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Structural and functional studies on recombinant non-collagenous carboxyl-terminal (NC1) domain of human type X collagen
Başlık:
Structural and functional studies on recombinant non-collagenous carboxyl-terminal (NC1) domain of human type X collagen
Yazar:
Gregory, Carl Austin, author.
ISBN:
9780438043145
Yazar Ek Girişi:
Fiziksel Tanımlama:
1 electronic resource (288 pages)
Genel Not:
Source: Dissertation Abstracts International, Volume: 76-08C.
Özet:
Type X collagen is a short-chain homotrimeric collagen expressed mainly at sites of endochondral ossification by hypertrophic chondrocytes. Although its precise function remains to be determined, mutations localised solely in the non-collagenous carboxyl-terminal (NCI) domain of collagen X have been shown to cause the bone developmental disorder, metaphyseal chondrodysplasia type Schmid (MCDS) in humans. With a view to characterising the overall structure of trimeric NCI domains and the molecular effects of MCDS mutations thereon, recombinant human collagen X NCI domain has been produced in both E. coli and in vitro based expression systems. Recombinant NCI domain of human collagen X has been expressed in HMS 174 (?DE3) E. coli. NCI monomers were found to spontaneously associate into a complex migrating with air apparent molecular mass of 54 kDa when analysed by SDS-PAGE. The complex was specifically recognised by the conformation-dependent anti-human collagen X NC1 monoclonal antibody X34. SDS-PAGE analysis of digestion intermediates during cleavage of the 54 kDa complex with factor Xa demonstrated that the complex is an aberrantly migrating trimer. The trimer is remarkably stable, even after incubation at 100°C for up to 1 hour in the presence of SDS. However, treatment of the trimeric NCI domains with 10% (w/v) trichloroacetic acid resulted in its complete dissociation into constituent 21 kDa monomers. CD analysis of the complex revealed that the trimeric NCI domains adopted a structure composed of beta-strands and loops. NCI monomers generated by cell-free translation were also found to associate into a trimeric complex which, when analysed by SDS-PAGE, migrated aberrantly with an apparent molecular mass of approximately 50 kDa. Although in v/fro-generated NCI domain harbouring mutations known to cause MCDS lose the ability to form stable trimers detectable by standard SDS-PAGE, monomers possessing the Y598D and S600P point mutations formed homotrimers and associate with wild type monomers into heterotrimers which could be visualised by SDS-PAGE in low SDS conditions. These observations suggest that in some cases, MCDS may be caused by the association of wild type pro-alpha-chains with mutant pro-alpha-chains, accounting for the dominant characteristics associated with the disease. Additional work has demonstrated that stable trimerisation is also dependent on the extreme carboxyl-terminus of the NCI domain and furthermore, the extreme amino-terminus of the NCI domain also contributes to thermal stability.
Notlar:
School code: 1543
Tüzel Kişi Ek Girişi:
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Yer Numarası | Demirbaş Numarası | Shelf Location | Lokasyon / Statüsü / İade Tarihi |
---|---|---|---|
XX(684251.1) | 684251-1001 | Proquest E-Tez Koleksiyonu | Arıyor... |
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