The Hinge Region of Bovine Zona Pellucida Glycoprotein ZP3 Is Involved in the Formation of the Sperm-Binding Active ZP3/ZP4 Complex
The zona pellucida (ZP) surrounds the mammalian oocyte and mediates species-selective sperm-oocyte interactions. Bovine ZP consists of glycoproteins ZP2, ZP3, and ZP4. Neither ZP3 nor ZP4 alone shows inhibitory activity for the binding of sperm to the ZP; however, this activity is seen with the ZP3/...
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doaj-2aa660e1bd964626b5007b7e168bbd4c2020-11-24T21:53:33ZengMDPI AGBiomolecules2218-273X2015-11-01543339335310.3390/biom5043339biom5043339The Hinge Region of Bovine Zona Pellucida Glycoprotein ZP3 Is Involved in the Formation of the Sperm-Binding Active ZP3/ZP4 ComplexKaori Suzuki0Nanami Tatebe1Sayuri Kojima2Ayumi Hamano3Misaki Orita4Naoto Yonezawa5Graduate School of Science, Chiba University, Chiba 263-8522, JapanGraduate School of Science, Chiba University, Chiba 263-8522, JapanGraduate School of Science, Chiba University, Chiba 263-8522, JapanGraduate School of Science, Chiba University, Chiba 263-8522, JapanGraduate School of Science, Chiba University, Chiba 263-8522, JapanGraduate School of Science, Chiba University, Chiba 263-8522, JapanThe zona pellucida (ZP) surrounds the mammalian oocyte and mediates species-selective sperm-oocyte interactions. Bovine ZP consists of glycoproteins ZP2, ZP3, and ZP4. Neither ZP3 nor ZP4 alone shows inhibitory activity for the binding of sperm to the ZP; however, this activity is seen with the ZP3/ZP4 heterocomplex. Here, we constructed a series of bovine ZP3 mutants to identify the ZP4-binding site on ZP3. Each ZP3 mutant was co-expressed with ZP4 using a baculovirus-Sf9 cell expression system and examined for interaction with ZP4 as well as inhibitory activity for sperm-ZP binding. N-terminal fragment Arg-32 to Arg-160 of ZP3 interacted with ZP4 and inhibited sperm-ZP binding, whereas fragment Arg-32 to Thr-155 showed much weaker interaction with ZP4. Mutation of N-glycosylated Asn-146 to Asp in the N-terminal fragment Arg-32 to Glu-178 of ZP3 did not interrupt the interaction of this fragment with ZP4, but it did reduce the inhibitory activity of the complex for sperm-ZP binding. In contrast, mutation of N-glycosylated Asn-124 to Asp did not significantly reduce the activity. Taken together, these results suggest that one of the ZP4 binding sites exists in the flexible hinge region of ZP3 and that the N-glycosylation in this region is involved in the sperm binding.http://www.mdpi.com/2218-273X/5/4/3339zona pellucidafertilizationglycoproteinbaculovirus-Sf9 cell |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Kaori Suzuki Nanami Tatebe Sayuri Kojima Ayumi Hamano Misaki Orita Naoto Yonezawa |
spellingShingle |
Kaori Suzuki Nanami Tatebe Sayuri Kojima Ayumi Hamano Misaki Orita Naoto Yonezawa The Hinge Region of Bovine Zona Pellucida Glycoprotein ZP3 Is Involved in the Formation of the Sperm-Binding Active ZP3/ZP4 Complex Biomolecules zona pellucida fertilization glycoprotein baculovirus-Sf9 cell |
author_facet |
Kaori Suzuki Nanami Tatebe Sayuri Kojima Ayumi Hamano Misaki Orita Naoto Yonezawa |
author_sort |
Kaori Suzuki |
title |
The Hinge Region of Bovine Zona Pellucida Glycoprotein ZP3 Is Involved in the Formation of the Sperm-Binding Active ZP3/ZP4 Complex |
title_short |
The Hinge Region of Bovine Zona Pellucida Glycoprotein ZP3 Is Involved in the Formation of the Sperm-Binding Active ZP3/ZP4 Complex |
title_full |
The Hinge Region of Bovine Zona Pellucida Glycoprotein ZP3 Is Involved in the Formation of the Sperm-Binding Active ZP3/ZP4 Complex |
title_fullStr |
The Hinge Region of Bovine Zona Pellucida Glycoprotein ZP3 Is Involved in the Formation of the Sperm-Binding Active ZP3/ZP4 Complex |
title_full_unstemmed |
The Hinge Region of Bovine Zona Pellucida Glycoprotein ZP3 Is Involved in the Formation of the Sperm-Binding Active ZP3/ZP4 Complex |
title_sort |
hinge region of bovine zona pellucida glycoprotein zp3 is involved in the formation of the sperm-binding active zp3/zp4 complex |
publisher |
MDPI AG |
series |
Biomolecules |
issn |
2218-273X |
publishDate |
2015-11-01 |
description |
The zona pellucida (ZP) surrounds the mammalian oocyte and mediates species-selective sperm-oocyte interactions. Bovine ZP consists of glycoproteins ZP2, ZP3, and ZP4. Neither ZP3 nor ZP4 alone shows inhibitory activity for the binding of sperm to the ZP; however, this activity is seen with the ZP3/ZP4 heterocomplex. Here, we constructed a series of bovine ZP3 mutants to identify the ZP4-binding site on ZP3. Each ZP3 mutant was co-expressed with ZP4 using a baculovirus-Sf9 cell expression system and examined for interaction with ZP4 as well as inhibitory activity for sperm-ZP binding. N-terminal fragment Arg-32 to Arg-160 of ZP3 interacted with ZP4 and inhibited sperm-ZP binding, whereas fragment Arg-32 to Thr-155 showed much weaker interaction with ZP4. Mutation of N-glycosylated Asn-146 to Asp in the N-terminal fragment Arg-32 to Glu-178 of ZP3 did not interrupt the interaction of this fragment with ZP4, but it did reduce the inhibitory activity of the complex for sperm-ZP binding. In contrast, mutation of N-glycosylated Asn-124 to Asp did not significantly reduce the activity. Taken together, these results suggest that one of the ZP4 binding sites exists in the flexible hinge region of ZP3 and that the N-glycosylation in this region is involved in the sperm binding. |
topic |
zona pellucida fertilization glycoprotein baculovirus-Sf9 cell |
url |
http://www.mdpi.com/2218-273X/5/4/3339 |
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