Atheromatous plaques contain various cell types, including macrophages, endothelial cells and smooth-muscle cells. To investigate the possible interactions between secreted matrix metalloproteinases and high-density lipoprotein (HDL) components, we tested the above cell types by culturing them for 24 h. HDL(3) (HDL subfractions with average sizes of between 8.44 nm for HDL(3A) and 7.62 nm for HDL(3C)) were then incubated in their cell-free conditioned media. Proteolytic degradation of apolipoprotein A-I was observed with macrophages, but not with endothelial-cell- or muscle-cell-conditioned supernatant. Absence of calcium or addition of EDTA to incubation media prevented all proteolytic processes. The identified apolipoprotein A-I fragments had sizes of 26, 22, 14 and 9 kDa. Two-dimensional electrophoresis and MS resolved the 26 and the 22 kDa components and identified peptides resulting from both N- and C-terminal cleavage of apolipoprotein A-I. The higher abundance of C- than N-terminally cleaved peptides agrees with data in the literature for a fully structured alpha-helix around Tyr(18) compared with an unstructured region around Gly(185) and Gly(186). The flexibility in the latter region of apolipoprotein A-I may explain its susceptibility to proteolysis. In our experimental set-up, HDL(3C) was more extensively degraded than the other HDL(3) subclasses (HDL(3A) and HDL(3B)). Proteolytic fragments produced by metalloproteinase action were shown by gel filtration and electrophoresis to be neither associated with lipids nor self-associated.
|Titolo:||Macrophage metalloproteinases degrade high-density-lipoprotein-associated apolipoprotein A-I at both the N- and C-termini|
|Autori interni:||GIANAZZA, ELISABETTA (Ultimo)|
CALABRESI, LAURA (Secondo)
EBERINI, IVANO (Primo)
PIRILLO, ANGELINA ANNA
SIRTORI, CESARE (Penultimo)
|Parole Chiave:||atherosclerosis ; mass spectrometry ; matrix metalloproteinase ; proteolysis ; two-dimensional electrophoresis|
|Settore Scientifico Disciplinare:||Settore BIO/10 - Biochimica|
Settore BIO/14 - Farmacologia
|Data di pubblicazione:||15-mar-2002|
|Digital Object Identifier (DOI):||10.1042/0264-6021:3620627|
|Appare nelle tipologie:||01 - Articolo su periodico|
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