Clin Orthop Surg.  2009 Dec;1(4):181-187. 10.4055/cios.2009.1.4.181.

Altered Synthesis of Cartilage-Specific Proteoglycans by Mutant Human Cartilage Oligomeric Matrix Protein

• Kwak YH
• Roh JY
• Lee KS
• Park HW
• Kim HW

• KMID: 999399
• DOI: http://doi.org/10.4055/cios.2009.1.4.181

Abstract

BACKGROUND
The mechanism by which mutant cartilage oligomeric matrix protein (COMP) induces a pseudoachondroplasia phenotype remains unknown, and the reason why a mutation of a minor protein of the growth plate cartilage causes total disruption of endochondral bone formation has not yet been determined. The current study was performed to investigate the effects of mutated COMP on the synthesis of the cartilage-specific major matrix proteins of Swarm rat chondrosarcoma chondrocytes.
METHODS
The Swarm rat chondrosarcoma chondrocytes transfected with a chimeric construct, which consisted of a mutant gene of human COMP and an amino acid FLAG tag sequence, were cultured in agarose gel. Formation of extracellular proteoglycan and type-II collagen by the cells was evaluated by immunohistochemical staining and measuring the (35)S-sulfate incorporation.
RESULTS
No difference was observed for the detection of type-II collagen among the cell lines expressing mutant COMP and the control cell lines. Histochemical staining of sulfated proteoglycans with safranin-O showed that lesser amounts of proteoglycans were incorporated into the extracellular matrix of the chondrocytes transfected with the mutant gene. (35)S-sulfate incorporation into the cell/matrix fractions demonstrated markedly lower radiolabel incorporation, as compared to that of the control cells.
CONCLUSIONS
Mutation of COMP has an important impact on the processing of proteoglycans, rather than type-II collagen, in the three-dimensional culture of Swarm rat chondrosarcoma chondrocytes.

Keyword

Mutant COMP; Extracellular matrix; Pseudoachondroplasia

MeSH Terms

Aggrecans/analysis/*biosynthesis
Animals
Cells, Cultured
Chondrocytes/*metabolism
Chondrosarcoma/metabolism
Collagen Type II/*biosynthesis
Extracellular Matrix/*metabolism
Extracellular Matrix Proteins/*genetics
Glycoproteins/*genetics
Humans
Mutation
Rats
Transfection

Figure

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  Fig. 1 Construction of a mutant hCOMPFLAG chimeric protein. A primer set consisting of an oligonucleotide sequence that encompasses the transcription start site and an oligonucleotide containing the 3' end of COMP with a mutation of the stop codon, plus a sequence that encodes for the 8 amino acid FLAG epitope was used to generate a 2348 nucleotide PCR product from the clone Mut3 that encodes for PSACH-linked ΔD469 hCOMP. The DNA fragment was inserted into the pcDNA 3.1 expression vector following ligation into the pCR2 vector blunt to obtain the Eco RI DNA restriction enzyme sites.

• 

  Fig. 2 Immunohistochemical staining of type II collagen at day 14. No differences for type II collagen were observed between the expressed mutant COMP cell line (C415) (A) and that of the antisense transfectant control cell line (C422) (B) (×200).

• 

  Fig. 3 Immunohistochemical identification of human PSACH-linked mutant COMP tagged with a selective immunoreactive sequence that was distinct from that of rat COMP in the Swarm rat chondrosarcoma cells cultured for 14 days. Brown staining identifies the retention of expressed mutant COMP within the cells (×1,500).

• 

  Fig. 4 35S-sulfate incorporation into the cell/matrix fractions of the cell lines cultured in 1% agarose for varying times (Days 1, 4, 7, and 14). The C415 cells expressing the mutant hCOMPFLAG , demonstrated markedly lower radiolabel incorporation compared to the C422 mutant hCOMPFLAG antisense control cells and the LTC cells. An asterisk indicates statistically significant difference at p < 0.05, as compared to the C415 cells.

• 

  Fig. 5 Histochemical staining of sulfated proteoglycans with safranin-O. Lesser amounts of proteoglycans are incorporated into the ECM in the C415 cell line (left panel) compared to the C422 cell line (right panel), suggesting the expression of the mutant COMP is altering the deposition of aggrecan in the ECM, as is similarly seen in PSACH patients (×200).

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