FIGURE 7.
Furin proteolysis of MBP and activation of the specific T cell clone. A, furin proteolysis of the MBP isoforms. MBP, J37, and BG21 were incubated for 3 h at 37 °C with furin at an enzyme/substrate molar ratio of 1:4. The digest reactions were analyzed by SDS-gel electrophoresis followed by Coomassie staining. Where indicated, dec-RVKR-cmk was added to the reactions. B, 1–171-residue sequence of human MBP (GenBankTM accession number AAH08749). The immunogenic regions are shown at the bottom of the panel using the MBP residue numbering. Following the MBP cleavage by MMPs, the mass and, consecutively, the sequence of the digest fragments was determined by MALDI-TOF MS. The italicized numbers indicate the positions of the cleavage sites. C, furin proteolysis of MBP generates the N-terminal peptide that stimulates the proliferation of the specific T cell clone. MBP, BG21, and J37 (5 μm each) were co-incubated for 3 h at 37 °C with furin at an enzyme/substrate molar ratio of 1:4. The irradiated splenocytes from B10.PL mice were incubated with the cleavage reactions. The PGPR7.5 T cells specific for the murine MBP-(1–15)-peptide and [3H]thymidine were then added to the reactions. The incorporation of the label into the T cells was measured by liquid scintillation counting. MBP, BG21, and J37 alone indicate intact MBP, BG21, and J37 (5 μm each) added to the cells. Cells alone indicates no peptide. Peptide, the 1–15-residue ASQKRPSQRSKYLATAS MBP peptide (5 μm) was used as a control (= 100%). The numbers show the percentage relative to the peptide control. The experiments were performed in triplicate and repeated twice. The data represent the mean ± S.E.