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Article: Selective removal of glycosylation sites from sex hormone-binding globulin by site-directed mutagenesis

TitleSelective removal of glycosylation sites from sex hormone-binding globulin by site-directed mutagenesis
Authors
Issue Date1992
PublisherThe Endocrine Society. The Journal's web site is located at http://endo.endojournals.org
Citation
Endocrinology, 1992, v. 131 n. 5, p. 2331-2336 How to Cite?
AbstractSex hormone-binding globulin (SHBG) is a homodimeric plasma glycoprotein with high affinity for sex steroid hormones. It contains two N-linked carbohydrate chains and one O-linked oligosaccharide per subunit, but their functional significance is not known. Site-directed mutagenesis of a human SHBG cDNA has enabled us to selectively disrupt the known glycosylation sites individually and in various combinations. The mutant cDNAs were expressed in Chinese hamster ovary (CHO) cells, and it was found that the presence of carbohydrates is not an absolute requirement for the secretion of SHBG from these cells, but the absence of both N-linked oligosaccharides reduced the amount of SHBG in the culture medium. In addition, the affinity and specificity of SHBG for steroid ligands was unaffected by the lack of one or more carbohydrate chains. Proportionally greater amounts (26-31%) of the mutants lacking a single N-linked carbohydrate chain failed to interact with Concanavalin-A (Con-A) compared to normal SHBG produced by CHO cells (15%). Western analysis demonstrated that both consensus sites for N-glycosylation are used and that the typical heavy [mol wt (M(r)), ~51,000] and light (M(r), ~47,000) subunit size-heterogeneity was maintained regardless of the absence of an O-linked carbohydrate at residue 7. Furthermore, the SHBG mutants containing only one N-linked oligosaccharide comprise only a single subunit with a M(r) of approximately 47,000. This suggests that the heavy subunit contains two N-linked oligosaccharides, while only one of these sites is used on the light subunit. The M(r) of the various SHBG mutants were also examined by gel filtration, and this indicated that they are all produced as homodimers and that carbohydrates are not involved in subunit association.
Persistent Identifierhttp://hdl.handle.net/10722/178524
ISSN
2015 Impact Factor: 4.159
2015 SCImago Journal Rankings: 2.363
ISI Accession Number ID

 

DC FieldValueLanguage
dc.contributor.authorBocchinfuso, WPen_US
dc.contributor.authorMa, KLen_US
dc.contributor.authorLee, WMen_US
dc.contributor.authorWarmelsRodenhiser, Sen_US
dc.contributor.authorHammond, GLen_US
dc.date.accessioned2012-12-19T09:48:12Z-
dc.date.available2012-12-19T09:48:12Z-
dc.date.issued1992en_US
dc.identifier.citationEndocrinology, 1992, v. 131 n. 5, p. 2331-2336en_US
dc.identifier.issn0013-7227en_US
dc.identifier.urihttp://hdl.handle.net/10722/178524-
dc.description.abstractSex hormone-binding globulin (SHBG) is a homodimeric plasma glycoprotein with high affinity for sex steroid hormones. It contains two N-linked carbohydrate chains and one O-linked oligosaccharide per subunit, but their functional significance is not known. Site-directed mutagenesis of a human SHBG cDNA has enabled us to selectively disrupt the known glycosylation sites individually and in various combinations. The mutant cDNAs were expressed in Chinese hamster ovary (CHO) cells, and it was found that the presence of carbohydrates is not an absolute requirement for the secretion of SHBG from these cells, but the absence of both N-linked oligosaccharides reduced the amount of SHBG in the culture medium. In addition, the affinity and specificity of SHBG for steroid ligands was unaffected by the lack of one or more carbohydrate chains. Proportionally greater amounts (26-31%) of the mutants lacking a single N-linked carbohydrate chain failed to interact with Concanavalin-A (Con-A) compared to normal SHBG produced by CHO cells (15%). Western analysis demonstrated that both consensus sites for N-glycosylation are used and that the typical heavy [mol wt (M(r)), ~51,000] and light (M(r), ~47,000) subunit size-heterogeneity was maintained regardless of the absence of an O-linked carbohydrate at residue 7. Furthermore, the SHBG mutants containing only one N-linked oligosaccharide comprise only a single subunit with a M(r) of approximately 47,000. This suggests that the heavy subunit contains two N-linked oligosaccharides, while only one of these sites is used on the light subunit. The M(r) of the various SHBG mutants were also examined by gel filtration, and this indicated that they are all produced as homodimers and that carbohydrates are not involved in subunit association.en_US
dc.languageengen_US
dc.publisherThe Endocrine Society. The Journal's web site is located at http://endo.endojournals.orgen_US
dc.relation.ispartofEndocrinologyen_US
dc.subject.meshAnimalsen_US
dc.subject.meshCho Cellsen_US
dc.subject.meshConcanavalin A - Pharmacologyen_US
dc.subject.meshCricetinaeen_US
dc.subject.meshDna - Geneticsen_US
dc.subject.meshElectrophoresis, Polyacrylamide Gelen_US
dc.subject.meshGlycosylationen_US
dc.subject.meshMutagenesis, Site-Directed - Physiologyen_US
dc.subject.meshOligosaccharides - Analysisen_US
dc.subject.meshSex Hormone-Binding Globulin - Chemistry - Genetics - Metabolismen_US
dc.titleSelective removal of glycosylation sites from sex hormone-binding globulin by site-directed mutagenesisen_US
dc.typeArticleen_US
dc.identifier.emailLee, WM: hrszlwm@hku.hken_US
dc.identifier.authorityLee, WM=rp00728en_US
dc.description.naturelink_to_subscribed_fulltexten_US
dc.identifier.doi10.1210/en.131.5.2331en_US
dc.identifier.pmid1425432-
dc.identifier.scopuseid_2-s2.0-0026463543en_US
dc.identifier.volume131en_US
dc.identifier.issue5en_US
dc.identifier.spage2331en_US
dc.identifier.epage2336en_US
dc.identifier.isiWOS:A1992JW33800045-
dc.publisher.placeUnited Statesen_US
dc.identifier.scopusauthoridBocchinfuso, WP=6602732269en_US
dc.identifier.scopusauthoridMa, KL=7202836855en_US
dc.identifier.scopusauthoridLee, WM=24799156600en_US
dc.identifier.scopusauthoridWarmelsRodenhiser, S=6602604086en_US
dc.identifier.scopusauthoridHammond, GL=7202011645en_US

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