Indicating sKl’s affinity for lipid rafts (83). F ster resonance energy transfer (FRET) and fluorescence lifetime imaging microscopy research demonstrated sKl alters lipid organization and decreases membrane order within rafts (83). Studies haveFrontiers in Endocrinology | www.frontiersin.orgshown that inhibition of PI3K-dependent TRPC6 function underlies cardioprotection by sKl (84). sKl also selectively downregulated development factor-driven PI3KAkt signaling and TRPC6 channel function in lipid rafts, but not in non-lipid raft regions (83). In vitro binding assays and competitors experiments working with TRPC6-based functional assays identified 2,3-sialyllactose inside the glycan of GM1 and GM3 gangliosides as the minimal motif essential for sKl binding and regulation of TRPC6 in lipid rafts (83). Additionally, these assays demonstrated that sKl affinity is 300-fold higher for clustered two,3-sialyllactose compared with cost-free two,3-sialyllactoses which supports the notion that lipid rafts enriched in 2,3-sialyllactose-containing GM1 and GM3 gangliosides are helpful targets for physiologically low circulating concentrations of sKl ( 30 pM) (83). Sialylated glycans bind especially to a variety of glycan-binding proteins, but these binding interactions tend to be of low affinity. The formation of glycan clusters can be a popular mechanism that generates high affinity biologically relevant binding internet sites for multivalent glycan-binding proteins (85). Furthermore, sKl is probably multivalent resulting from the fact that sKl forms dimers and every unit includes two hugely homologous KL1 and KL2 domains with prospective glycan-binding activity (86). The multimeric nature of sKl and also the clustering of gangliosides most likely clarify why circulating sKl preferentially targets GM1 and GM3 clustered in lipid rafts instead of un-clustered GM1 and GM3 present in non-raft membranes or isolated two,3-sialyllactose residues present in glycoproteins (Figure 1). The concept of sKl particularly binding lipid rafts was further supported by FRET experiments in live cells that showed sKl selectively interacts with lipid raft-associated GM1, as well as permeation experiments Algo bio Inhibitors Related Products utilizing hexyltriphenylphosphonium (C6TPP) showing sKl has no effect on disordered membranes (i.e., non-lipid raft membrane regions) (83). The in vivo relevance of those findings was confirmed by the discovery that raft-dependent PI3K signaling is upregulated in klotho– mouse hearts compared with WT mouse hearts (83). By contrast, PI3K signaling in non-raft membranes is not diverse involving WT and klotho– mouse hearts (83). To additional support the notion that sKl binds sialogangliosides in lipid rafts to regulate TRPC6 and cardioprotection, the investigators determined a modeled structure of sKl by homology modeling and utilized docking protocols to examine the potential binding sites in sKl for two,3-sialyllactose (87). It was shown that Arg148, His246, plus the 465EWHR468 motif identified within the KL1 domain of sKl are important for binding 2,3-sialyllactose (87). Binding experiments employing biolayer inferometry showed the KL1 domain alone indeed binds 2,3-sialyllactose having a Kd worth that is comparable to that reported for the entire ectodomain of sKl (83, 87). Ultimately, purified recombinant KL1 domain inhibits TRPC6 in cultured cells and protects against stress-induced cardiac hypertrophy in mice (87). Overall, these research present compelling evidence supporting that sialogangliosides GM1 and GM3 and lipid rafts can serve as membrane receptors for.
