Primate recombinant zona pellucida proteins expressed in Escherichia coli bind to spermatozoa
To delineate the role of individual zona pellucida (ZP) glycoproteins during sperm–oocyte interaction, bonnet monkey (bm; Macaca radiata) ZPA (bmZPA), ZPB (bmZPB), and ZPC (bmZPC) have been cloned without native signal sequence and transmembrane-like domain, and expressed in Escherichia coli. Recomb...
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| Veröffentlicht in: | Journal of reproductive immunology 2002, Vol.53 (1), p.67-77 |
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| creator | Gahlay, Gagandeep K Srivastava, Neelu Govind, Chhabi K Gupta, Satish K |
| description | To delineate the role of individual zona pellucida (ZP) glycoproteins during sperm–oocyte interaction, bonnet monkey (bm;
Macaca radiata) ZPA (bmZPA), ZPB (bmZPB), and ZPC (bmZPC) have been cloned without native signal sequence and transmembrane-like domain, and expressed in
Escherichia coli. Recombinant proteins have been purified from the inclusion bodies in presence of low concentration of chaotropic agent (2 M urea) and high pH (pH 12), and subsequently refolded in presence of oxidized and reduced glutathione. Binding of the recombinant refolded zona proteins to bonnet monkey spermatozoa in an indirect immunofluorescence assay revealed that recombinant bmZPC binds to the head region of the capacitated spermatozoa but does not bind to the acrosome reacted spermatozoa. Recombinant bmZPB binds to the principal segment of the acrosomal cap of capacitated bonnet monkey spermatozoa. After induction of acrosome reaction by calcium ionophore A23187, the binding of recombinant bmZPB shifts to the equator, post-acrosome and midpiece of the spermatozoa. bmZPA binds to the principal segment of capacitated spermatozoa but the binding shifts to the equatorial segment, tip of the inner acrosomal membrane and midpiece in acrosome reacted spermatozoa. These studies suggest that polypeptide backbone is sufficient for the binding of ZPA, ZPB and ZPC to spermatozoa in non-human primates. Further studies with recombinant glycosylated zona proteins will help in delineating the role of carbohydrate moieties for higher affinity binding of the ligand to spermatozoa and subsequent signal transduction pathways. |
| doi_str_mv | 10.1016/S0165-0378(01)00083-3 |
| format | Article |
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Macaca radiata) ZPA (bmZPA), ZPB (bmZPB), and ZPC (bmZPC) have been cloned without native signal sequence and transmembrane-like domain, and expressed in
Escherichia coli. Recombinant proteins have been purified from the inclusion bodies in presence of low concentration of chaotropic agent (2 M urea) and high pH (pH 12), and subsequently refolded in presence of oxidized and reduced glutathione. Binding of the recombinant refolded zona proteins to bonnet monkey spermatozoa in an indirect immunofluorescence assay revealed that recombinant bmZPC binds to the head region of the capacitated spermatozoa but does not bind to the acrosome reacted spermatozoa. Recombinant bmZPB binds to the principal segment of the acrosomal cap of capacitated bonnet monkey spermatozoa. After induction of acrosome reaction by calcium ionophore A23187, the binding of recombinant bmZPB shifts to the equator, post-acrosome and midpiece of the spermatozoa. bmZPA binds to the principal segment of capacitated spermatozoa but the binding shifts to the equatorial segment, tip of the inner acrosomal membrane and midpiece in acrosome reacted spermatozoa. These studies suggest that polypeptide backbone is sufficient for the binding of ZPA, ZPB and ZPC to spermatozoa in non-human primates. Further studies with recombinant glycosylated zona proteins will help in delineating the role of carbohydrate moieties for higher affinity binding of the ligand to spermatozoa and subsequent signal transduction pathways.</description><identifier>ISSN: 0165-0378</identifier><identifier>EISSN: 1872-7603</identifier><identifier>DOI: 10.1016/S0165-0378(01)00083-3</identifier><identifier>PMID: 11730905</identifier><identifier>CODEN: JRIMDR</identifier><language>eng</language><publisher>Shannon: Elsevier Ireland Ltd</publisher><subject>Acrosome - metabolism ; Amino Acid Sequence ; Animals ; Base Sequence ; Biological and medical sciences ; DNA, Complementary - genetics ; E. coli expression ; Egg Proteins - genetics ; Egg Proteins - isolation & purification ; Egg Proteins - metabolism ; Escherichia coli - genetics ; Female ; Fundamental and applied biological sciences. Psychology ; In Vitro Techniques ; Macaca radiata ; Male ; Mammalian reproduction. General aspects ; Membrane Glycoproteins - genetics ; Membrane Glycoproteins - isolation & purification ; Membrane Glycoproteins - metabolism ; Protein Binding ; Receptors, Cell Surface ; Recombinant Proteins - genetics ; Recombinant Proteins - isolation & purification ; Recombinant Proteins - metabolism ; Recombinant zona pellucida glycoproteins ; Sperm binding ; Sperm Capacitation ; Sperm-Ovum Interactions - physiology ; Spermatozoa - metabolism ; Vertebrates: reproduction ; Zona Pellucida Glycoproteins</subject><ispartof>Journal of reproductive immunology, 2002, Vol.53 (1), p.67-77</ispartof><rights>2002 Elsevier Science Ireland Ltd</rights><rights>2002 INIST-CNRS</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c391t-ef286d455076baa5d7739a53f9a04e8502dfdd2827588322e81fbcf10e4e603c3</citedby><cites>FETCH-LOGICAL-c391t-ef286d455076baa5d7739a53f9a04e8502dfdd2827588322e81fbcf10e4e603c3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktohtml>$$Uhttps://www.sciencedirect.com/science/article/pii/S0165037801000833$$EHTML$$P50$$Gelsevier$$H</linktohtml><link.rule.ids>309,310,314,776,780,785,786,3537,4010,4036,4037,23909,23910,25118,27900,27901,27902,65306</link.rule.ids><backlink>$$Uhttp://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=13420770$$DView record in Pascal Francis$$Hfree_for_read</backlink><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/11730905$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Gahlay, Gagandeep K</creatorcontrib><creatorcontrib>Srivastava, Neelu</creatorcontrib><creatorcontrib>Govind, Chhabi K</creatorcontrib><creatorcontrib>Gupta, Satish K</creatorcontrib><title>Primate recombinant zona pellucida proteins expressed in Escherichia coli bind to spermatozoa</title><title>Journal of reproductive immunology</title><addtitle>J Reprod Immunol</addtitle><description>To delineate the role of individual zona pellucida (ZP) glycoproteins during sperm–oocyte interaction, bonnet monkey (bm;
Macaca radiata) ZPA (bmZPA), ZPB (bmZPB), and ZPC (bmZPC) have been cloned without native signal sequence and transmembrane-like domain, and expressed in
Escherichia coli. Recombinant proteins have been purified from the inclusion bodies in presence of low concentration of chaotropic agent (2 M urea) and high pH (pH 12), and subsequently refolded in presence of oxidized and reduced glutathione. Binding of the recombinant refolded zona proteins to bonnet monkey spermatozoa in an indirect immunofluorescence assay revealed that recombinant bmZPC binds to the head region of the capacitated spermatozoa but does not bind to the acrosome reacted spermatozoa. Recombinant bmZPB binds to the principal segment of the acrosomal cap of capacitated bonnet monkey spermatozoa. After induction of acrosome reaction by calcium ionophore A23187, the binding of recombinant bmZPB shifts to the equator, post-acrosome and midpiece of the spermatozoa. bmZPA binds to the principal segment of capacitated spermatozoa but the binding shifts to the equatorial segment, tip of the inner acrosomal membrane and midpiece in acrosome reacted spermatozoa. These studies suggest that polypeptide backbone is sufficient for the binding of ZPA, ZPB and ZPC to spermatozoa in non-human primates. Further studies with recombinant glycosylated zona proteins will help in delineating the role of carbohydrate moieties for higher affinity binding of the ligand to spermatozoa and subsequent signal transduction pathways.</description><subject>Acrosome - metabolism</subject><subject>Amino Acid Sequence</subject><subject>Animals</subject><subject>Base Sequence</subject><subject>Biological and medical sciences</subject><subject>DNA, Complementary - genetics</subject><subject>E. coli expression</subject><subject>Egg Proteins - genetics</subject><subject>Egg Proteins - isolation & purification</subject><subject>Egg Proteins - metabolism</subject><subject>Escherichia coli - genetics</subject><subject>Female</subject><subject>Fundamental and applied biological sciences. Psychology</subject><subject>In Vitro Techniques</subject><subject>Macaca radiata</subject><subject>Male</subject><subject>Mammalian reproduction. General aspects</subject><subject>Membrane Glycoproteins - genetics</subject><subject>Membrane Glycoproteins - isolation & purification</subject><subject>Membrane Glycoproteins - metabolism</subject><subject>Protein Binding</subject><subject>Receptors, Cell Surface</subject><subject>Recombinant Proteins - genetics</subject><subject>Recombinant Proteins - isolation & purification</subject><subject>Recombinant Proteins - metabolism</subject><subject>Recombinant zona pellucida glycoproteins</subject><subject>Sperm binding</subject><subject>Sperm Capacitation</subject><subject>Sperm-Ovum Interactions - physiology</subject><subject>Spermatozoa - metabolism</subject><subject>Vertebrates: reproduction</subject><subject>Zona Pellucida Glycoproteins</subject><issn>0165-0378</issn><issn>1872-7603</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2002</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFkU1PxCAQhonR6PrxEzRcNHqoDqUUejLG-JWYaKIeDWFhGjHdskLXqL9e1t3o0QtweGaY9xlCdhkcM2D1yUM-RAFcqkNgRwCgeMFXyIgpWRayBr5KRr_IBtlM6RWASWjYOtlgTHJoQIzI8330EzMgjWjDZOx70w_0K_SGTrHrZta7_IphQN8nih_TiCmho76nF8m-YPT2xRtqQ-dpLnZ0CDRNMeaW4SuYbbLWmi7hzvLeIk-XF4_n18Xt3dXN-dltYXnDhgLbUtWuEgJkPTZGOCl5YwRvGwMVKgGla50rVSmFUrwsUbF2bFsGWGFOavkWOVj0zaO-zTANeuKTzQFMj2GWtGScNxWrMygWoI0hpYitns7zx0_NQM-96h-vei5NA9M_XjXPdXvLD2bjCbq_qqXIDOwvAZOs6dpoeuvTH8erEqSEzJ0uOMw63j1GnazH3qLzeQODdsH_M8o31euVRA</recordid><startdate>2002</startdate><enddate>2002</enddate><creator>Gahlay, Gagandeep K</creator><creator>Srivastava, Neelu</creator><creator>Govind, Chhabi K</creator><creator>Gupta, Satish K</creator><general>Elsevier Ireland Ltd</general><general>Elsevier Science</general><scope>IQODW</scope><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7X8</scope></search><sort><creationdate>2002</creationdate><title>Primate recombinant zona pellucida proteins expressed in Escherichia coli bind to spermatozoa</title><author>Gahlay, Gagandeep K ; Srivastava, Neelu ; Govind, Chhabi K ; Gupta, Satish K</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c391t-ef286d455076baa5d7739a53f9a04e8502dfdd2827588322e81fbcf10e4e603c3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2002</creationdate><topic>Acrosome - metabolism</topic><topic>Amino Acid Sequence</topic><topic>Animals</topic><topic>Base Sequence</topic><topic>Biological and medical sciences</topic><topic>DNA, Complementary - genetics</topic><topic>E. coli expression</topic><topic>Egg Proteins - genetics</topic><topic>Egg Proteins - isolation & purification</topic><topic>Egg Proteins - metabolism</topic><topic>Escherichia coli - genetics</topic><topic>Female</topic><topic>Fundamental and applied biological sciences. Psychology</topic><topic>In Vitro Techniques</topic><topic>Macaca radiata</topic><topic>Male</topic><topic>Mammalian reproduction. General aspects</topic><topic>Membrane Glycoproteins - genetics</topic><topic>Membrane Glycoproteins - isolation & purification</topic><topic>Membrane Glycoproteins - metabolism</topic><topic>Protein Binding</topic><topic>Receptors, Cell Surface</topic><topic>Recombinant Proteins - genetics</topic><topic>Recombinant Proteins - isolation & purification</topic><topic>Recombinant Proteins - metabolism</topic><topic>Recombinant zona pellucida glycoproteins</topic><topic>Sperm binding</topic><topic>Sperm Capacitation</topic><topic>Sperm-Ovum Interactions - physiology</topic><topic>Spermatozoa - metabolism</topic><topic>Vertebrates: reproduction</topic><topic>Zona Pellucida Glycoproteins</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Gahlay, Gagandeep K</creatorcontrib><creatorcontrib>Srivastava, Neelu</creatorcontrib><creatorcontrib>Govind, Chhabi K</creatorcontrib><creatorcontrib>Gupta, Satish K</creatorcontrib><collection>Pascal-Francis</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>MEDLINE - Academic</collection><jtitle>Journal of reproductive immunology</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Gahlay, Gagandeep K</au><au>Srivastava, Neelu</au><au>Govind, Chhabi K</au><au>Gupta, Satish K</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Primate recombinant zona pellucida proteins expressed in Escherichia coli bind to spermatozoa</atitle><jtitle>Journal of reproductive immunology</jtitle><addtitle>J Reprod Immunol</addtitle><date>2002</date><risdate>2002</risdate><volume>53</volume><issue>1</issue><spage>67</spage><epage>77</epage><pages>67-77</pages><issn>0165-0378</issn><eissn>1872-7603</eissn><coden>JRIMDR</coden><abstract>To delineate the role of individual zona pellucida (ZP) glycoproteins during sperm–oocyte interaction, bonnet monkey (bm;
Macaca radiata) ZPA (bmZPA), ZPB (bmZPB), and ZPC (bmZPC) have been cloned without native signal sequence and transmembrane-like domain, and expressed in
Escherichia coli. Recombinant proteins have been purified from the inclusion bodies in presence of low concentration of chaotropic agent (2 M urea) and high pH (pH 12), and subsequently refolded in presence of oxidized and reduced glutathione. Binding of the recombinant refolded zona proteins to bonnet monkey spermatozoa in an indirect immunofluorescence assay revealed that recombinant bmZPC binds to the head region of the capacitated spermatozoa but does not bind to the acrosome reacted spermatozoa. Recombinant bmZPB binds to the principal segment of the acrosomal cap of capacitated bonnet monkey spermatozoa. After induction of acrosome reaction by calcium ionophore A23187, the binding of recombinant bmZPB shifts to the equator, post-acrosome and midpiece of the spermatozoa. bmZPA binds to the principal segment of capacitated spermatozoa but the binding shifts to the equatorial segment, tip of the inner acrosomal membrane and midpiece in acrosome reacted spermatozoa. These studies suggest that polypeptide backbone is sufficient for the binding of ZPA, ZPB and ZPC to spermatozoa in non-human primates. Further studies with recombinant glycosylated zona proteins will help in delineating the role of carbohydrate moieties for higher affinity binding of the ligand to spermatozoa and subsequent signal transduction pathways.</abstract><cop>Shannon</cop><pub>Elsevier Ireland Ltd</pub><pmid>11730905</pmid><doi>10.1016/S0165-0378(01)00083-3</doi><tpages>11</tpages></addata></record> |
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| subjects | Acrosome - metabolism Amino Acid Sequence Animals Base Sequence Biological and medical sciences DNA, Complementary - genetics E. coli expression Egg Proteins - genetics Egg Proteins - isolation & purification Egg Proteins - metabolism Escherichia coli - genetics Female Fundamental and applied biological sciences. Psychology In Vitro Techniques Macaca radiata Male Mammalian reproduction. General aspects Membrane Glycoproteins - genetics Membrane Glycoproteins - isolation & purification Membrane Glycoproteins - metabolism Protein Binding Receptors, Cell Surface Recombinant Proteins - genetics Recombinant Proteins - isolation & purification Recombinant Proteins - metabolism Recombinant zona pellucida glycoproteins Sperm binding Sperm Capacitation Sperm-Ovum Interactions - physiology Spermatozoa - metabolism Vertebrates: reproduction Zona Pellucida Glycoproteins |
| title | Primate recombinant zona pellucida proteins expressed in Escherichia coli bind to spermatozoa |
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