Abstract
Glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-APs) are anchored at the surface of mammalian blood and tissue cells through a carboxy-terminal GPI glycolipid. Eventually, they are released into incubation medium in vitro and blood in vivo and subsequently inserted into neighboring cells, potentially leading to inappropriate surface expression or lysis. To obtain first insight into the potential (patho)physiological relevance of intercellular GPI-AP transfer and its biochemical characterization, a cell-free chip- and microfluidic channel-based sensing system was introduced. For this, rat or human adipocyte or erythrocyte plasma membranes (PM) were covalently captured by the TiO2 chip surface operating as the acceptor PM. To measure transfer between PM, donor erythrocyte or adipocyte PM were injected into the channels of a flow chamber, incubated, and washed out, and the type and amount of proteins which had been transferred to acceptor PM evaluated with specific antibodies. Antibody binding was detected as phase shift of horizontal surface acoustic waves propagating over the chip surface. Time- and temperature-dependent transfer, which did not rely on fusion of donor and acceptor PM, was detected for GPI-APs, but not typical transmembrane proteins. Transfer of GPI-APs was found to be prevented by α-toxin, which binds to the glycan core of GPI anchors, and serum proteins in concentration-dependent fashion. Blockade of transfer, which was restored by synthetic phosphoinositolglycans mimicking the glycan core of GPI anchors, led to accumulation in the chip channels of full-length GPI-APs in association with phospholipids and cholesterol in non-membrane structures. Strikingly, efficacy of transfer between adipocytes and erythrocytes was determined by the metabolic state (genotype and feeding state) of the rats, which were used as source for the PM and sera, with upregulation in obese and diabetic rats and counterbalance by serum proteins. The novel chip-based sensing system for GPI-AP transfer may be useful for the prediction and stratification of metabolic diseases as well as elucidation of the putative role of intercellular transfer of cell surface proteins, such as GPI-APs, in (patho)physiological mechanisms.
Highlights
Following removal of Ca2+ by EGTA and injection of NaCl to avoid fusion of the subsequently injected donor plasma membranes (PM) with the acceptor PM as well as their unspecific binding to the chip surface, respectively, the chips were ready for use as acceptor for Glycosylphosphatidylinositol-anchored proteins (GPI-APs) in case of their putative transfer
Restoration of GPLD1-blocked GPI-AP transfer by PIG41 was observed with each of the six donor–acceptor PM combinations and found to depend on the concentration with half-maximal effect at 7–10 μM (Figure 8d). These findings strongly suggested that (i) GPLD1 represents one of the serum proteins interfering with transfer of GPI-APs and (ii) its inhibitory potency is due to interaction with the core glycan rather than lipolytic cleavage of the GPI anchor of GPI-APs (Figure 1c)
The cell-free chip-based sensing assay for the transfer of full-length GPI-anchored cell surface proteins between PM, introduced in the present study, demonstrated its dependence on the metabolic state of the donor organism and its control by serum proteins
Summary
Glycosylphosphatidylinositol-anchored proteins (GPI-APs) represent a specific class of cell surface proteins, which lack proteinaceous transmembrane domains and in humans encompass about 150 members ([7], Uniprot database). They are constituted by a hydrophilic protein moiety of variable size (1.5–200 kDa) and a glycosylphosphatidylinositol (GPI) moiety [8,9,10]. This amphiphilic GPI moiety consists of phosphatidylinositol and the core glycan, which is conserved from yeast to man and modified by additional glycan side chains [11]. Of particular relevance is the possibility of intercellular transfer (i.e., from the PM of donor cells to the PM of acceptor cells), which relies on the presence of the full-length GPI anchor (i.e., including its diacylglycerol/phosphatidate moiety) and the resulting biophysical consequences
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