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Matrix metalloproteinase proteolysis of the myelin basic protein isoforms is a source of immunogenic peptides in autoimmune multiple sclerosis.

Shiryaev SA, Savinov AY, Cieplak P, Ratnikov BI, Motamedchaboki K, Smith JW, Strongin AY - PLoS ONE (2009)

Bottom Line: As a result, we identified the MMP cleavage sites and the sequence of the resulting fragments.MT6-MMP (initially called leukolysin), however, was superior over all of the other MMPs in cleaving the MBP isoforms.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.

View Article: PubMed Central - PubMed

Affiliation: Inflammatory and Infectious Disease Center, Burnham Institute for Medical Research, La Jolla, California, United States of America.

ABSTRACT

Background: 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.

Methodology/principal findings: 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.

Conclusions/significance: 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.

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Related in: MedlinePlus

MMPs cleave MBP, BG21 and J37.A, Gel-electrophoresis of the digest samples. MBP, BG21 and J37 were incubated alone for 60 min at 37°C or co-incubated with the individual MMPs at the indicated enzyme-substrate molar ratio. Where indicated, GM6001 was added to the reactions to block MMPs. B, The MBP isoforms are inefficiently cleaved by certain MMPs. MBP, BG21 and J37 were each co-incubated for 60 min at 37°C with the indicated MMPs at a 1∶100 enzyme-substrate ratio.
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pone-0004952-g002: MMPs cleave MBP, BG21 and J37.A, Gel-electrophoresis of the digest samples. MBP, BG21 and J37 were incubated alone for 60 min at 37°C or co-incubated with the individual MMPs at the indicated enzyme-substrate molar ratio. Where indicated, GM6001 was added to the reactions to block MMPs. B, The MBP isoforms are inefficiently cleaved by certain MMPs. MBP, BG21 and J37 were each co-incubated for 60 min at 37°C with the indicated MMPs at a 1∶100 enzyme-substrate ratio.

Mentions: MBP was co-incubated for 1 h at 37°C with the individual MMPs. The digests were separated by SDS-PAGE (Fig. 2). Where indicated, the samples included GM6001 (a potent, broad-range inhibitor of MMPs). MBP was sensitive to proteolysis by many individual MMPs. MMP-2, MMP-10 and MT6-MMP, however, were the most efficient in cleaving MBP while MT2-MMP, MT3-MMP, MT4-MMP and MT5-MMP were the least efficient. Similarly, MMP-12 and especially MT6-MMP were the most efficient in the proteolysis of the BG21 and J37 constructs while MT5-MMP was the least efficient (Fig. 2). We determined that MT6-MMP was the most efficient among all of the individual MMPs we tested for cleaving the MBP, BG21 and J37 constructs.


Matrix metalloproteinase proteolysis of the myelin basic protein isoforms is a source of immunogenic peptides in autoimmune multiple sclerosis.

Shiryaev SA, Savinov AY, Cieplak P, Ratnikov BI, Motamedchaboki K, Smith JW, Strongin AY - PLoS ONE (2009)

MMPs cleave MBP, BG21 and J37.A, Gel-electrophoresis of the digest samples. MBP, BG21 and J37 were incubated alone for 60 min at 37°C or co-incubated with the individual MMPs at the indicated enzyme-substrate molar ratio. Where indicated, GM6001 was added to the reactions to block MMPs. B, The MBP isoforms are inefficiently cleaved by certain MMPs. MBP, BG21 and J37 were each co-incubated for 60 min at 37°C with the indicated MMPs at a 1∶100 enzyme-substrate ratio.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2654159&req=5

pone-0004952-g002: MMPs cleave MBP, BG21 and J37.A, Gel-electrophoresis of the digest samples. MBP, BG21 and J37 were incubated alone for 60 min at 37°C or co-incubated with the individual MMPs at the indicated enzyme-substrate molar ratio. Where indicated, GM6001 was added to the reactions to block MMPs. B, The MBP isoforms are inefficiently cleaved by certain MMPs. MBP, BG21 and J37 were each co-incubated for 60 min at 37°C with the indicated MMPs at a 1∶100 enzyme-substrate ratio.
Mentions: MBP was co-incubated for 1 h at 37°C with the individual MMPs. The digests were separated by SDS-PAGE (Fig. 2). Where indicated, the samples included GM6001 (a potent, broad-range inhibitor of MMPs). MBP was sensitive to proteolysis by many individual MMPs. MMP-2, MMP-10 and MT6-MMP, however, were the most efficient in cleaving MBP while MT2-MMP, MT3-MMP, MT4-MMP and MT5-MMP were the least efficient. Similarly, MMP-12 and especially MT6-MMP were the most efficient in the proteolysis of the BG21 and J37 constructs while MT5-MMP was the least efficient (Fig. 2). We determined that MT6-MMP was the most efficient among all of the individual MMPs we tested for cleaving the MBP, BG21 and J37 constructs.

Bottom Line: As a result, we identified the MMP cleavage sites and the sequence of the resulting fragments.MT6-MMP (initially called leukolysin), however, was superior over all of the other MMPs in cleaving the MBP isoforms.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.

View Article: PubMed Central - PubMed

Affiliation: Inflammatory and Infectious Disease Center, Burnham Institute for Medical Research, La Jolla, California, United States of America.

ABSTRACT

Background: 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.

Methodology/principal findings: 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.

Conclusions/significance: 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.

Show MeSH
Related in: MedlinePlus