LRP1 and, in particular the region that spans the C-terminal half of domain CR9 (from Gly1127 to Cys1140), named LP3, is essential for aggregated LDL internalization and human coronary vascular smooth muscle cells (hcVSMC)-cholesterol loading. Here, we investigated whether LP3 and its retro-enantio version (DP3) are protective against sphingomyelinase (SMase) and phospholipase 2 (PLA2)-induced LDL aggregation, the structural basis underlying this protection and the impact that LRP1-derived peptides might have on hcVSMC-cholesterol loading and cholesterol-modulated signaling pathways. For this purpose, biochemical, biophysical, molecular, proteomic, and cellular experiments were performed. Turbidimetry measurements show that LP3 and DP3 inhibit LDL aggregation induced by SMase and PLA2 in a dose-dependent manner, although the efficacy of DP3 is higher. Gel filtration chromatography (GFC) and transmission electron microscopy (TEM) show that LP3, and more efficiently DP3, almost completely counteract the increased percentage of aggregated LDL induced by both SMase and PLA2, respectively. Native polyacrilamide gradient gel electrophoresis (GGE), agarose gel electrophoresis (AGE) and high-performance thin layer chromatography (HPTLC) partitioning of LDL phospholipids indicated that LP3 and DP3 prevent SMase-induced alterations in LDL size, electric charge and phospholipid content but not those induced by PLA2. In contrast, LP3 and DP3 show high efficacy to counteract changes in ApoB-100 conformation induced by both SMase and PLA2. Together, these results indicate that LRP1-derived peptides protect LDL against aggregation induced by SMase and PLA2 through a common mechanism based on their capacity to prevent ApoB-100 conformational changes. Proteomics and computational modeling methods suggest that LRP1 derived peptides are able to establish strong electrostatic interactions with a specific ApoB-100 basic region. TLC and confocal microscopy show that DP3 with higher efficacy than LP3 significantly reduce intracellular cholesteryl ester accumulation induced by SMase-LDL in hcVSMC. Moreover, proteomics studies evidence several signaling pathways modulated by SMase-LDL that are counteracted specifically by DP3. These findings demonstrate that LRP1 derivative peptides protect against LDL aggregation and preserve vascular cells against cholesterol loading and associated alterations in critical signal pathways

Four LDL samples were prepared: 1. LDL, 2. LDL exposed to SMase in absence of peptide or 3. in the presence of DP3 or 4. IP321, and samples were subjected to limited trypsin digestion for 1h or 3h, and tryptic peptides were analyzed by shotgun LC-MSMS using a LTQ-Orbitrap Velos Pro mass spectrometer.

Different LDL samples were prepared: 1. LDL, 2. LDL exposed to SMase in absence of peptide or 3. in the presence of DP3 or 4. IP321. Aggregation of LDL was verified by turbidimetry before cell exposure to LDL, and samples were directly subjected to limited trypsin digestion (1 µg, 37ºC) for 1h or 3h, and tryptic peptides were desalted using a C18 column, and evaporated to dryness. The different LDL samples were analyzed using a LTQ-Orbitrap Velos Pro mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA) coupled to an EasyLC (Thermo Fisher Scientific (Proxeon), Odense, Denmark). Peptides were separated by reverse-phase chromatography using a 25-cm column with an inner diameter of 75 μm, packed with 5 μm C18 particles (Nikkyo Technos Co., Ltd. Japan). Chromatographic gradients started at 93% buffer A and 7% buffer B and gradually increased to 65% buffer A / 35% buffer B in 60 min at a flow rate of 250 nl/min. The mass spectrometer was operated in positive ionization mode with nanospray voltage set at 2.2 kV and source temperature at 250°C. Ultramark 1621 for the FT mass analyzer was used for external calibration prior the analyses, and an internal calibration was performed using the background polysiloxane ion signal at m/z 445.1200. The acquisition was performed in data-dependent acquisition mode and full MS scans with 1 micro scans at resolution of 60,000 were used over a mass range of m/z 350-2000 with detection in the Orbitrap. Auto gain control (AGC) was set to 1E6, dynamic exclusion (60 seconds) and charge state filtering disqualifying singly charged peptides was activated. In each cycle of data-dependent acquisition analysis, following each survey scan, the top ten most intense ions with multiple charged ions above a threshold ion count of 5000 were selected for fragmentation at normalized collision energy of 35%. Fragment ion spectra produced via collision-induced dissociation (CID) were acquired in the Ion Trap, AGC was set to 5e4, isolation window of 2.0 m/z, activation time of 0.1ms and maximum injection time of 100 ms was used. All data were acquired with Xcalibur software v2.2. Acquired spectra for the different LDL samples and tryptic peptides derived from hcVSMC cells were analyzed using the Proteome Discoverer software suite (v2.0, Thermo Fisher Scientific) and the Mascot search engine (v2.5, Matrix Science). The data were searched against the Swiss-Prot human database (v.2017/10). At the MS1 level, a precursor ion mass tolerance of 7 ppm was used, and up to three missed cleavages were allowed. The fragment ion mass tolerance was set to 0.5 Da for MS2 spectra. Oxidation of methionine, and N-terminal protein acetylation were defined as variable modifications whereas carbamidomethylation on cysteines was set as a fixed modification. False discovery rate (FDR) in peptide identification was limited to a maximum of 5% by using a decoy database. Quantitation data were retrieved from the “Precursor ion area detector” node from Proteome Discoverer (v2.0) using 2 ppm mass tolerance for the peptide extracted ion current (XIC). Finally, for the different LDL samples, the Skyline software v4.1 was used to build a spectral library for the ApoB100 protein with the corresponding identified spectra, and to extract all its peptide MS1 areas.