Age-dependent membrane release and degradation of full-length glycosylphosphatidylinositol-anchored proteins in rats

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

“…Principle: Three different modes for capture by the chip surface of bAChE complexes, TX-100 bAChE micelles, or bAChE liposomes (injection of 75 µL of suspension containing identical AChE volume activity at a flow rate of 15 µL/min), which had been prepared as described previously [29], as putative interaction partners for GPLD1 as well as other serum proteins were used: For covalent capture via the protein moiety of bAChE, the microfluidic channels of uncoated chips were primed by three injections of 150 µL each of immobilization buffer at a flow rate of 45 µL/min. Then the chip surface was activated by a 150 µL injection of 0.2 M EDC and 0.05 M Sulfo-NHS (mixed from 2x-stock solutions right before injection) at a flow rate of 45 µL/min.…”

“…After a waiting period of 3 min, subsequent injection of the suspension, an additional waiting period (flow rate 0) for 2 min and final washing of the channels with 300 µL of PBST at a flow rate of 180 µL/min, the residual activated groups on the chip surface were capped by injecting 200 µL of 1 M ethanolamine (pH 8.5) at a flow rate of 60 µL/min. For capture by α-toxin via the GPI glycan core of bAChE, α-toxin-coated plain AU chips were used as described previously [29]. For ionic capture, uncoated negatively charged and highly hydrophilic TiO 2 chips were used.…”

“…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].…”