Structure of the Main Saccharide Chain in the Acrosome Reaction-inducing Substance of the Starfish, Asterias amurensis

Abstract

The structure of the main saccharide chain of the acrosome reaction-inducing substance in the egg jelly coat of the starfish, Asterias amurensis, is composed of the following pentasaccharide repeating units (Structure I). A polymer consisting of 10 -11 repeating units has been observed to induce the acrosome reaction in starfish sperm at high calcium concentrations. STRUCTURE IThe identities and linkage positions of constituent sugars were established using sugar, methylation, and sulfate analyses together with one-and two-dimensional nmr spectroscopy. The structure was supported by the data obtained for desulfation products and the Smith degradation of the polysaccharide.The acrosome reaction is a prerequisite for fertilization in various animals including mammals. In starfish, three components of the egg jelly, namely a highly sulfated glycoprotein of an extremely large molecular size named acrosome reactioninducing substance (ARIS), 1 a steroid saponin named Co-ARIS, and a sperm-activating peptide (asterosap) are responsible for triggering the acrosome reaction (1). Three saponins consisting of a sulfated steroid and a pentasaccharide chain (Co-ARIS 1-3) were isolated from the egg jelly and structurally characterized (2). A mixture of glutamine-rich peptides consisting of 34 amino acid residues was isolated as asterosap, and the structures of 11 of them were determined (3). ARIS alone induces the acrosome reaction only in high calcium or high pH seawater, whereas either Co-ARIS or asterosap is also required in normal seawater (3)(4)(5)23). A tripartite of ARIS, Co-ARIS, and asterosap provides the best condition for the induction of the acrosome reaction in normal seawater. A Pronase digest of ARIS (P-ARIS) shows similar activity to that of ARIS (23).Previous work in our laboratory resulted in the isolation (as ARIS) of a sulfated glycoprotein of an apparent molecular mass of 10 7 daltons (23). Recent work involved isolation of a trisaccharide Xyl133Gal13(SO 3 Ϫ )3,4Fuc from P-ARIS after mild acid hydrolysis (6). Since the protein content of ARIS was reduced by about 50% by the Pronase digestion, which made it easier to handle in comparison with ARIS, P-ARIS was used as the starting material in this analysis. This study was carried out in an attempt to understand the structure-activity relationship of ARIS, and we now report the structural study of the main saccharide chain of P-ARIS. EXPERIMENTAL PROCEDURESGeneral Methods-Water was purified with a Milli-Q reagent water system (Millipore Corp.). Sample concentrations were performed under diminished pressure at Ͻ40°C or in a stream of nitrogen. For gas-liquid chromatography, a Shimadzu GC-7A instrument fitted with a flame ionization detector was used. Gas-liquid chromatography-mass spectrometry was performed on a Shimadzu GC-14A instrument coupled with a Shimadzu GCMS-QP2000A instrument. A Dionex HPAEC-PAD was used to determine sugar compositions. Alditol acetates and partially O-methylated alditol acetates were analyzed on a HP-5 capillary column using a temp...

Characterization of the Sperm Receptor for Acrosome Reaction-Inducing Substance of the Starfish, Asterias Amurensis

“…We have recently found that sugar chain fragments liberated from ARIS or P-ARIS by ultra-sonication retains the biological activity of ARIS to an appreciable extent. The fragments are composed of 10 repeats or so of the following pentasaccharide unit and collectively named Fragment 1 (Koyota et al ., 1997):…”

Sulfated O‐linked glycans of the vitelline coat as ligands in gamete interaction in the ascidian, Halocynthia roretzi

“…Therefore, besides sulfation per se, the proper spacing of sulfate groups seems essential for biological activity. A similar situation is suggested for ARIS (Koyota et al 1997). Also, as has been pointed out previously (Dhume & Lennarz 1995), sulfate groups might contribute to the unique properties of glycans and could probably amplify their recognition specificity.…”

“…While Xyl is sometimes found at terminal positions (both reducing and non-reducing) of glycans from animal glycoproteins, the presence of inner Xyl residues, deduced here from methylation analysis data, is rather unusual. Recently described acrosome reaction-inducing substance (ARIS) of the starfish Asterias amurensis is another example of glycan involved in fertilization and containing inner Xyl residues (Koyota et al 1997). Also, there have been a few reports of the occurrence of Ara or Rha in animal glycoproteins.…”

High Molecular Mass Egg Fucose Sulfate Polymer Is Required for Opening Both Ca2+ Channels Involved in Triggering the Sea Urchin Sperm Acrosome Reaction

“…In starfish, the giant pentasaccharide repeat polymer of EJ contains two sulfate groups per repeat. Solvolysis of the polymer shows that the loss of one sulfate per repeat greatly decreases its ability to induce the AR of starfish sperm (33).…”