Matrix Metalloproteinase Proteolysis of the Myelin Basic Protein Isoforms Is a Source of Immunogenic Peptides in Autoimmune Multiple Sclerosis

Matrix metalloproteinases (MMPs) play a significant role in the fragmentation of myelin basic protein (MBP) and demyelination leading to autoimmune multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE). The classic MBP isoforms are predominantly expressed in the oligodendrocytes of the CNS. The splice variants of the single MBP gene (Golli-MBP BG21 and J37) are widely expressed in the neurons and also in the immune cells. The relative contribution of the individual MMPs to the MBP cleavage is not known.To elucidate which MMP plays the primary role in cleaving MBP, we determined the efficiency of MMP-2, MMP-8, MMP-9, MMP-10, MMP-12, MT1-MMP, MT2-MMP, MT3-MMP, MT4-MMP, MT5-MMP and MT6-MMP in the cleavage of the MBP, BG21 and J37 isoforms in the in vitro cleavage reactions followed by mass-spectroscopy analysis of the cleavage fragments. As a result, we identified the MMP cleavage sites and the sequence of the resulting fragments. We determined that MBP, BG21 and J37 are highly sensitive to redundant MMP proteolysis. MT6-MMP (initially called leukolysin), however, was superior over all of the other MMPs in cleaving the MBP isoforms. Using the mixed lymphocyte culture assay, we demonstrated that MT6-MMP proteolysis of the MBP isoforms readily generated, with a near quantitative yield, the immunogenic N-terminal 1-15 MBP peptide. This peptide selectively stimulated the proliferation of the PGPR7.5 T cell clone isolated from mice with EAE and specific for the 1-15 MBP fragment presented in the MHC H-2(U) context.In sum, our biochemical observations led us to hypothesize that MT6-MMP, which is activated by furin and associated with the lipid rafts, plays an important role in MS pathology and that MT6-MMP is a novel and promising drug target in MS especially when compared with other individual MMPs

Leucine zipper sequences.

<p>Sequences of mutant (-pII) leucine zipper motifs that have been linked <i>via</i> linker to the TRAIL sequence are separated by heptad repeats. “a-g” positions in the heptad sequence are shown in the top row. Mutagenized amino acid residues are underlined.</p><p>Leucine zipper sequences.</p

Stability of leucine zipper-TRAIL fusion proteins.

<p>The ratio of dead cells was determined as described in the legend for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122980#pone.0122980.t002" target="_blank">Table 2</a>. Residual cytotoxic activity of the TRAIL samples after 20 min treatment at 70°C was calculated for 3 and 10 μl of 100-fold diluted TRAIL samples.</p><p>Stability of leucine zipper-TRAIL fusion proteins.</p

Construction of leucine zipper-TRAIL.

<p>LZ, leucine zipper.</p

Ratio of dead cells as compared to untreated cells after TRAIL treatment.

<p>Conditioned medium from TRAIL-producing <i>P</i>. <i>pastoris</i> clones was 100-fold diluted in DMEM/FBS. Subconfluent prostate carcinoma PPC-1 in a 96-well plate (100 μl/well) were incubated with 1 and 3 μl of the diluted TRAIL samples for 24 h. At the end of the treatment, the ratio of dead cells was determined by an ATPLite reagent. The conditioned medium obtained from non-transfected yeast cells was not cytotoxic to PPC-1 cells. The cytotoxic activity of the most efficient clones for each TRAIL construct is shown.</p><p>Ratio of dead cells as compared to untreated cells after TRAIL treatment.</p

ATF7-TRAIL apoptotic activity.

<p>Human breast carcinoma MDA-MB-231, prostate carcinoma PPC-1, lung carcinoma SK-MES-1 cells, and human primary hepatocytes were incubated for 24 h with the increasing concentrations of ATF7-TRAIL. Cytotoxicity (A) and caspase-3/7 activity (B) were determined by ATP-Lite reagent and caspase-3/7 activity assay, respectively. <i>P</i> < 0.05.</p

ATF7-TRAIL exhibits a high anti-tumor activity in mice.

<p>Left panel, tumor volume and weight of the vehicle-treated and experimental mice that received PBS and 5 mg/kg/day ATF7-TRAIL, respectively. Tumor incidence per group is shown below the bars. *<i>P</i> = 0.004. Right panel, tumors of the control and experimental mice.</p