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Glycobiology Advance Access published online on April 17, 2003
Glycobiology, doi:10.1093/glycob/cwg070
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Submitted on January 18, 2003
Revised on March 12, 2003
Accepted on March 13, 2003

© 2003 Oxford University Press

ORIGINAL ARTICLES

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

H.M.M. Jayantha Gunaratne 1, Tohru Yamagaki 2, Midori Matsumoto 3, Motonori Hoshi 3*

1 Department of Biological Science, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259, Nagatsuta, Midori-ku, Yokohama, Japan
2 Department of Chemistry, School of Science, The University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo, Japan
3 Laboratory of Developmental and Reproductive Biology, Department of Biosciences and Informatics, Keio University, 3-14-1, Hiyoshi, Kohoku-ku, Yokohama

* To whom correspondence should be addressed. E-mail: hoshim{at}bio.keio.ac.jp.

Abstract

The inception of 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 acrosome reaction-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, (Koyota et al., 1996). 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 Fragment 2 (Fr. 2) from the sonicated products of pronase digest of ARIS. Fr. 2, which retains AR inducing activity to an admirable extent, having an apparent molecular size of 400kDa, 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 (di-sulfated), 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 absolutely new structure of inner sugar chains of ARIS was elucidated by using electrospray ionization mass spectrometry along with tandem mass analysis, sugar composition and methylation analysis of the sugar fragments obtained by acid-catalyzed resin-based partial hydrolysis of Fr. 2. Further, this study corroborated 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.


Acrosome Reaction, ARIS, CID MS-MS, Egg Jelly Coat, Sulfated Glycans
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