Mapping the dynamic cell surface interactome of high-density lipoprotein reveals Aminopeptidase N as modulator of its endothelial uptake

Heterogeneous high-density lipoprotein (HDL) particles, which can contain hundreds of proteins, affect human health and disease through dynamic molecular interactions with cell surface proteins. How HDL mediates its long-range signaling functions and interactions with various cell types is largely unknown. Due to the complexity of HDL, we hypothesize that multiple receptors engage with HDL particles resulting in condition-dependent receptor-HDL interaction clusters at the cell surface. Here we used the mass spectrometry-based and light-controlled proximity labeling strategy LUX-MS in a discovery-driven manner to decode HDL-receptor interactions. Surfaceome nanoscale organization analysis of hepatocytes and endothelial cells using LUX-MS revealed that the previously known HDL-binding protein scavenger receptor SCRB1 is embedded in a cell surface protein community, which we term HDL synapse. Modulating the endothelial HDL synapse, composed of 60 proteins, by silencing individual members showed that the HDL synapse can be assembled in the absence of SCRB1 and that the members are interlinked. The aminopeptidase AMPN (also known as CD13) was identified as an HDL synapse member that directly influences HDL uptake into the primary human aortic endothelial cells (HAECs). Our data indicate that preformed cell surface residing protein complexes modulate HDL function and suggest new theragnostic opportunities. ### Competing Interest Statement The authors have declared no competing interest. * (auto-CSC) : Automated Cell Surface Capturing (Human endothelial somatic hybrid cells) : EA.hy926 (human hepatocellular carcinoma) : HEPG2 (HDL) : High-density lipoproteins (HAEC) : Human aortic endothelial cells (SOG) : Singlet oxygen generators (siRNA) : Small interfering RNA * ALCAM : CD166 antigen (CD166) ANPEP : Aminopeptidase N (AMPN/ CD13) APOA1 : Apolipoprotein A (APOA1) ASGR1 : Asialoglycoprotein receptor 1 (ASGR1) ASGR2 : Asialoglycoprotein receptor 1 (ASGR2) ATP1B1 : Sodium/potassium-transporting ATPase subunit beta-1 (AT1B1) ATP1B3 : Sodium/potassium-transporting ATPase subunit beta-1 (AT1B3) AXL : Tyrosine-protein kinase receptor UFO (UFO) DAG1 : Dystroglycan (DAG1) ESAM : endothelial cell-selective adhesion molecule (ESAM) F2R : Proteinase-activated receptor 1 (PAR1) ICAM1 : Intercellular adhesion molecule 1 (ICAM1) KDR : Vascular endothelial growth factor receptor 2 (VGFR2) LDLR : Low-density lipoprotein receptor (LDLR) MMRN2 : Multimerin-2 (MMRN2) NECTIN2 : Nectin-2 (NECT2/ PVRL2) NRP1 : Neuropilin-1 (NRP1) PECAM1 : Platelet endothelial cell adhesion molecule (PECA1) PROCR : Endothelial protein C receptor (EPCR) PTPRB : Receptor-type tyrosine-protein phosphatase beta (PTPRB) PTPRF : Receptor-type tyrosine-protein phosphatase F (PTPRF) PVR : Poliovirus receptor (PVR) S1PR1 : Sphingosine-1-phosphate receptor 1 (S1PR1) S1PR3 : Sphingosine 1-phosphate receptor 3 (S1PR3) SCARB1 : Scavenger receptor B1 (SCRB1) SCARF1 : Scavenger receptor class F member 1 (SREC) SERPINE1 : plasminogen activator inhibitor 1 (PAI1) SIRPA : Tyrosine-protein phosphatase non-receptor type substrate 1 (SHPS1) SLC3A2 : 4F2 cell-surface antigen heavy chain (4F2) SLITRK1 : SLIT and NTRK-like protein 1 (SLIK1) TEK : Angiopoietin-1 receptor (TIE2) THBD : Thrombomodulin (TRBM) TIE1 : Tyrosine-protein kinase receptor Tie-1 (TIE1) VEGFA : vascular endothelial growth factor A (VEGF-A)