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Article: Rabbit sex hormone binding globulin: Primary structure, tissue expression, and structure/function analyses by expression in Escherichia coli

TitleRabbit sex hormone binding globulin: Primary structure, tissue expression, and structure/function analyses by expression in Escherichia coli
Authors
Issue Date1997
PublisherSociety for Endocrinology. The Journal's web site is located at http://joe.endocrinology-journals.org
Citation
Journal Of Endocrinology, 1997, v. 153 n. 3, p. 373-384 How to Cite?
AbstractSex hormone binding globulin (SHBG) is a homodimeric plasma protein found in mammals that binds sex steroids with high affinity and regulates their bioavailability. The protein is identical in structure and properties to the androgen binding protein (ABP) found in the male reproductive tract. We have isolated a 1245-base pair rabbit SHBG cDNA encoding a reading frame for a signal peptide followed by a protein of 367 amino acids, which shares 79.0, 68.1 and 63.2% amino acid identity with the corresponding human, rat and mouse proteins respectively. Northern blot and host-nested PCR analyses indicated that rabbit SHBG is produced from a 1.6 kilobase mRNA in the liver of both sexes and in the testis. The rabbit SHBG cDNA was inserted into pGEX- 1λT for expression of glutathione S-transferase/SHBG fusion protein in Escherichia coli. The bacterial product bound 5α-dihydrotestoterone (DHT) in the same manner as the corresponding protein in serum. The dissociation constants (K(d)) for rabbit and human SHBGs produced in E. coli were 11.1 ± 1.1 nM and 2.1 ± 0.6 nM respectively, and rabbit SHBG formed a less stable protein-steroid complex (t( 1/4 )= 5 min) that human SHBG (t( 1/4 )>60 min). Unlike human SHBG, rabbit SHBG does not bind estradiol with high affinity. To aid in the identification of differences in the sequences of rabbit and human SHBG, which determine species differences in steroid-binding affinity and specificity, chimeras containing the 5'-terminal half of SHBG from one species and 3'-terminal half of SHBG from the other species were constructed and expressed. It was found that the chimeric proteins assumed similar steroid-binding affinity and specificity as the wild-type proteins when the amino (N)-terminal half of SHBG was derived from the same species. Replacement of the carboxyl (C)-terminal half of rabbit SHBG by the corresponding region of the human molecule increased the integrity of its steroid-protein complex. This supports the concept that amino acids within the N-terminal half of SHBG constitute the steroid-binding domain while the C-terminal half of the molecule may provide structural stability to the protein and its steroid-binding site.
Persistent Identifierhttp://hdl.handle.net/10722/84845
ISSN
2015 Impact Factor: 4.498
2015 SCImago Journal Rankings: 1.910
ISI Accession Number ID
References

 

DC FieldValueLanguage
dc.contributor.authorLee, WMen_HK
dc.contributor.authorWong, ASTen_HK
dc.contributor.authorTu, AWKen_HK
dc.contributor.authorCheung, CHen_HK
dc.contributor.authorLi, JCHen_HK
dc.contributor.authorHammond, GLen_HK
dc.date.accessioned2010-09-06T08:57:47Z-
dc.date.available2010-09-06T08:57:47Z-
dc.date.issued1997en_HK
dc.identifier.citationJournal Of Endocrinology, 1997, v. 153 n. 3, p. 373-384en_HK
dc.identifier.issn0022-0795en_HK
dc.identifier.urihttp://hdl.handle.net/10722/84845-
dc.description.abstractSex hormone binding globulin (SHBG) is a homodimeric plasma protein found in mammals that binds sex steroids with high affinity and regulates their bioavailability. The protein is identical in structure and properties to the androgen binding protein (ABP) found in the male reproductive tract. We have isolated a 1245-base pair rabbit SHBG cDNA encoding a reading frame for a signal peptide followed by a protein of 367 amino acids, which shares 79.0, 68.1 and 63.2% amino acid identity with the corresponding human, rat and mouse proteins respectively. Northern blot and host-nested PCR analyses indicated that rabbit SHBG is produced from a 1.6 kilobase mRNA in the liver of both sexes and in the testis. The rabbit SHBG cDNA was inserted into pGEX- 1λT for expression of glutathione S-transferase/SHBG fusion protein in Escherichia coli. The bacterial product bound 5α-dihydrotestoterone (DHT) in the same manner as the corresponding protein in serum. The dissociation constants (K(d)) for rabbit and human SHBGs produced in E. coli were 11.1 ± 1.1 nM and 2.1 ± 0.6 nM respectively, and rabbit SHBG formed a less stable protein-steroid complex (t( 1/4 )= 5 min) that human SHBG (t( 1/4 )>60 min). Unlike human SHBG, rabbit SHBG does not bind estradiol with high affinity. To aid in the identification of differences in the sequences of rabbit and human SHBG, which determine species differences in steroid-binding affinity and specificity, chimeras containing the 5'-terminal half of SHBG from one species and 3'-terminal half of SHBG from the other species were constructed and expressed. It was found that the chimeric proteins assumed similar steroid-binding affinity and specificity as the wild-type proteins when the amino (N)-terminal half of SHBG was derived from the same species. Replacement of the carboxyl (C)-terminal half of rabbit SHBG by the corresponding region of the human molecule increased the integrity of its steroid-protein complex. This supports the concept that amino acids within the N-terminal half of SHBG constitute the steroid-binding domain while the C-terminal half of the molecule may provide structural stability to the protein and its steroid-binding site.en_HK
dc.languageengen_HK
dc.publisherSociety for Endocrinology. The Journal's web site is located at http://joe.endocrinology-journals.orgen_HK
dc.relation.ispartofJournal of Endocrinologyen_HK
dc.rightsJournal of Endocrinology. Copyright © Society for Endocrinology.en_HK
dc.titleRabbit sex hormone binding globulin: Primary structure, tissue expression, and structure/function analyses by expression in Escherichia colien_HK
dc.typeArticleen_HK
dc.identifier.openurlhttp://library.hku.hk:4550/resserv?sid=HKU:IR&issn=0022-0795&volume=153&spage=373&epage=384&date=1997&atitle=Rabbit+sex+hormone+binding+globulin:+primary+structure,+tissue+expression,+and+structure/function+analyses+by+expression+in+Escherichia+colien_HK
dc.identifier.emailLee, WM: hrszlwm@hku.hken_HK
dc.identifier.emailWong, AST: awong1@hkucc.hku.hken_HK
dc.identifier.authorityLee, WM=rp00728en_HK
dc.identifier.authorityWong, AST=rp00805en_HK
dc.description.naturelink_to_subscribed_fulltext-
dc.identifier.pmid9203991-
dc.identifier.scopuseid_2-s2.0-0030923944en_HK
dc.identifier.hkuros22389en_HK
dc.relation.referenceshttp://www.scopus.com/mlt/select.url?eid=2-s2.0-0030923944&selection=ref&src=s&origin=recordpageen_HK
dc.identifier.volume153en_HK
dc.identifier.issue3en_HK
dc.identifier.spage373en_HK
dc.identifier.epage384en_HK
dc.identifier.isiWOS:A1997XD47900005-
dc.publisher.placeUnited Kingdomen_HK
dc.identifier.scopusauthoridLee, WM=24799156600en_HK
dc.identifier.scopusauthoridWong, AST=23987963300en_HK
dc.identifier.scopusauthoridTu, AWK=35874586000en_HK
dc.identifier.scopusauthoridCheung, CH=36902188100en_HK
dc.identifier.scopusauthoridLi, JCH=7410060637en_HK
dc.identifier.scopusauthoridHammond, GL=7202011645en_HK

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