title: β-hexosaminidase from Xenopus laevis eggs and oocytes: from gene to immunochemical characterization.
abstract: Glycosidases are present both in sperm and eggs in vertebrates and have been associated with different fertilization steps as gamete binding, egg coat penetration, and polyspermy prevention. In this manuscript, we have analyzed the activity of different glycosidases of Xenopus laevis eggs. The main activity corresponded to N-acetyl-β-D-glucosaminidase (Hex), which was reported to participate both in gamete binding and polyspermy prevention among phylogenetically distant animals. We have raised homologous antibodies against a recombinant N-terminal fragment of a X. laevis Hex, and characterized egg's Hex both by Western blot and immunohistochemical assays. Noteworthy, Hex was mainly localized to the cortex of animal hemisphere of full-grown oocytes and oviposited eggs, and remained unaltered after fertilization. Hex is constituted by different pair arrangements of two subunits (α and β), giving rise to three possible Hex isoforms: A (αβ), B (ββ), and S (αα). However, no information was available regarding molecular identity of Hex in amphibians. We present for the first time the primary sequences of two isoforms of X. laevis Hex. Interestingly, our results suggest that α- and β-like subunits that constitute Hex isoforms could be synthesized from a same gene in Xenopus, by alternative exon use. This finding denotes an evolutionary divergence with mammals, where α and β Hex subunits are synthesized from different genes on different chromosomes.
authors: Instituto de Biología Molecular y Celular de Rosario (CONICET-UNR) and Área Biología, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Rosario S2002LRK, Argentina.
authors: Enrique S
authors: ES
authors: Morales
authors: Darío
authors: D
authors: Krapf
authors: Pablo E
authors: PE
authors: Botta
authors: Marcelo O
authors: MO
authors: Cabada
authors: Silvia E
authors: SE
authors: Arranz
mesh_terms: D000118:Acetylglucosaminidase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D020134:Catalytic Domain; D003001:Cloning, Molecular; D017124:Conserved Sequence; D018076:DNA, Complementary / Q000235:genetics / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D057075:Enzyme Assays; D005091:Exons; D005260:Female; D007150:Immunohistochemistry / Q000379:methods*; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009865:Oocytes / Q000201:enzymology*; D010063:Ovum / Q000201:enzymology*; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D029867:Xenopus Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D014982:Xenopus laevis / Q000235:genetics / Q000378:metabolism*
chemical_list: D018076:DNA, Complementary; D007527:Isoenzymes; D011994:Recombinant Proteins; D029867:Xenopus Proteins; D000118:Acetylglucosaminidase