ± 49.93) (P<0.05). This decrease was in line with the increased activity of α-L-fucosidase in the cauda fluid. In sperm from the cauda, the defucosylation occurred in some proteins, whereas others showed higher fucosylation rates. A significant decrease of fucose in the gametes was observed upon incubation of crude cauda fluid with caput spermatozoa (from R.F.I.= 1.45 ± 0.08 to 1.06 ± 0.03) (P<0.05) indicating that the α-L-fucosidase present in the epididymal fluid is active on spermatozoa. Moreover, this effect was blocked with specific enzyme inhibitors. These results provide direct evidence that the α-Lfucosidase from epididymal fluid participates in the fucose removal from spermatozoa, as a step of sperm maturation in the bull epididymis.
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.
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.