Unprocessed serum glycosylphosphatidylinositol-anchored proteins are correlated to metabolic states

Abstract

Glycosylphosphatidylinositol-anchored proteins (GPI-AP), which represent about 1% of all proteins in eukaryotes, are constituted by a highly conserved hydrophobic glycolipidic membrane anchor (GPI) and variable large hydrophilic protein moieties 1-3 . On basis of their amphiphilic nature, GPI-AP equipped with the complete GPI anchor together with exogenous lipids (phospholipids, cholesterol), presumably required to shield their fatty acyl moieties from the aqueous environment, into extracellular complexes (GLEC) may be regarded as candidates for release from the extracellular face of plasma membranes upon exposure towards endogenous or exogenous cues, such as metabolites and mechanical forces. The putative release of GLEC was first studied with adipocytes since their plasma membranes undergo extensive stretching upon lipid filling and are in intimate contact with serum albumin and fatty acids. To avoid isolation of the presumably labile GLEC, a chipbased sensor was developed. It relies on specific capturing of the GLEC streaming through the microfluidic channels of the chip by their gold surface coated with α-toxin.Coating with α-toxin, which binds to the glycan core of the GPI anchor 4 , was performed with conventional coupling chemistry ( Supplementary Fig. 1). Any (covalent or secondary) interaction of materials with the chip surface will lead to right-ward shifts in phase and/or reductions in amplitude of the horizontal surface acoustic waves (SAW) propagating along the chip surface. This reflects mass loading and/or increased viscosity, respectively, exerted by the interacting materials 5-7 . Consequently, the coating with α-toxin per se (Supplementary Fig. 1) and the capturing of GLEC can be monitored by chipbased sensing (see below).Initially, the sensor was developed and validated using so-called extracellular vesicles (EV) as analytes. EV are membrane vesicles which are released from most cell types 8 , in particular upon challenge with exogenous stressors 9 . A subset of EV released from adipocytes into the incubation medium are known to harbor complete GPI-AP at the outer surface of their phospholipid bilayer 10,11 and consequently can be regarded as subtype of GLEC. Injection of EV isolated from rat adipocyte incubation medium into α-toxin-coated, but not albumin-coated chips caused volume-dependent phases shifts of the SAW (Fig. 1a). Depletion of the GPI-AP-harboring EV upon adsorption to α-toxin-coupled magnetic beads or cleavage of the GPI anchor by bacterial PI-PLC prior to injection (partially) prevented phase shift. The presence of GPI-AP, such as CD73, and phospholipids in the EV was shown by sequential binding "in sandwich" of anti-CD73 antibodies and the Ca 2+dependent phospholipid-sequestering protein annexin-V (in the presence of Ca 2+ , but not EGTA) to the chip (Fig. 1b). The specificity of detection of GPI-AP in association with lipids was confirmed by the lack of SAW phase shift using (i) chips (non-covalently) coated with rat serum albumin, (ii) EV depleted from GPI-AP or (iii) antib...

Transfer of membrane(s) matter(s)—non-genetic inheritance of (metabolic) phenotypes?

“…Specificity is guaranteed by various distinct mechanisms operating at the level of both acceptor and donor cells: With regard to acceptor cells, i) transfer of GPI-APs via EVs or micelle-like complexes seems to be restricted to very short distances, i.e., within the same tissue depot at a paracrine level, e.g., from large to small adipocytes at the immediate neighborhood (Müller, 2011), ii) endocrine transfer of GPI-APs via EVs or micellelike complexes escaping into the circulation is apparently hampered by cleavage through GPI-PLD, a major component of mammalian serum, as well as the interaction with GPI anchor-binding proteins, among them as a minor component serum albumin, and certain GPI-PLCs, which become converted from a cleaving-into a bindingprotein upon chelation of Ca 2+ /Mg 2+ (Müller et al, 2019;Müller et al, 2021a), iii) the interactions prevalent during ii) are presumably modulated by a complex and competitive interplay between the phosphoinositolglycan portion of full-length GPI-APs which become released via EVs or micelle-like complexes, and phosphoinositolglycan moieties of anchor-less GPI-APs which are generated by certain GPI-PLCs in response to insulin or sulfonylureas (Müller et al, 2005;Müller and Müller et al, 2023c).…”

“…It may be of considerable relevance that we and others have previously shown that certain environmental factors, such as hormones, nutrients, drugs, mechanical stress, serum proteins, metabolites, are able to modulate the topography (composition, arrangement) of non-genetic matter, encompassing micelle-like complexes and EVs, as well as the efficiency of their release from donor cells/tissues and their transfer to acceptor cells/tissues (Müller and Müller, 2023c). For example, serum levels of micelle-like GPI-AP complexes have been found to be significantly lower in high-fat fed and genetically predisposed obese rats, as well as in diabetic and obese humans (Müller et al, 2019). This effect was found to be even more pronounced with concomitant hyperinsulinemia and increasing age.…”

“…Finally, the view is explained that both intercellular (via both intra-and extracellular paths) and intergenerational transfer of nongenetic matter, encompassing MELs and EVs as well as micelle-like GPI-AP complexes carrying them, complements the repertoire of epigenetic mechanisms (for a review, see Dupont et al, 2012;Feil and Fraga, 2012). The explanation of phenotypic plasticity and the inheritance of acquired traits that is not based on the modifications of DNA or DNA-associated proteins has only recently become accessible to new technologies (e.g., Andrä et al, 2008;Müller et al, 2019). One reason for this is believed to rely on the adherence to the DNA-centric view of heredity that has persisted for almost a century.…”