New tools for the study of an old collagen : characterization of the human COL9A1, COL9A2 and COL9A3 genes and production of human type IX collagen as a recombinant protein
|Organizations:||University of Oulu, Collagen Research Unit
University of Oulu, Biocenter Oulu
University of Oulu, Faculty of Medicine, Department of Medical Biochemistry
|Online Access:||PDF Full Text (PDF, 0.9 MB)|
|Persistent link:|| http://urn.fi/urn:isbn:9514257359
|Publish Date:|| 2000-08-18
|Thesis type:||Doctoral Dissertation
|Defence Note:||Academic Dissertation to be presented with the assent of the Faculty of Medicine, University of Oulu, for public discussion in the Auditorium of the Department of Medical Biochemistry, on September 15th, 2000, at 1 p.m.
Professor Raili Myllylä
Professor Aleksander L. Sieron
Type IX collagen is a quantitatively minor component of cartilage collagen fibrils. Although a few mutations have been associated with multiple epiphyseal dysplasia, recent evidence suggests involvement of type IX collagen in a wider spectrum of phenotypes. The functional role of this molecule remains undetermined, in part due to difficulties in obtaining high amounts of intact protein.
To facilitate more efficient mutation screening and comparison of the genomic organization of the human genes encoding the α1(IX), α2(IX) and α3(IX) polypeptides, their genomic structures were characterized. Complete nucleotide sequences were determined for the COL9A2 and COL9A3 genes along with sequences for all the exon boundaries in the COL9A1 gene. Putative transcription control elements were identified and the alternative promoter region was characterized in the human and mouse COL9A1 genes. Mutation screening was performed for the COL9A3 gene and two apparently neutral 9-bp deletions within the COL1 domain were identified. These are the first deletions within a triple-helical domain of any collagen that are not associated with a disease phenotype.
An insect cell expression system with an exogenous source of prolyl 4-hydroxylase was used to produce heterotrimeric human type IX collagen. The recombinant protein consisted of the three a chains in a 1:1:1 ratio and showed correct folding and high thermal stability. Up to 10 mg of secreted protein could be purified from a litre of culture medium. The expression system was used to analyze the chain association of type IX collagen in cellulo. Although the chains are capable of homotrimerization, a preference for heterotrimer formation was noted.The neutral deletion was characterized further using the insect cell system. Mutant α3(IX) chains carrying a deletion of one Gly-X-Y triplet were shown to form correctly folded heterotrimers with the wild-type α1(IX) and α2(IX) chains. The results suggest a function for the NC2 domain in neutralizing the effect of the deletion.
This work provides a novel means for the analysis of type IX collagen mutations and their protein-level effects, and should enable future studies to be made of the structure-function relationship in type IX collagen.
Acta Universitatis Ouluensis. D, Medica
|Copyright information:||This publication is copyrighted. You may download, display and print it for your own personal use. Commercial use is prohibited.|