TY - JOUR
T1 - Glycoprotein constituents of the vitelline coat of Phallusia mammillata (Ascidiacea) with fertilization inhibiting activity
AU - Litscher, Eveline
AU - Honegger, Thomas G.
N1 - Funding Information:
We are grateful to all members of the laboratories of Professor P. Tardent, Professor E. Kubli, and Professor H. Briegel, who supplied materials, technical assistance, and helpful cooperation during the course of this research. We thank Dr. L. Meijer and his group at the “Station Biologique de Roseoff” and the staff and colleagues at the “Laboratoire Arago” for their continuous assistance. Thanks are also due to Professor D. Rast, Dr. L. Hoesch, and Dr. H. Ruffner for providing equipment and advice for GLC analysis, and to Dr. M. Hunziker for carrying out AAA analyses. Dr. V. Vacquier’s valuable advice and encouragement during the late period of this study and Dr. R. Stid-will’s constructive criticism of the manuscript are gratefully acknowledged. This study was supported by grants from the Swiss National Science Foundation (31.9128~87), the Foundation for Scientific Research at the University of Zurich, and the G. and A. Claraz Donation.
PY - 1991/12
Y1 - 1991/12
N2 - Vitelline coats (VCs) of Phallusia mammillata were isolated and purified following homogenization of live eggs in order to investigate the molecular basis of sperm-egg recognition. Clean VCs were partly solubilized by sonication in H2O and the soluble fraction (SFVC), derived from the outer surface of VCs, was used for further characterization. Electrophoretic analyses of radioiodinated VCs revealed that SFVC consists of two major glycoprotein components with apparent average Mr's of 450,000 and 180,000, respectively. The 450,000 Mr component is composed of several charge isomers, whereas the 180,000 Mr component is supposed to consist of two oligomers, both with acidic pI, held together by a disulfide linkage(s). Each of the two components possesses WGA-binding sites as shown by transblotting followed by WGA-peroxidase treatment. The amino acid composition of SFVC was determined after acid hydrolysis and its carbohydrate composition was analyzed and quantified by GLC. GlcNAc and GalNAc were found to predominate with 86% by weight of total sugar content and fucose, mannose, and glucose accounted for the remaining 14%. The susceptibility of SFVC to enzymatic (N-glycosidase F) and chemical (TFMS) deglycosylation as well as to protease (trypsin and chymotrypsin) digestion was investigated. Furthermore, sperm receptor activity of SFVC was tested in a fertilization assay. The fertilization rate decreased in a concentration-dependent manner when sperm were preincubated with SFVC. Additionally, sperm treated with SFVC showed binding for FITC-WGA or WGA-gold at the apical portion of the sperm head. Therefore, we strongly assume that one or both of the identified glycoprotein macromolecules of SFVC are involved in sperm-egg recognition.
AB - Vitelline coats (VCs) of Phallusia mammillata were isolated and purified following homogenization of live eggs in order to investigate the molecular basis of sperm-egg recognition. Clean VCs were partly solubilized by sonication in H2O and the soluble fraction (SFVC), derived from the outer surface of VCs, was used for further characterization. Electrophoretic analyses of radioiodinated VCs revealed that SFVC consists of two major glycoprotein components with apparent average Mr's of 450,000 and 180,000, respectively. The 450,000 Mr component is composed of several charge isomers, whereas the 180,000 Mr component is supposed to consist of two oligomers, both with acidic pI, held together by a disulfide linkage(s). Each of the two components possesses WGA-binding sites as shown by transblotting followed by WGA-peroxidase treatment. The amino acid composition of SFVC was determined after acid hydrolysis and its carbohydrate composition was analyzed and quantified by GLC. GlcNAc and GalNAc were found to predominate with 86% by weight of total sugar content and fucose, mannose, and glucose accounted for the remaining 14%. The susceptibility of SFVC to enzymatic (N-glycosidase F) and chemical (TFMS) deglycosylation as well as to protease (trypsin and chymotrypsin) digestion was investigated. Furthermore, sperm receptor activity of SFVC was tested in a fertilization assay. The fertilization rate decreased in a concentration-dependent manner when sperm were preincubated with SFVC. Additionally, sperm treated with SFVC showed binding for FITC-WGA or WGA-gold at the apical portion of the sperm head. Therefore, we strongly assume that one or both of the identified glycoprotein macromolecules of SFVC are involved in sperm-egg recognition.
UR - http://www.scopus.com/inward/record.url?scp=0026346112&partnerID=8YFLogxK
U2 - 10.1016/0012-1606(91)90272-5
DO - 10.1016/0012-1606(91)90272-5
M3 - Article
C2 - 1660420
AN - SCOPUS:0026346112
SN - 0012-1606
VL - 148
SP - 536
EP - 551
JO - Developmental Biology
JF - Developmental Biology
IS - 2
ER -