Abstract
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.
| Identifer | oai:union.ndltd.org:oulo.fi/oai:oulu.fi:isbn951-42-5735-9 |
| Date | 18 August 2000 |
| Creators | Pihlajamaa, T. (Tero) |
| Publisher | University of Oulu |
| Source Sets | University of Oulu |
| Language | English |
| Detected Language | English |
| Type | info:eu-repo/semantics/doctoralThesis, info:eu-repo/semantics/publishedVersion |
| Format | application/pdf |
| Rights | info:eu-repo/semantics/openAccess, © University of Oulu, 2000 |
| Relation | info:eu-repo/semantics/altIdentifier/pissn/0355-3221, info:eu-repo/semantics/altIdentifier/eissn/1796-2234 |
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