By means of a degradomic approach applying proteomic techniques, we previously suggested that apolipoprotein E (apoE) is a substrate of matrix metalloproteinase-14 (MMP-14). Here we confirm that apoE is, in fact, a substrate of MMP-14 and also of MMP-7 and MMP-2 to a lesser extent. The 34 kDa apoE protein was initially processed by MMP-14 into fragments with molecular masses of 28, 23, 21, and 11 kDa. MMP-14 cleavage sites within the apoE protein were determined by C-terminal labeling of MMP-14-digested apoE fragments with isotope (18O/16O = 1:1) and identification of the doublet fragments or peptides showing 2 Da difference by MS, along with N-terminal sequencing of the fragments. It was determined that the primary MMP-14 cleavage sites were A176-I177, P183-L184, P202-L203, and Q249-T250. The MMP-14-mediated cleavage of apoE was consistent regardless of whether apoE existed in its lipid-bound or lipid-free form. Upon digestion with MMP-14, apoE loses its ability to suppress the platelet-derived growth factor-induced migration of rat vascular smooth muscle cells. Considering the important role of apoE for lipid metabolism and atherosclerosis protection, our findings suggest that MMP-14 plays an essential role for the development of hyperlipidemia and atherosclerosis as a result of degradation of apoE.
All Science Journal Classification (ASJC) codes
- Molecular Biology