Cleavage and functional loss of human apolipoprotein E by digestion of matrix metalloproteinase-14.
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 ((18)O/(16)O = 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 A(176)-I(177), P(183)-L(184), P(202)-L(203), and Q(249)-I(250). 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.
Park
Jun Hyoung
JH
Department of Biochemistry, College of Life Science and Biotechnology, Protein Network Research Center, Yonsei University, Seoul, Korea.
Park
Sung-Min
SM
Park
Sun-Hyun
SH
Cho
Kyung-Hyun
KH
Lee
Seung-Taek
ST
D000818:Animals; D001057:Apolipoproteins E / Q000037:antagonists & inhibitors / Q000378:metabolism* / Q000502:physiology; D050197:Atherosclerosis / Q000201:enzymology; D002460:Cell Line; D006801:Humans; D006868:Hydrolysis; D006949:Hyperlipidemias / Q000201:enzymology; D053511:Matrix Metalloproteinase 14 / Q000378:metabolism* / Q000502:physiology; D051381:Rats; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D018411:Spodoptera; D013379:Substrate Specificity
D001057:Apolipoproteins E; C507086:MMP14 protein, human; D053511:Matrix Metalloproteinase 14