Molecular cloning and sequence analysis of an ascidian egg β‐N‐acetylhexosaminidase with a potential role in fertilization

Abstract

␤ -N-Acetylhexosaminidase, which is found almost ubiquitously in sperm of invertebrates and vertebrates, supposedly mediates a carbohydrate-based transient sperm-egg coat binding. In ascidians and mammals, ␤ -hexosaminidase released at fertilization from eggs has been proposed to modify sperm receptor glycoproteins of the egg envelope, thus setting up a block to polyspermy. Previously, it was shown that in potential sperm receptor glycoproteins of the ascidian Phallusia mammillata , N-acetylglucosamine is the prevailing glycoside residue and that the egg harbors three active molecular forms of ␤ -hexosaminidase. In the present study, P. mammillata ␤ -hexosaminidase cDNA was isolated from an ovarian cDNA library and characterized. The deduced amino acid sequence showed a high similarity with other known ␤ -hexosaminidases; however, P. mammillata ␤ -hexosaminidase had a unique potential N-glycosylation site. A phylogenetic analysis suggested that P. mammillata ␤ -hexosaminidase developed independently after having branched off from the common ancestor gene of the chordate enzyme before two isoforms of the mammalian enzyme appeared. In situ hybridization revealed stage-specific expression of ␤ -hexosaminidase mRNA during oogenesis in the oocyte and in the accessory test and follicle cells. This suggests that the three egg ␤ -hexosaminidase forms are specific for the oocyte, test cells and follicle cells.

Identification and expression profiling of Ceratitis capitata genes coding for β-hexosaminidases

“…It has been demonstrated, both in vertebrates and invertebrates, that sperm glycosidases and glycosyltransferases are the first receptors for the carbohydrates of the egg surface in early gamete recognition (Martinez et al, 2000;Mengerink and Vacquier, 2001;Koyanagi and Honegger, 2003;Zitta et al, 2006;Lyng and Shur, 2007). It is known that several other proteins of the sperm surface, such as proteases, tyrosine kinases and phospholipases, may also function as receptors for the egg coat(s) in mammals (Mengerink and Vacquier, 2001;Wassarman et al, 2001;Miller and Pitnick, 2002;Talbot et al, 2003;Shur et al, 2006).…”

Drosophila sperm surface alpha-l-fucosidase interacts with the egg coats through its core fucose residues

“…The mechanism of recognition and binding between gametes is multiphase and involves several components and, sometimes, the interactions of carbohydrate-binding proteins of spermatozoa to oligosaccharide chains of the egg (Tulsiani et al, 1997;Martinez et al, 2000;Miranda et al, 2000;Mengerink and Vacquier, 2001;Koyanagi and Honegger, 2003;Venditti et al, 2010;Clark, 2013Clark, , 2014. Proteinecarbohydrate complementarity appears to be conserved in fertilization processes throughout the whole evolutionary scale (Dale, 1991).…”

“…It has been well documented that fertilization relies primarily on carbohydrate-mediated events in both vertebrate and invertebrate organisms (Dale, 1991;Tulsiani et al, 1997;Martinez et al, 2000;Miranda et al, 2000;Mengerink and Vacquier, 2001;Koyanagi and Honegger, 2003;Venditti et al, 2010;Clark, 2013Clark, , 2014. Gamete binding in many species has been proposed to involve interactions between proteins on sperm and glycoconjugates on the egg surface (Mengerink and Vacquier, 2001;Evans, 2012;Clark, 2013Clark, , 2014.…”

The ascidian egg envelope in fertilization: structural and molecular features

“…Within seconds after fertilization an increased β-hexosaminidase activity is present in the surrounding seawater (Lambert, 1986;Lambert, 1989;Matsuura et al, 1993;McDougall et al, 1995). However, the situation is complicated by the fact that sperm, eggs, test cells (Koyanagi and Honegger, 2003) and the follicle cells (Honegger, 1992) contain β-hexosaminidase. The supernatant of separate batches of sperm or eggs or isolated FCs always exhibits glycosidase activity.…”

“…Although approaches to purify and characterize egg glycosidase were successful, isolation of an appropriate amount of enzyme for tests and the production of specific antibodies failed (Eisenhut, 2001). To circumvent this problem, we started a molecular approach by cloning β-hexosaminidase from P. mammillata (Koyanagi and Honegger, 2003) followed by the preparation of a recombinant GFP-fusion protein. This protein exhibits specific binding to the FCs (Fig.…”