Characterization of the sperm receptor for acrosome reaction-inducing substance of the starfish, Asterias amurensis.
Acrosome reaction-inducing substance (ARIS) in the jelly coat of starfish eggs is a highly sulfated proteoglycan-like molecule of an apparent molecular size over 10(4) kDa and plays a pivotal role in the induction of acrosome reaction in homologous spermatozoa. It is known in Asterias amurensis that ARIS binds to a restricted area of the anterior portion of sperm head, and that a glycan fragment of ARIS, named Fragment 1, consisting of 10 repeats or so of a pentasaccharide unit retains the biological activity of ARIS to an appreciable extent. In this report, we have shown the binding of Fragment 1, a relatively small pure glycan fragment of ARIS, to the putative ARIS receptor on the sperm surface by three independent methods. First, the specific binding of P-ARIS to isolated sperm membranes was monitored in real-time by using a surface plasmon resonance detector, namely a Biacore sensor system. The specific and quantitative binding of Fragment 1 to the intact sperm and to isolated sperm membranes was similarly monitored. Secondly, the binding of 125I-labeled Fragment 1 to the intact sperm was stoichiometrically measured, for which we had developed a unique procedure for radioiodination of saccharide chains. It is found that Fragment 1 competes with P-ARIS for the binding to ARIS-receptor, suggesting that Fragment 1 is a useful ligand in the search for ARIS receptor protein(s). Thirdly, the putative receptor molecules were specifically labeled by using Fragment 1 as a ligand for photoaffinity crosslink technique. Taking these results into account, we conclude that starfish sperm have the ARIS receptor, which consists most probably of 50 to 60 kDa proteins, of reasonably high affinity (for Fragment 1, Kd = 15 microM, Bmax = 8.4 x 10(4) per cell).
Kawamura
Mayu
M
Department of Bioscience, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta, Yokohama 226-8501.
Matsumoto
Midori
M
Hoshi
Motonori
M
D000818:Animals; D015374:Biosensing Techniques; D005260:Female; D006023:Glycoproteins / Q000378:metabolism*; D036341:Intercellular Signaling Peptides and Proteins; D007457:Iodine Radioisotopes; D008024:Ligands; D008297:Male; D015394:Molecular Structure; D010446:Peptide Fragments / Q000302:isolation & purification / Q000378:metabolism; D019920:Photoaffinity Labels; D011485:Protein Binding; D011956:Receptors, Cell Surface / Q000302:isolation & purification / Q000378:metabolism*; D013077:Sperm Head / Q000378:metabolism; D013094:Spermatozoa / Q000378:metabolism*; D013215:Starfish / Q000502:physiology*
C067898:ARIS protein, Asterias amurensis; D006023:Glycoproteins; D036341:Intercellular Signaling Peptides and Proteins; D007457:Iodine Radioisotopes; D008024:Ligands; D010446:Peptide Fragments; D019920:Photoaffinity Labels; D011956:Receptors, Cell Surface