Identification of the salusin-β receptor using proteoliposomes embedded with endogenous membrane proteins

Abstract

Although orphan G protein-coupled receptors (GPCRs) have been used as targets to discover unidentified natural ligands, increasing numbers of non-GPCRs have been found to mediate important biological functions. Bioinformatics of genome and cDNA resources predict putative bioactive peptides, demanding an alternative approach to efficiently unravel cell surface targets. In silico analysis of a full-length cDNA library previously allowed us to identify salusin-β, a parasympathomimetic/pro-atherosclerotic peptide with unique physicochemical properties. Here, we show that the β-chain of ATP synthase is a cell surface receptor for salusin-β by utilizing artificial liposomes embedded with endogenous membrane proteins directly transferred from animal tissues while retaining the ligand-binding capability. Conventional techniques using detergents identified a β-actin-profilin complex as membrane-associated salusin-β-binding proteins, but failed to identify the cell surface receptor. Since the α-chain of ATP synthase is a principal cell surface target for angiostatin, a potent endogenous angiogenesis inhibitor, we investigated whether salusin-β modulates angiogenesis. Salusin-β inhibited cell surface ATP synthase activity and prevented sarcoma cell-induced angiogenesis in an in vivo mouse air sac model. Therefore, salusin-β binds to membrane-bound ATP synthase and acts as an angiogenesis inhibitor. The current methodology allows the identification of novel cell surface targets, irrespective of the receptor structure.

Suprabasin-derived bioactive peptides identified by plasma peptidomics

“…Cells were then washed twice with PBS and fixed with 4% paraformaldehyde for 30 min. The nuclei were counterstained using DAPI Fluoromount-G (SouthernBiotech, Birmingham, AL, USA) and fluorescence was detected using an LSM710 confocal microscope (Carl Zeiss) as described 53 , 54 . For quantification of SBSN_HUMAN[225–237]- and SBSN_HUMAN[243–259]-binding to cultured cells, confluent cultures of A10 cells and HAoSMCs in non-coated 96 well black plates were incubated with serum-starved medium for 16 h, and further incubated for 5, 15, 30, 45, 60, 75, 90, or 105 min after addition of 10 –6 M FAM-labeled SBSN_HUMAN[225–237] or SBSN_HUMAN[243–259].…”

“…Cells were washed three times with HBSS and fluorescence was measured using a POWERSCAN HT plate reader (BioTek Instruments). Cell-bound-specific fluorescence was calculated by subtracting non-specific adherent fluorescence arising from peptide material bound to the inside wall of the 96 well plate from the total fluorescence detected in each well 53 .…”

“…Western blotting was performed essentially as described 53 , 59 . Confluent HAoSMCs (1.2 × 10 6 cells) deprived of serum for 16 h were incubated with SBSN_HUMAN[225–237] or SBSN_HUMAN[243–259] for the indicated time, washed three times with phosphate-buffered saline (PBS) and solubilized in RIPA Lysis and Extraction Buffer (Thermo Fisher Scientific) containing 10 μM protein inhibitors (Thermo Fisher Scientific).…”

“…Cell nuclei were counterstained using DAPI Fluoromount-G (SouthernBiotech). Laser scanning confocal microscopy was performed using an LSM710 confocal microscope (Carl Zeiss) 53 , 54 .…”

Molecular form and concentration of serum α2-macroglobulin in diabetes

“…SDS-PAGE was performed using purified human α 2 -macroglobulin protein and human serum essentially as described 61,62 except for the following modifications. Ten-microliters of fresh human serum diluted to 1:80 and 0.5 µg of purified α 2 -macroglobulin protein pretreated with or without DTT were subjected to 4–20% gradient gel electrophoresis using Mini-PROTEAN ® TGX™ and subsequent immunoblotting using human α 2 -macroglobulin antibody.…”