Purification and properties of N‐acetylglucosaminidase from eggs of the ascidian, Halocynthia roretzi

Abstract

In several ascidians, beta-D-N-acetylglucosaminidase (GlcNAcase) released from eggs following fertilization is proposed to play a key role in polyspermy block through its binding to the sperm receptor on the vitelline coat [C. C. Lambert (1989) Development 105, 415-420]. In the ascidian Halocynthia roretzi, GlcNAc-specific lectins inhibited the fertilization most strongly among various lectins. Furthermore, GlcNAcase activity was released from the eggs in response to the egg activation. The GlcNAcase was purified from the eggs to apparent homogeneity by chromatographies on DEAE-Toyopearl, SP-Toyopearl, Toyopearl HW-65, and Mono S. The purified enzyme gave a single band on isoelectric focusing with an isoelectric point of 7.0. It gave two bands on SDS/PAGE: the molecular masses of the bands were estimated to be 65 kDa/66 kDa, and 84 kDa/85 kDa under reducing/non-reducing conditions, respectively. The two bands were found to converge to a single band of 56 kDa after deglycosylation, which suggests microheterogeneity in the sugar moiety. The enzyme showed an oligomeric structure with an apparent molecular mass of 520 kDa, estimated by gel filtration. The optimum pH of the activity was around 4.5. The enzyme hydrolyzed both 4-methylumbelliferyl-GlcNAc and 4-methylumbelliferyl-GalNAc, suggesting that it should be characterized as a beta-D-N-acetylhexosaminidase, with Km values of 1.2 mM and 0.52 mM, respectively. The purified enzyme was found to be capable of binding to the vitelline coat in a GlcNAc-specific manner. Immunoblot analysis using antibody raised against the purified GlcNAcase revealed that the enzyme itself is released from the eggs upon fertilization. Thus, the GlcNAcase purified in this study is released from eggs following fertilization and bound to the vitelline coat in order to function in the polyspermy blocking mechanism.