Biochemical characterization of inner sugar chains of acrosome reaction-inducing substance in jelly coat of starfish eggs.

Abstract

The inception of the acrosome reaction (AR) in the starfish Asterias amurensis is perceived to be strongly associated with sulfated polysaccharide chains derived from an extremely large proteoglycan-like molecule called AR-inducing substance (ARIS), in which one of the sugar fragments, named fragment 1 (Fr. 1), was composed of the repeating units of [-->4]-beta-D-Xylp-(1-->3)-alpha-D-Galp-(1-->3)-alpha-L-Fucp-4 (SO3-)-(1-->3)- alpha-L-Fucp-4(SO3-)-(1-->4)-alpha-L-Fucp-(1-->)n. In the current study, this sugar chain is inferred to link to the peptide part by O-glycosidic linkage through a sugar chain with different structural features from Fr. 1. This inner sugar portion of ARIS was isolated as Fr. 2 from the sonicated products of pronase digest of ARIS. Fr. 2, which retains AR-inducing activity to an admirable extent and has an apparent molecular size of 400 kDa, is composed of Gal, Xyl, Fuc, GalNAc, and GlcNAc in a molar ratio of 5:1:5:4:2 with O-sulfate substitutions at Gal-4, Gal-2, Gal-2,3 and Gal-2,4 (disulfated), Fuc-4, and GlcNAc-6. The study of Fr. 2 revealed that the major portion of the inner sugar chain of ARIS is composed of the heptasaccharide units of -->3)-Galp-(1-->3)-Fucp-(1-->3)-Galp-(1-->4)-GalNAcp-(1-->4)-GlcNAcp-6(SO3-)-(1-->6)-Galp-4(SO3-)-(1-->4)-GalNAcp-(1-->. This new structure of inner sugar chains of ARIS is elucidated by using electrospray ionization MS along with tandem mass analysis, sugar composition analysis, and methylation analysis of the sugar fragments obtained by acid-catalyzed resin-based partial hydrolysis of Fr. 2. Furthermore, this study corroborates that the sulfate groups are solely liable to the anionic character of ARIS, which ought to be present in the sugar chains of ARIS for its biological activity.