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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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MT6-MMP proteolysis of MBP generates highly specific immunogenic peptides which efficiently stimulate the proliferation of the specific T cell clone.MBP, BG21 and J37 (5 µM each) were cleaved by MT6-MMP (an enzyme-substrate ratio of 1∶100). The irradiated splenocytes from B10.PL mice were co-incubated with the digest reactions. The CD4+ T cells (clone PGPR7.5) specific for the murine 1–15 MBP peptide presented in the MHC H-2U context were then added to the reactions. H3-thymidine was then added to the cells. The incorporation of the label into the T cells was measured by liquid scintillation counting. MBP alone, BG21 alone and J37 alone - intact MBP, BG21 and J37 (5 µM each) were added to the cells. Cells alone, no peptide. The 1–15 ASQKRPSQRSKYLATAS MBP peptide (5 µM) was used as a control ( = 100%). The numbers show the percentage relative to the peptide control.
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pone-0004952-g005: MT6-MMP proteolysis of MBP generates highly specific immunogenic peptides which efficiently stimulate the proliferation of the specific T cell clone.MBP, BG21 and J37 (5 µM each) were cleaved by MT6-MMP (an enzyme-substrate ratio of 1∶100). The irradiated splenocytes from B10.PL mice were co-incubated with the digest reactions. The CD4+ T cells (clone PGPR7.5) specific for the murine 1–15 MBP peptide presented in the MHC H-2U context were then added to the reactions. H3-thymidine was then added to the cells. The incorporation of the label into the T cells was measured by liquid scintillation counting. MBP alone, BG21 alone and J37 alone - intact MBP, BG21 and J37 (5 µM each) were added to the cells. Cells alone, no peptide. The 1–15 ASQKRPSQRSKYLATAS MBP peptide (5 µM) was used as a control ( = 100%). The numbers show the percentage relative to the peptide control.

Mentions: To directly show that MT6-MMP proteolysis generates the immunogenic 1–15 peptide, we used the digests to stimulate, in the mixed lymphocyte cultures, the proliferation of the murine T cells clone which is specific to the 1–15 fragment of MBP. The PGPR7.5 clone which is specific for the murine MBP 1–15 peptide presented in the MHC H-2U context was isolated from EAE mice [22]. MBP, BG21 and J37 (5 µM each) were cleaved by MT6-MMP. The irradiated splenocytes from B10.PL mice were co-incubated with the digests. The PGPR7.5 T cells and H3-thymidine were then added to the reactions. The incorporation of the label into the T cells was measured by liquid scintillation counting. The digest products were internalized and presented by the splenocytes and then the presented peptides stimulated the proliferation of the specific PGPR7.5 T cells. As a control, we used the synthetic ASQKRPSQRSKYLATAS peptide (5 µM) that corresponded to the 2–18 sequence of murine MBP and the stimulatory effect of which we took as 100%. The MT6-MMP digest of MBP resulted in a 44% level of proliferation of PGPR7.5 T cells compared to the equimolar amount of the ASQKRPSQRSKYLATAS peptide, thus suggesting a high yield of the specific immunogenic fragment in the digest (Fig. 5).


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)

MT6-MMP proteolysis of MBP generates highly specific immunogenic peptides which efficiently stimulate the proliferation of the specific T cell clone.MBP, BG21 and J37 (5 µM each) were cleaved by MT6-MMP (an enzyme-substrate ratio of 1∶100). The irradiated splenocytes from B10.PL mice were co-incubated with the digest reactions. The CD4+ T cells (clone PGPR7.5) specific for the murine 1–15 MBP peptide presented in the MHC H-2U context were then added to the reactions. H3-thymidine was then added to the cells. The incorporation of the label into the T cells was measured by liquid scintillation counting. MBP alone, BG21 alone and J37 alone - intact MBP, BG21 and J37 (5 µM each) were added to the cells. Cells alone, no peptide. The 1–15 ASQKRPSQRSKYLATAS MBP peptide (5 µM) was used as a control ( = 100%). The numbers show the percentage relative to the peptide control.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2654159&req=5

pone-0004952-g005: MT6-MMP proteolysis of MBP generates highly specific immunogenic peptides which efficiently stimulate the proliferation of the specific T cell clone.MBP, BG21 and J37 (5 µM each) were cleaved by MT6-MMP (an enzyme-substrate ratio of 1∶100). The irradiated splenocytes from B10.PL mice were co-incubated with the digest reactions. The CD4+ T cells (clone PGPR7.5) specific for the murine 1–15 MBP peptide presented in the MHC H-2U context were then added to the reactions. H3-thymidine was then added to the cells. The incorporation of the label into the T cells was measured by liquid scintillation counting. MBP alone, BG21 alone and J37 alone - intact MBP, BG21 and J37 (5 µM each) were added to the cells. Cells alone, no peptide. The 1–15 ASQKRPSQRSKYLATAS MBP peptide (5 µM) was used as a control ( = 100%). The numbers show the percentage relative to the peptide control.
Mentions: To directly show that MT6-MMP proteolysis generates the immunogenic 1–15 peptide, we used the digests to stimulate, in the mixed lymphocyte cultures, the proliferation of the murine T cells clone which is specific to the 1–15 fragment of MBP. The PGPR7.5 clone which is specific for the murine MBP 1–15 peptide presented in the MHC H-2U context was isolated from EAE mice [22]. MBP, BG21 and J37 (5 µM each) were cleaved by MT6-MMP. The irradiated splenocytes from B10.PL mice were co-incubated with the digests. The PGPR7.5 T cells and H3-thymidine were then added to the reactions. The incorporation of the label into the T cells was measured by liquid scintillation counting. The digest products were internalized and presented by the splenocytes and then the presented peptides stimulated the proliferation of the specific PGPR7.5 T cells. As a control, we used the synthetic ASQKRPSQRSKYLATAS peptide (5 µM) that corresponded to the 2–18 sequence of murine MBP and the stimulatory effect of which we took as 100%. The MT6-MMP digest of MBP resulted in a 44% level of proliferation of PGPR7.5 T cells compared to the equimolar amount of the ASQKRPSQRSKYLATAS peptide, thus suggesting a high yield of the specific immunogenic fragment in the digest (Fig. 5).

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