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Article: Site-directed mutagenesis of human type X collagen. Expression of α1(X) NC1, NC2, and helical mutations in vitro and in transfected cells

TitleSite-directed mutagenesis of human type X collagen. Expression of α1(X) NC1, NC2, and helical mutations in vitro and in transfected cells
Authors
Issue Date1996
PublisherAmerican Society for Biochemistry and Molecular Biology, Inc. The Journal's web site is located at http://www.jbc.org/
Citation
Journal Of Biological Chemistry, 1996, v. 271 n. 23, p. 13566-13572 How to Cite?
AbstractType X collagen is a short chain collagen expressed in the hypertrophic zone of calcifying cartilage during skeletal development and bone growth. The α1(X) homotrimer consists of three protein domains, a short triple helix (COL1) flanked by nonhelical amino-terminal (NC2) and carboxyl-terminal (NC1) domains. While mutations of the NC1 domain result in Schmid metaphyseal chondrodysplasia, which suggests a critical role for this protein domain, little biochemical detail is known about type X collagen synthesis, secretion, and the mechanisms of molecular assembly. To study these processes, a range of mutations were produced in human α1 (X) cDNA and the biochemical consequences determined by in vitro expression, using T7-driven coupled transcription and translation, and by transient transfection of cells. Three NC1 mutants, which were designed to be analogous to Schmid mutations (1952delC, 1963del10, and Y598D), were unable to assemble into type X collagen homotrimers in vitro, but the mutant chains did not associate with, or interfere with, the efficiency of normal chain assembly in co- translations with a normal construct. Expression in transiently transfected cells confirmed that mutant type X collagen assembly was also compromised in vivo. The mutant chains were not secreted from the cells but did not accumulate intracellularly, suggesting that the unassociated mutant chains were rapidly degraded. In-frame deletions within the helix (amino acid residues 72-354) and the NC2 domain (amino acid residues 21-54) were also produced. In contrast to the NC1 mutations, these mutations did not prevent assembly. Mutant homotrimers and mutant-normal heterotrimers were formed in vitro, and the mutant homotrimers formed in transiently transfected cells had assembled into pepsin-stable triple helical molecules which were secreted.
Persistent Identifierhttp://hdl.handle.net/10722/147409
ISSN
2015 Impact Factor: 4.258
2015 SCImago Journal Rankings: 3.151
ISI Accession Number ID

 

DC FieldValueLanguage
dc.contributor.authorChan, Den_US
dc.contributor.authorYi Ma Wengen_US
dc.contributor.authorHocking, AMen_US
dc.contributor.authorGolub, Sen_US
dc.contributor.authorMcquillan, DJen_US
dc.contributor.authorBateman, JFen_US
dc.date.accessioned2012-05-29T06:03:31Z-
dc.date.available2012-05-29T06:03:31Z-
dc.date.issued1996en_US
dc.identifier.citationJournal Of Biological Chemistry, 1996, v. 271 n. 23, p. 13566-13572en_US
dc.identifier.issn0021-9258en_US
dc.identifier.urihttp://hdl.handle.net/10722/147409-
dc.description.abstractType X collagen is a short chain collagen expressed in the hypertrophic zone of calcifying cartilage during skeletal development and bone growth. The α1(X) homotrimer consists of three protein domains, a short triple helix (COL1) flanked by nonhelical amino-terminal (NC2) and carboxyl-terminal (NC1) domains. While mutations of the NC1 domain result in Schmid metaphyseal chondrodysplasia, which suggests a critical role for this protein domain, little biochemical detail is known about type X collagen synthesis, secretion, and the mechanisms of molecular assembly. To study these processes, a range of mutations were produced in human α1 (X) cDNA and the biochemical consequences determined by in vitro expression, using T7-driven coupled transcription and translation, and by transient transfection of cells. Three NC1 mutants, which were designed to be analogous to Schmid mutations (1952delC, 1963del10, and Y598D), were unable to assemble into type X collagen homotrimers in vitro, but the mutant chains did not associate with, or interfere with, the efficiency of normal chain assembly in co- translations with a normal construct. Expression in transiently transfected cells confirmed that mutant type X collagen assembly was also compromised in vivo. The mutant chains were not secreted from the cells but did not accumulate intracellularly, suggesting that the unassociated mutant chains were rapidly degraded. In-frame deletions within the helix (amino acid residues 72-354) and the NC2 domain (amino acid residues 21-54) were also produced. In contrast to the NC1 mutations, these mutations did not prevent assembly. Mutant homotrimers and mutant-normal heterotrimers were formed in vitro, and the mutant homotrimers formed in transiently transfected cells had assembled into pepsin-stable triple helical molecules which were secreted.en_US
dc.languageengen_US
dc.publisherAmerican Society for Biochemistry and Molecular Biology, Inc. The Journal's web site is located at http://www.jbc.org/en_US
dc.relation.ispartofJournal of Biological Chemistryen_US
dc.subject.meshAnimalsen_US
dc.subject.meshBase Sequenceen_US
dc.subject.meshCell Lineen_US
dc.subject.meshCollagen - Chemistry - Genetics - Metabolismen_US
dc.subject.meshDna Primers - Geneticsen_US
dc.subject.meshDna, Complementary - Geneticsen_US
dc.subject.meshGene Expressionen_US
dc.subject.meshHumansen_US
dc.subject.meshMolecular Sequence Dataen_US
dc.subject.meshMolecular Structureen_US
dc.subject.meshMutagenesis, Site-Directeden_US
dc.subject.meshMutationen_US
dc.subject.meshOsteochondrodysplasias - Geneticsen_US
dc.subject.meshPoint Mutationen_US
dc.subject.meshProtein Biosynthesisen_US
dc.subject.meshProtein Structure, Secondaryen_US
dc.subject.meshRatsen_US
dc.subject.meshSequence Deletionen_US
dc.subject.meshTranscription, Geneticen_US
dc.subject.meshTransfectionen_US
dc.titleSite-directed mutagenesis of human type X collagen. Expression of α1(X) NC1, NC2, and helical mutations in vitro and in transfected cellsen_US
dc.typeArticleen_US
dc.identifier.emailChan, D:chand@hkucc.hku.hken_US
dc.identifier.authorityChan, D=rp00540en_US
dc.description.naturelink_to_subscribed_fulltexten_US
dc.identifier.doi10.1074/jbc.271.23.13566en_US
dc.identifier.pmid8662807-
dc.identifier.scopuseid_2-s2.0-0029946910en_US
dc.identifier.volume271en_US
dc.identifier.issue23en_US
dc.identifier.spage13566en_US
dc.identifier.epage13572en_US
dc.identifier.isiWOS:A1996UP38500041-
dc.publisher.placeUnited Statesen_US
dc.identifier.scopusauthoridChan, D=7402216545en_US
dc.identifier.scopusauthoridYi Ma Weng=7409810641en_US
dc.identifier.scopusauthoridHocking, AM=7004556812en_US
dc.identifier.scopusauthoridGolub, S=35609444800en_US
dc.identifier.scopusauthoridMcQuillan, DJ=7003319485en_US
dc.identifier.scopusauthoridBateman, JF=16135557700en_US

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