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Marco Moracci - One of the best experts on this subject based on the ideXlab platform.
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probing the catalytically essential residues of the α l Fucosidase from the hyperthermophilic archaeon sulfolobus solfataricus
Biochemistry, 2005Co-Authors: Beatrice Cobucciponzano, Mose Rossi, Marialuisa Mazzone, Marco MoracciAbstract:Retaining glycosidases promote the hydrolysis of the substrate by following a double-displacement mechanism involving a covalent intermediate. The catalytic residues are a general acid/base catalyst and the nucleophile. Experimental identification of these residues in a specific glycosidase allows for the assigning of the corresponding residues in all of the other enzymes belonging to the same family. By means of sequence alignment, mutagenesis, and detailed kinetic studies of the alpha-Fucosidase from Sulfolobus solfataricus (Ssalpha-fuc) (family 29), we show here that the residues, invariant in this family, have the function inferred from the analysis of the 3D structure of the enzyme from Thermotoga maritima (Tmalpha-fuc). These include in Ssalpha-fuc the substrate-binding residues His46 and His123 and the nucleophile of the reaction, previously described. The acid/base catalyst could be assigned less easily. The k(cat) of the Ssalpha-fucGlu292Gly mutant, corresponding to the acid/base catalyst of Tmalpha-fuc, is reduced by 154-fold but could not be chemically rescued. Instead, the Ssalpha-fucGlu58Gly mutant revealed a 4000-fold reduction of k(cat)/K(M) if compared to the wild-type and showed the rescue of the k(cat) by sodium azide at wild-type levels. Thus, our data suggest that a catalytic triad, namely, Glu58, Glu292, and Asp242, is involved in catalysis. Glu58 and Glu292 cooperate in the role of acid/base catalyst, while Asp242 is the nucleophile of the reaction. Our data suggest that in glycosidase family 29 alpha-Fucosidases promoting the retaining mechanism with slightly different catalytic machineries coexist.
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structural characterization of the nonameric assembly of an archaeal α l Fucosidase by synchrotron small angle x ray scattering
Biochemical and Biophysical Research Communications, 2004Co-Authors: Camillo Rosano, Marco Moracci, Beatrice Cobucciponzano, Mose Rossi, Marialuisa Mazzone, Simone Zuccotti, Maxim V Petoukhov, Dmitri I SvergunAbstract:alpha-l-Fucosidase is a lysosomal enzyme responsible for hydrolyzing the alpha-1,6-linked fucose joined to the reducing-end N-acetylglucosamine of carbohydrate moieties in glycoproteins. The first alpha-l-Fucosidase from Archaea was recently identified in the genome of the hyperthermophile Sulfolobus solfataricus; the enzyme is encoded by two open reading frames separated by a -1 frameshift. A preliminary biochemical and biophysical characterization of this extremophile enzyme has been carried out both in solution, through small angle X-ray scattering experiments, and in the crystalline state, showing an unusual oligomeric assembly resulting from the association of nine subunits, endowed with 3-fold molecular symmetry.
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identification of the catalytic nucleophile of the family 29 α l Fucosidase from sulfolobus solfataricus via chemical rescue of an inactive mutant
Biochemistry, 2003Co-Authors: Beatrice Cobucciponzano, Assunta Giordano, Antonio Trincone, Mose Rossi, Marco MoracciAbstract:We have recently reported that a functional alpha-L-Fucosidase could be expressed by a single insertional mutation in the region of overlap between the ORFs SSO11867 and SSO3060 of the hyperthermophilic Archaeon Sulfolobus solfataricus [Cobucci-Ponzano et al. J. Biol. Chem. (2003) 278, 14622-14631]. This enzyme, belonging to glycoside hydrolase family 29 (GH29), showed micromolar specificity for p-nitrophenyl-alpha-L-fucoside (pNp-Fuc) and promoted transfucosylation reactions by following a reaction mechanism in which the products retained the anomeric configuration of the substrate. The active site residues in GH29 enzymes are still unknown. We describe here the identification of the catalytic nucleophile of the reaction in the alpha-L-Fucosidase from S. solfataricus by reactivation with sodium azide of the mutant Asp242Gly that shows a 10(3)-fold activity reduction on pNp-Fuc. The detailed stereochemical analysis of the fucosyl-azide produced by the mutant reactivated on pNp-Fuc revealed its inverted (beta-fucosyl azide) configuration compared with the substrate. This allows for the first time the unambiguous assignment of Asp242, and its homologous residues, as the nucleophilic catalytic residues of GH29 alpha-L-Fucosidases. This is the first time that this approach is used for alpha-L-glycosidases, widening the applicability of this method.
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identification of an archaeal α l Fucosidase encoded by an interrupted gene production of a functional enzyme by mutations mimicking programmed 1 frameshifting
Journal of Biological Chemistry, 2003Co-Authors: Beatrice Cobucciponzano, Assunta Giordano, Antonio Trincone, Marco MoracciAbstract:The analysis of the complete genome of the thermoacidophilic Archaeon Sulfolobus solfataricus revealed two open reading frames (ORF), named SSO11867 and SSO3060, interrupted by a -1 frameshift and encoding for the N- and the C-terminal fragments, respectively, of an alpha-l-Fucosidase. We report here that these ORFs are actively transcribed in vivo, and we confirm the presence of the -1 frameshift between them at the cDNA level, explaining why we could not find alpha-Fucosidase activity in S. solfataricus extracts. Detailed analysis of the region of overlap between the two ORFs revealed the presence of the consensus sequence for a programmed -1 frameshifting. Two specific mutations, mimicking this regulative frameshifting event, allow the expression, in Escherichia coli, of a fully active thermophilic and thermostable alpha-l-Fucosidase (EC ) with micromolar substrate specificity and showing transfucosylating activity. The analysis of the fucosylated products of this enzyme allows, for the first time, assigning a retaining reaction mechanism to family 29 of glycosyl hydrolases. The presence of an alpha-Fucosidase putatively regulated by programmed -1 frameshifting is intriguing both with respect to the regulation of gene expression and, in post-genomic era, for the definition of gene function in Archaea.
Jean-bernard Behr - One of the best experts on this subject based on the ideXlab platform.
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A second-generation ferrocene-iminosugar hybrid with improved Fucosidase binding properties
Bioorganic and Medicinal Chemistry Letters, 2016Co-Authors: Audrey Hottin, Amandine Scandolera, Laurent Duca, Daniel W Wright, Gideon J Davies, Jean-bernard BehrAbstract:The synthesis and the biological evaluation of a new ferrocenyl-iminosugar conjugate designed for Fucosidase inhibitory and anticancer activity is described. The compound showed strong affinity for Fucosidase from bovine kidney (Ki = 23 nM) and from Bacteroides thetaiotaomicron (Ki = 150 nM), displaying a 10-fold tighter binding affinity for these enzymes than the previous analogs. The interaction pattern that improves binding has been evaluated through structural analysis of the inhibitor–enzyme complex. The ferrocenyl-iminosugar exhibits significant anticancer activity on MDA-MB-231 and SK-MEL28 cell lines at 100 μM.
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Exploring the divalent effect in Fucosidase inhibition with stereoisomeric pyrrolidine dimers
Organic and Biomolecular Chemistry, 2016Co-Authors: Audrey Hottin, Daniel W Wright, Gideon J Davies, Elena Moreno-clavijo, Antonio Moreno-vargas, Jean-bernard BehrAbstract:Multi-valent inhibitors offer promise for the enhancement of therapeutic compounds across a range of chemical and biological processes. Here, a significant increase in enzyme-inhibition potencies was observed with a dimeric iminosugar-templated Fucosidase inhibitor (IC50 = 0.108 μM) when compared to its monovalent equivalent (IC50 = 2.0 μM). Such a gain in binding is often attributed to a “multivalent effect” rising from alternative recapture of the scaffolded binding epitopes. The use of control molecules such as the meso analogue (IC50 = 0.365 μM) or the enantiomer (IC50 = 569 μM), as well as structural analysis of the Fucosidase-inhibitor complex, allowed a detailed analysis of the possible mechanism of action, at the molecular level. Here, the enhanced binding affinity of the dimer over the monomer can be attributed to additional interactions in non-catalytic sites as also revealed in the 3-D structure of a bacterial Fucosidase inhibitor complex.
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Perfluoroalkylation of Nitrones for the Synthesis of a Series of Fucosidase Inhibitors
European Journal of Organic Chemistry, 2015Co-Authors: Jadwiga Paszkowska, Olalla Novo Fernandez, Ilona Wandzik, Stéphanie Boudesoque, Laurent Dupont, Richard Plantier-royon, Jean-bernard BehrAbstract:Fucosidase inhibitors represent captivating targets for various biological applications. Iminosugars featuring a fluoroalkyl chain in the pseudoanomeric position were synthesized by nucleophilic addition of a perfluoroalkyl Grignard reagent to a nitrone. Reduction of the N–O bond of the hydroxylamine was achieved by treatment with an aqueous solution of sulfur dioxide. The pKa = 4.5 of the target pyrrolidine reflected a strong electronic effect of the fluoroalkyl chain. Inhibitory potencies of the fluorinated iminosugars and their corresponding hydroxylamines were evaluated against α-LFucosidase at various pH values.
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α-L-Fucosidase inhibition by pyrrolidine-ferrocene hybrids: rationalization of ligand-binding properties by structural studies.
Chemistry - A European Journal, 2013Co-Authors: Audrey Hottin, Daniel W Wright, Gideon J Davies, Agata Steenackers, Philippe Delannoy, Faustine Dubar, Christophe Biot, Jean-bernard BehrAbstract:Enhanced metabolism of fucose through Fucosidase overexpression is a signature of some cancer types, thus suggesting that Fucosidase-targetted ligands could play the role of drug-delivery vectors. Herein, we describe the synthesis of a new series of pyrrolidine-ferrocene conjugates, consisting of a L-fuco-configured dihydroxypyrrolidine as the Fucosidase ligand armed with a cytotoxic ferrocenylamine moeity. Three-dimensional structures of several of these Fucosidase inhibitors reveal transition-state-mimicking (3)E conformations. Elaboration with the ferrocenyl moiety results in sub-micromolar inhibitors of both bovine and bacterial Fucosidases, with the 3D structure of the latter revealing electron density indicative of highly mobile alkylferrocene compounds. The best compounds show a strong antiproliferative effect, with up to 100% inhibition of the proliferation of MDA-MB-231 cancer cells at 50 μM.
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synthesis and l Fucosidase inhibitory activity of a new series of cyclic sugar imines in situ formation and assay of their saturated counterparts
Tetrahedron Letters, 2009Co-Authors: Jean-bernard BehrAbstract:Abstract The synthesis of a series of aryl-substituted cyclic sugar imines was performed via a tandem nucleophilic addition/substitution reaction. The so-obtained ketimines displayed Fucosidase inhibitory activities (IC 50 = 46–556 μM). Their reduced counterparts were prepared and assayed after addition of sodium borohydride to the enzymatic assay stock solution. The pyrrolidines strongly inhibit Fucosidase (IC 50 = 0.65–150 μM).
Beatrice Cobucciponzano - One of the best experts on this subject based on the ideXlab platform.
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probing the catalytically essential residues of the α l Fucosidase from the hyperthermophilic archaeon sulfolobus solfataricus
Biochemistry, 2005Co-Authors: Beatrice Cobucciponzano, Mose Rossi, Marialuisa Mazzone, Marco MoracciAbstract:Retaining glycosidases promote the hydrolysis of the substrate by following a double-displacement mechanism involving a covalent intermediate. The catalytic residues are a general acid/base catalyst and the nucleophile. Experimental identification of these residues in a specific glycosidase allows for the assigning of the corresponding residues in all of the other enzymes belonging to the same family. By means of sequence alignment, mutagenesis, and detailed kinetic studies of the alpha-Fucosidase from Sulfolobus solfataricus (Ssalpha-fuc) (family 29), we show here that the residues, invariant in this family, have the function inferred from the analysis of the 3D structure of the enzyme from Thermotoga maritima (Tmalpha-fuc). These include in Ssalpha-fuc the substrate-binding residues His46 and His123 and the nucleophile of the reaction, previously described. The acid/base catalyst could be assigned less easily. The k(cat) of the Ssalpha-fucGlu292Gly mutant, corresponding to the acid/base catalyst of Tmalpha-fuc, is reduced by 154-fold but could not be chemically rescued. Instead, the Ssalpha-fucGlu58Gly mutant revealed a 4000-fold reduction of k(cat)/K(M) if compared to the wild-type and showed the rescue of the k(cat) by sodium azide at wild-type levels. Thus, our data suggest that a catalytic triad, namely, Glu58, Glu292, and Asp242, is involved in catalysis. Glu58 and Glu292 cooperate in the role of acid/base catalyst, while Asp242 is the nucleophile of the reaction. Our data suggest that in glycosidase family 29 alpha-Fucosidases promoting the retaining mechanism with slightly different catalytic machineries coexist.
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structural characterization of the nonameric assembly of an archaeal α l Fucosidase by synchrotron small angle x ray scattering
Biochemical and Biophysical Research Communications, 2004Co-Authors: Camillo Rosano, Marco Moracci, Beatrice Cobucciponzano, Mose Rossi, Marialuisa Mazzone, Simone Zuccotti, Maxim V Petoukhov, Dmitri I SvergunAbstract:alpha-l-Fucosidase is a lysosomal enzyme responsible for hydrolyzing the alpha-1,6-linked fucose joined to the reducing-end N-acetylglucosamine of carbohydrate moieties in glycoproteins. The first alpha-l-Fucosidase from Archaea was recently identified in the genome of the hyperthermophile Sulfolobus solfataricus; the enzyme is encoded by two open reading frames separated by a -1 frameshift. A preliminary biochemical and biophysical characterization of this extremophile enzyme has been carried out both in solution, through small angle X-ray scattering experiments, and in the crystalline state, showing an unusual oligomeric assembly resulting from the association of nine subunits, endowed with 3-fold molecular symmetry.
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identification of the catalytic nucleophile of the family 29 α l Fucosidase from sulfolobus solfataricus via chemical rescue of an inactive mutant
Biochemistry, 2003Co-Authors: Beatrice Cobucciponzano, Assunta Giordano, Antonio Trincone, Mose Rossi, Marco MoracciAbstract:We have recently reported that a functional alpha-L-Fucosidase could be expressed by a single insertional mutation in the region of overlap between the ORFs SSO11867 and SSO3060 of the hyperthermophilic Archaeon Sulfolobus solfataricus [Cobucci-Ponzano et al. J. Biol. Chem. (2003) 278, 14622-14631]. This enzyme, belonging to glycoside hydrolase family 29 (GH29), showed micromolar specificity for p-nitrophenyl-alpha-L-fucoside (pNp-Fuc) and promoted transfucosylation reactions by following a reaction mechanism in which the products retained the anomeric configuration of the substrate. The active site residues in GH29 enzymes are still unknown. We describe here the identification of the catalytic nucleophile of the reaction in the alpha-L-Fucosidase from S. solfataricus by reactivation with sodium azide of the mutant Asp242Gly that shows a 10(3)-fold activity reduction on pNp-Fuc. The detailed stereochemical analysis of the fucosyl-azide produced by the mutant reactivated on pNp-Fuc revealed its inverted (beta-fucosyl azide) configuration compared with the substrate. This allows for the first time the unambiguous assignment of Asp242, and its homologous residues, as the nucleophilic catalytic residues of GH29 alpha-L-Fucosidases. This is the first time that this approach is used for alpha-L-glycosidases, widening the applicability of this method.
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identification of an archaeal α l Fucosidase encoded by an interrupted gene production of a functional enzyme by mutations mimicking programmed 1 frameshifting
Journal of Biological Chemistry, 2003Co-Authors: Beatrice Cobucciponzano, Assunta Giordano, Antonio Trincone, Marco MoracciAbstract:The analysis of the complete genome of the thermoacidophilic Archaeon Sulfolobus solfataricus revealed two open reading frames (ORF), named SSO11867 and SSO3060, interrupted by a -1 frameshift and encoding for the N- and the C-terminal fragments, respectively, of an alpha-l-Fucosidase. We report here that these ORFs are actively transcribed in vivo, and we confirm the presence of the -1 frameshift between them at the cDNA level, explaining why we could not find alpha-Fucosidase activity in S. solfataricus extracts. Detailed analysis of the region of overlap between the two ORFs revealed the presence of the consensus sequence for a programmed -1 frameshifting. Two specific mutations, mimicking this regulative frameshifting event, allow the expression, in Escherichia coli, of a fully active thermophilic and thermostable alpha-l-Fucosidase (EC ) with micromolar substrate specificity and showing transfucosylating activity. The analysis of the fucosylated products of this enzyme allows, for the first time, assigning a retaining reaction mechanism to family 29 of glycosyl hydrolases. The presence of an alpha-Fucosidase putatively regulated by programmed -1 frameshifting is intriguing both with respect to the regulation of gene expression and, in post-genomic era, for the definition of gene function in Archaea.
Stephen G Withers - One of the best experts on this subject based on the ideXlab platform.
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identity and role of the non conserved acid base catalytic residue in the gh29 Fucosidase from the spider nephilingis cruentata
Glycobiology, 2018Co-Authors: Natalia N Perrella, Stephen G Withers, Adriana R LopesAbstract:α-l-Fucosidases are widely occurring enzymes that remove fucose residues from N- and O-fucosylated glycoproteins. Comparison of amino acid sequences of Fucosidases reveals that although the nucleophile is conserved among all α-l-Fucosidases, the position of the acid/base residue is quite variable. Although several site-directed mutation studies have previously been performed on bacterial Fucosidases, the only eukaryotic Fucosidase so studied was the human Fucosidase. Recent alignments indicate that human and Arthropoda α-l-Fucosidases share at least 50% identity and the acid/base residue seems to be conserved among them suggesting a common acid/base residue in Metazoa. Here we describe the cloning and expression in Pichia pastoris of a very active α-l-Fucosidase from the spider Nephilingis cruentata (NcFuc) with a Km value for pNPFuc of 0.4 mM. NcFuc hydrolyzed fucoidan, 2´fucosyllactose and also lacto-N-difucohexaose II. Mutants modified at the conserved residues D214N, E209A, E59A were expressed and characterized. The 500-fold lower kcat of D214N than the wild type was consistent with a role in catalysis, as was the 8000-fold lower kcat value of E59A. This was supported by the 57-fold increase in the kcat of E59A upon addition of azide. A complex pH/rate profile was seen for the wild-type and mutant forms of NcFuc, similar to those measured previously for the Sulfolobus Fucosidase. The non-conservative catalytic structure and distinct active site organization reinforce the necessity of structural studies of new Fucosidases.
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the mirror image postulate as a guide to the selection and evaluation of pyrrolidines as α l Fucosidase inhibitors
Carbohydrate Research, 2013Co-Authors: Bridget L Stocker, Stephen G Withers, Seino A K Jongkees, Anna L Winmason, Emma M Dangerfield, Mattie S M TimmerAbstract:Abstract The ability of a series of pyrrolidines to inhibit several glycosidases was investigated. Using Fleet’s ‘mirror-image postulate’, it was proposed that enantiomeric derivatives of 1,4-dideoxy-1,4-imino- d -lyxitol (a known α- d -galactosidase inhibitor) would show inhibitory activity against α- l -Fucosidases. Some modest α- l -Fucosidase inhibitory activity was observed for selected compounds (particularly an aminomethyl pyrrolidine) and it was proposed that better activity could be obtained by modifying the C-2 side chain of the pyrrolidine core. The d - galacto carbamate scaffold also exhibited somewhat selective, albeit modest, α- l -Fucosidase inhibitory activity and may prove to be an interesting scaffold for further development.
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crystal structure of thermotoga maritima alpha l Fucosidase insights into the catalytic mechanism and the molecular basis for fucosidosis
Journal of Biological Chemistry, 2004Co-Authors: Gerlind Sulzenbacher, Stephen G Withers, Christophe Bignon, Takeshi Nishimura, Chris A Tarling, Bernard Henrissat, Yves BourneAbstract:Fucosylated glycoconjugates are involved in numerous biological events, and alpha-l-Fucosidases, the enzymes responsible for their processing, are therefore of crucial importance. Deficiency in alpha-l-Fucosidase activity is associated with fucosidosis, a lysosomal storage disorder characterized by rapid neurodegeneration, resulting in severe mental and motor deterioration. To gain insight into alpha-l-Fucosidase function at the molecular level, we have determined the crystal structure of Thermotoga maritima alpha-l-Fucosidase. This enzyme assembles as a hexamer and displays a two-domain fold, composed of a catalytic (beta/alpha)(8)-like domain and a C-terminal beta-sandwich domain. The structures of an enzyme-product complex and of a covalent glycosyl-enzyme intermediate, coupled with kinetic and mutagenesis studies, allowed us to identify the catalytic nucleophile, Asp(244), and the Bronsted acid/base, Glu(266). Because T. maritima alpha-l-Fucosidase occupies a unique evolutionary position, being far more closely related to the mammalian enzymes than to any other prokaryotic homolog, a structural model of the human enzyme was built to document the structural consequences of the genetic mutations associated with fucosidosis.
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identification of the catalytic nucleophile of the family 29 α l Fucosidase from thermotoga maritima through trapping of a covalent glycosyl enzyme intermediate and mutagenesis
Journal of Biological Chemistry, 2003Co-Authors: Chris A Tarling, Gerlind Sulzenbacher, Christophe Bignon, Bernard Henrissat, Yves Bourne, Stephen G WithersAbstract:Fucose-containing glycoconjugates are key antigenic determinants in many biological processes. A change in expression levels of the enzymes responsible for tailoring these glycoconjugates has been associated with many pathological conditions and it is therefore surprising that little information is known regarding the mechanism of action of these important catabolic enzymes. Thermotoga maritima, a thermophilic bacterium, produces a wide range of carbohydrate-processing enzymes including a 52-kDa alpha-L-Fucosidase that has 38% sequence identity and 56% similarity to human Fucosidases. The catalytic nucleophile of this enzyme was identified to be Asp-224 within the peptide sequence 222WNDMGWPEKGKEDL235 using the mechanism-based covalent inactivator 2-deoxy-2-fluoro-alpha-L-fucosyl fluoride. The 10(4)-fold lower activity (kcat/Km) of the site-directed mutant D224A, and the subsequent rescue of activity upon addition of exogenous nucleophiles, conclusively confirms this assignment. This article presents the first direct identification of the catalytic nucleophile of an alpha-L-Fucosidase, a key step in the understanding of these important enzymes.
Jack A Alhadeff - One of the best experts on this subject based on the ideXlab platform.
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altered isoenzyme patterns of liver α l Fucosidase in cystic fibrosis
Clinical Genetics, 2008Co-Authors: Jack A Alhadeff, Linda Tennant, John S ObrienAbstract:The isoenzyme pattern of alpha-L-Fucosidase was studied by isoelectric focusing in livers from seven patients with cystic fibrosis and in normal and pathological (GM1-gangliosidosis, Type II and Sanfilippo disease) controls. The controls had very reproducible patterns consisting of seven isoenzymes of alpha-L-Fucosidase with the most neutral from (I) representing a small proportion of the total activity. All seven of the cystic fibrosis livers had altered alpha-L-Fucosidase isoenzyme patterns. The chemical relationship of the seven isoenzymes of normal liver alpha-L-Fucosidase was investigated using neuramindase. The five most acidic forms of alpha-L-Fucosidase appear to be related to the most neutral form by sialic acid residues. Since the isoenzymes of liver alpha-L-Fucosidase appear to be related by sialic acid residues, it is possible that the altered alpha-L-Fucosidase isoenzyme patterns found in cystic fibrosis livers may result from aberrant sialylation.
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purification and characterization of plasma membrane associated human sperm α l Fucosidase
Biology of Reproduction, 2003Co-Authors: Sumpars Khunsook, Barry S Bean, Susan Rose Mcgowan, Jack A AlhadeffAbstract:Abstract Detergent and salt extraction studies, as well as cytochemical localization with fluorescein isothiocyanate-bovine serum albumin-l-fucose, have provided further evidence for the plasma membrane association of a novel human sperm, α-l-Fucosidase. This α-l-Fucosidase has been solubilized and purified 8600-fold to high specific activity (35 000 U/mg protein) by affinity chromatography on agarose-C24-fucosylamine. To our knowledge, this is the first report concerning the purification and characterization of a mammalian plasma membrane-associated α-l-Fucosidase. Both SDS-PAGE and Western blot analysis indicated the α-l-Fucosidase is highly purified and contains a single subunit with a molecular mass of 51 kDa. N-glycanase studies indicated the subunit contains N-glycans, and lectin blot analysis detected the presence of mannose, but no terminal galactose or sialic acid residues. Isoelectric focusing indicated the presence of two major α-l-Fucosidase isoforms (pIs 6.5 and 6.7) and a possible minor isof...
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immunocytochemical localization and biochemical characterization of a novel plasma membrane associated neutral ph optimum alpha l Fucosidase from rat testis and epididymal spermatozoa
Biochemical Journal, 1996Co-Authors: Manuel Aviles, Jack A Alhadeff, Irene Abascal, Jose A Martinezmenarguez, M T Castells, Sheri R Skalaban, J BallestaAbstract:1. Immunocytochemical and biochemical techniques have been used to localize and characterize a novel plasma membrane-associated, neutral-pH-optimum alpha-L-Fucosidase from rat spermatozoa. Light and electron microscopy specifically localized the Fucosidase on the plasma membrane of the convex region of the principal segment of testicular and cauda epididymal sperm heads. Immunoreactivity for alpha-L-Fucosidase was also detected in the Golgi apparatus of spermatocytes and spermatids but no immunoreactivity was observed in the acrosome. 2. Fractionation of epididymal sperm homogenates indicated that over 90% of the alpha-L-Fucosidase activity was associated with the 48,000 g pellet. This pellet-associated activity could be solubilized with 0.5 M NaCl but not with 0.5% Triton X-100, suggesting that Fucosidase is peripherally associated with membranes. Sucrose-density-gradient centrifugation of sperm homogenates indicated that Fucosidase was enriched in the plasma membrane-enriched fraction. Analysis of alpha-L-Fucosidase on intact epididymal sperm indicated that the enzyme was active, displayed linear kinetics and had a pH-activity curve (with an optimum near 7) which was comparable to that of Fucosidase from epididymal sperm extracts. These results further suggest that Fucosidase is associated with plasma membranes, and that its active site is accessible to fucoconjugates. Evidence that most of the Fucosidase is associated with the exterior of the plasma membrane came from studies in which intact sperm had Fucosidase activity comparable to that of sperm sonicates, and from studies in which approx. 90% of the Fucosidase activity on intact sperm could be released from the sperm by gentle shaking with 0.5 M NaCl. Isoelectric focusing indicated that the NaCl-solubilized epididymal sperm Fucosidase appears to have one major and one minor isoform with pIs near 7.2 and 5.2, respectively. SDS/PAGE and Western blotting indicated that the NaCl-solubilized extract of epididymal sperm contains two protein bands of 54 and 50 kDa which were highly immunoreactive with the IgG fraction of anti-Fucosidase antibodies. Although the function of the novel sperm Fucosidase is not known, its specific localization to the plasma membrane of the region of the rat sperm head involved in sperm-egg binding and its high enzymic activity at neutral pH on intact sperm suggest that this enzyme may have a role in sperm-egg interactions.
