Background Collagenase-3 (COL3) is a matrix metalloproteinase abnormally over-expressed in pathological processes . During OA, COL3 together with interstitial collagenase MMP-1, are responsible for type II collagen digestion due to their ability to open the triple helix of collagen and allow the action of other collagenases. Different COL3 transcripts have been described to be expressed in human cells. In this study, we focused our interest on COL3DEL, a small transcript of 2.2/2.0 kb that presents an identical sequence to the original COL3, but with a deletion of 88 amino acids affecting the C-terminal domain. The mutation introduces 4 new amino acids and a new termination codon . The C-terminal hemopexin-like domain is essential for the substrate recognition and collagenolytic activity . We hypothesize that the COL3DEL isoform may present different collagenolytic activities due to the mutation in its hemopexin-like domain
Objectives This study aimed to characterize COL3DEL isoform levels in human OA samples and analyze its collagenolytic activity
Methods Samples of OA cartilage were obtained from patients undergoing arthroplasty and healthy cartilage from hip fractures of osteoporotic patients. ECM proteins were extracted by the Guanidium Chloride method. The COL3DEL isoform was detected by SDS-PAGE and 2D gel electrophoresis using a specific polyclonal antibody produced by our team. On the other hand, in order to analyse COL3DEL collagenolytic activity, we cloned the COL3DEL isoform with pRC-CMV2 plasmids that were amplified and purified from E. coli. Afterwards, HeLa cells were transfected with this plasmid and the collagenolytic activity from the isoform COL3 was analyzed by collagen zymography.
Results The COL3DEL isoform was detected only in ECM from OA cartilage but not from healthy cartilage. Due to similarities in COL3DEL and COL3 molecular weight, we performed a 2D gel electrophoresis and both isoforms were identified by different isoelectric points. COL3DEL production and secretion to the extracellular medium by transfected HeLa cells was identified by zymography. This technique was also useful to demonstrate collagenolytic activity of the COL3DEL isoform. COL3DEL mRNA expression was confirmed in HeLa cells after transfection by PCR
Conclusions The C-terminal domain is necessary to open native triple helical collagens and provide substrate specificity and affinity. The expression of a COL3 isoform with a different C-terminal domain may have clinical implications in OA development. This isoform can present different substrate recognition and/or speed of degradation, explaining some differences observed in the pathophysiology of the disease
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Disclosure of Interest None declared
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