Figure 3

MMP-9 proteolysis yields MBP epitopes. (A) In vitro cleavage of MBP (18 kDa) by MMP-9. Human MBP (4 μg; 11 μM) was co-incubated with MMP-9 at an enzyme-substrate ratio of 1:100–1:10,000 in 50 mM HEPES, pH 6.8, 10 mM CaCl₂ and 50 μM ZnCl₂ for 60 min at 37 °C. Where indicated, GM6001 was added to the reactions. The digests were analyzed using SDS-PAGE. M, molecular weight markers. (B) Representative MALDI-TOF MS spectra of the MBP samples. MBP (11 μM) was co-incubated for 60 min at 37 °C with MMP-9 (10 nM). The digests were analyzed using a Bruker Daltonics Autoflex II MALDI TOF mass spectrometer to determine the molecular mass of the resulting peptides. The high and low molecular mass fragments are shown in the top and bottom panels, respectively. The spectra represent arbitrary units (AU) for the MBP digest (magenta), the intact MBP (red), and the buffer alone (green). The numbers in parentheses show the numbering of the peptide in the MBP sequence. (C) Western blotting for MBP in the sciatic nerves at 1 day and week 1 post-crush or contralateral (normal) nerves in MMP-9 null (^−/−) or a wild-type (WT) mice (pooled from n = 3 mice/lane). β-actin, protein loading control. (D) Immunostaining of MBP69-86 using a specific antibody (AB5864, Millipore, red) and MHCII (green, top) or intact MBP (green, bottom) in the myelinating Schwann cells (arrows) and other cells (arrowheads) at day 1 post-CCI. DAPI, blue. Representative of n = 3/group. Scale bar, 10 μm.