Chip-based sensing for release of unprocessed cell surface proteins in vitro and in serum and its (patho)physiological relevance

Abstract

To study the possibility that certain components of eukaryotic plasma membranes are released under certain (patho)physiological conditions, a chip-based sensor was developed for the detection of cell surface proteins, which are anchored at the outer leaflet of eukaryotic plasma membranes by a covalently attached glycolipid, exclusively, and might be prone to spontaneous or regulated release on the basis of their amphiphilic character. For this, unprocessed, full-length glycosylphosphatidylinositol-anchored proteins (GPI-AP), together with associated phospholipids, were specifically captured and detected by a chip- and microfluidic channel-based sensor, leading to changes in phase and amplitude of surface acoustic waves (SAW) propagating over the chip surface. Unprocessed GPI-AP in complex with lipids were found to be released from rat adipocyte plasma membranes immobilized on the chip, which was dependent on the flow rate and composition of the buffer stream. The complexes were identified in the incubation medium of primary rat adipocytes, in correlation to the cell size, and in rat as well as human serum. With rats, the measured changes in SAW phase shift, reflecting specific mass/size or amount of the unprocessed GPI-AP in complex with lipids, and SAW amplitude, reflecting their viscoelasticity, enabled the differentiation between the lean and obese (high-fat diet) state, and the normal (Wistar) and hyperinsulinemic (Zucker fatty) as well as hyperinsulinemic hyperglycemic (Zucker diabetic fatty) state. Thus chip-based sensing for complexes of unprocessed GPI-AP and lipids reveals the inherently labile anchorage of GPI-AP at plasma membranes and their susceptibility for release in response to (intrinsic/extrinsic) cues of metabolic relevance and may, therefore, be useful for monitoring of (pre-)diabetic disease states.

Interaction of Full-Length Glycosylphosphatidylinositol-Anchored Proteins with Serum Proteins and Their Translocation to Cells In Vitro Depend on the (Pre-)Diabetic State in Rats and Humans

“…In agreement with previous studies [28,30], rat adipocytes that had been incubated in the absence (Figure 7a-f, black curves) or presence of micelle-like complexes (Figure 7a-f, blue curves), liposomes (data not shown), and reconstituted HDLs (Figure 7d, grey curve, recHDL) released phospholipids, bAChE, CD73, CD55, and aP into micelle-like complexes. The efficacies were comparable as revealed by the very similar stepwise increases in the phase shift upon consecutive injection of the adipocyte-free infranatant, annexin-V, anti-AChE, anti-rCD73 (specifically recognizing rat CD73), anti-CD55, and anti-aP antibodies into α-toxin-coated chips (Figure 7a,d).…”

“…The protocol encompasses four steps: (i) the translocation per se in the course of an initial incubation of primary rat adipocytes with micelle-like complexes, liposomes, or HDLs reconstituted with bAChE or hCD73 in the absence or presence of serum pro-teins, (ii) the recovery of the adipocytes harboring the translocated bAChE or hCD73 by centrifugation of the incubation medium through dinonylphtalate, (iii) the spontaneous release of the translocated bAChE or hCD73 from the washed adipocytes in the course of a subsequent incubation as a measure for bAChE or hCD73 translocated during the initial incubation, and (iv) the measurement of the amount of released (and initially translocated) bAChE or hCD73 by chip-based sensing. (ad i) Primary rat adipocytes were prepared from epididymal fat pads of male Wistar rats (140-160 g, fed ad libitum) as described previously [28], thereafter suspended in 2.5 mL of adipocyte buffer (20 mM Hepes/KOH, pH 7.4, 140 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl 2 , 1.2 mM MgSO 4 , 1.2 mM KH 2 PO 4 , 2% [w/v] BSA, 100 µg/mL gentamycin, 1 mM sodium pyruvate, 5.5 mM glucose) at a lipocrit of 0.1% and then incubated (under conditions as indicated in the figures) with various amounts of micelle-like complexes, liposomes or recHDL containing (or lacking) bAChE or hCD73 (prepared as described in [29] and the Supplementary Materials) in the presence or absence of serum samples (diluted 10-fold with PBS) in 20 mL plastic vessels in a shaking water bath (100 cycles/min) under constant bubbling with 5% CO 2 /95% O 2 . (ad ii) Thereafter, three 350-µL portions of the total mixtures were transferred to microfuge tubes (Beckman Inc., Krefeld, Germany) prefilled with 100 µL of dinonylphtalate and then centrifuged (1000× g, 1 min, 20 • C).…”

“…Chemical synthesis of PIG41, protein determination, preparation of primary rat adipocytes, assay for AChE enzymic activity, preparation of α-toxin from the culture supernatant of Clostridium septicum and bAChE from bovine erythrocytes, coupling of α-toxin to the gold chip surface of long-chain 3D CM-dextran sam ® 5 chips using conventional EDC/NHS-based protocol in a SamX instrument (SAW/Nanotemper, Bonn/Munich, Germany), and SAW measurement, instrumentation and evaluation were performed as has been described in detail previously [28][29][30]50]. Before injection of serum samples into CM-dextran chips, 0.1 vol.…”

“…GPI-APs that had retained the complete GPI moiety were identified in multimeric high-molecular aggregates in body fluids, such as seminal plasma [18]; microvesicles and exosomes released from blood and tissue cells [19][20][21][22][23]; lipoprotein-like particules, such as milk fat globules [24] and intestinal surfactant-like particles [25]; and lipoproteins [26,27]. Recently, full-length GPI-APs were detected in the incubation medium of isolated rat adipocyte plasma membranes and cultured rat adipocytes in vitro, as well as in serum of metabolically deranged rats and humans [28] or old rats [29]. Importantly, they were found to be embedded together with (lyso)phospholipids and cholesterol in micelle-like complexes, called micelle-like GPI-AP complexes, rather than in lipid-free aggregates, vesicles, or lipoproteins [30].…”

“…One protective mechanism may rely on the cleavage of the GPI anchor by GPI-specific phospholipase D (GPI-PLD) activity, which in mammalian serum is constituted solely by GPI-PLD1 (GPLD1), according to our current knowledge [36][37][38]. Recent studies revealed that the serum concentration of full-length GPI-APs is correlated to serum GPLD1 activity and blood glucose/plasma insulin levels in diabetic rats and human patients [28,30]. This was interpreted as counterregulation between the release of full-length GPI-APs into the circulation due to metabolic stress and their lipolytic degradation.…”