Background Collagenase-3 (MMP-13) is a human matrix metalloproteinase highly overexpressed in diseases where tissue repair and remodelling is needed, such as rheumatoid arthritis (RA), osteoarthritis (OA) and some cancers. However, its expression in normal tissues is limited to fetal ossification processes1. MMP-13 can digest fibrillar collagen, preferentially type II collagen, but also glomerular collagens and other components of the extracellular matrix (ECM) 2. MMP-13 structures is composed of 4 domains: a hydrophobic pre-domine necessary for the secretion, a pro-domain involved in enzyme latency, a catalytic domain, and a hemopexin-like domain This last is essential for the substrate recognition and collagenolytic activity3 Three different MMP-13 transcripts of 3.0, 2.5 and 2.2/2.0 kb have been described to be expressed in human cells. The 2.5kb transcript (isoform 205) corresponds to the MMP-13 original form, while the 3.0kb transcript (isoform 344) presents an insertion at the C-terminal produced by lack of exon 9B splicing. Finally, the smaller transcript of 2.2/2.0kb (isoform 209) presents a deletion (probably due to alternative splicing) of 88 amino acids, also affecting the C-terminal domain4.
Objectives Our objective is to demonstrate the presence of these three MMP13 protein isoforms in human OA samples.
Methods A specific peptide from each isoform was synthesized and two rabbits were immunized with each peptide. The antibodies recognizing each isoform were purified from the total antiserum by sulfolink column. On the other hand MMP-13 isoforms were synthesized in vitro in a Sf9 insect cells system, with a recombinant baculovirus containing MMP-13 transcripts cloned. The insect cells were culture to produce the respective isoforms, and they were purified by molecular exclusion chromatography. These proteins were used to test the specificity of the polyclonal antibodies obtained by Western Blot. Finally, ECM proteins were extracted from cartilage samples obtained from knee and hip arthrosic patients undergoing arthroplasty, using Guanidinium chloride (4M) method. The presence of the 3 isoforms in these samples was assay by western blot with the specific isoform polyclonal antibodies obtained.
Results The purified polyclonal antibodies show immunoreactivity only in front of its specific isoform, showing that they are useful for the detection of each specific isoform in the samples. In all the samples tested, different intensities of the bands corresponding of each antibody were detected. No relative quantification could be performed due to the lack of a standard of each MMP-13 isoforms that allows us to compare the affinity of each polyclonal antibody to each specific isoform.
Conclusions The MMP-13 mRNA isoforms are translated to protein in cartilage OA samples. The transcripts containing the deleted and the alternatively spliced exons differ from the original sequence in the region coding for the hemopexin-like domain5. This domain is necessary to cleave native triple helical collagens and provide to the protease its substrate specificity and affinity. The expression of 209 and 344 isoform could have clinical implications in OA if they present different subtracted recognition and/or speed of degradation, explaining some differences observed in the pathophysiology of the disease.
Mitchell PG et al (1996)
Knauper V et al (1996)
Freije JM et al (1994)
Tardif G et al(2003)
Vera Knauper et al(1997)
Disclosure of Interest None Declared