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). Research 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 Petunidin (chloride) web PI3KAkt signaling and TRPC6 channel function in lipid rafts, but not in non-lipid raft regions (83). In vitro binding assays and competition experiments making use of TRPC6-based functional assays identified two,3-sialyllactose in the glycan of GM1 and GM3 gangliosides because the minimal motif required for sKl binding and regulation of TRPC6 in lipid rafts (83). In addition, these assays demonstrated that sKl affinity is 300-fold greater for clustered 2,3-sialyllactose compared with free two,3-sialyllactoses which supports the notion that lipid rafts enriched in 2,3-sialyllactose-containing GM1 and GM3 gangliosides are efficient targets for physiologically low Algo bio Inhibitors medchemexpress circulating concentrations of sKl ( 30 pM) (83). Sialylated glycans bind particularly to a number of glycan-binding proteins, but these binding interactions have a tendency to become of low affinity. The formation of glycan clusters is usually a popular mechanism that generates higher affinity biologically relevant binding web sites for multivalent glycan-binding proteins (85). In addition, sKl is probably multivalent because of the truth that sKl types dimers and every single 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 explain why circulating sKl preferentially targets GM1 and GM3 clustered in lipid rafts rather than 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 specifically binding lipid rafts was additional supported by FRET experiments in reside cells that showed sKl selectively interacts with lipid raft-associated GM1, as well as permeation experiments using hexyltriphenylphosphonium (C6TPP) displaying sKl has no impact on disordered membranes (i.e., non-lipid raft membrane regions) (83). The in vivo relevance of these 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 just isn’t different between WT and klotho– mouse hearts (83). To further help 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 prospective binding web sites in sKl for two,3-sialyllactose (87). It was shown that Arg148, His246, and also the 465EWHR468 motif identified inside the KL1 domain of sKl are vital for binding 2,3-sialyllactose (87). Binding experiments applying biolayer inferometry showed the KL1 domain alone indeed binds two,3-sialyllactose with a Kd value that’s comparable to that reported for the complete ectodomain of sKl (83, 87). Finally, purified recombinant KL1 domain inhibits TRPC6 in cultured cells and protects against stress-induced cardiac hypertrophy in mice (87). Overall, these studies supply compelling proof supporting that sialogangliosides GM1 and GM3 and lipid rafts can serve as membrane receptors for.
