Modeled structural basis for the recognition of α2–3‐sialyllactose by soluble Klotho

Abstract

Soluble Klotho (sKlotho) is the shed ectodomain of antiaging membrane Klotho that contains 2 extracellular domains KL1 and KL2, each of which shares sequence homology to glycosyl hydrolases. sKlotho elicits pleiotropic cellular responses with a poorly understood mechanism of action. Notably, in injury settings, sKlotho confers cardiac and renal protection by down-regulating calcium-permeable transient receptor potential canonical type isoform 6 (TRPC6) channels in cardiomyocytes and glomerular podocytes. Inhibition of PI3K-dependent exocytosis of TRPC6 is thought to be the underlying mechanism, and recent studies showed that sKlotho interacts with α2-3-sialyllactose-containing gangliosides enriched in lipid rafts to inhibit raft-dependent PI3K signaling. However, the structural basis for binding and recognition of α2-3-sialyllactose by sKlotho is unknown. Using homology modeling followed by docking, we identified key protein residues in the KL1 domain that are likely involved in binding sialyllactose. Functional experiments based on the ability of Klotho to down-regulate TRPC6 channel activity confirm the importance of these residues. Furthermore, KL1 domain binds α2-3-sialyllactose, down-regulates TRPC6 channels, and exerts protection against stress-induced cardiac hypertrophy in mice. Our results support the notion that sialogangliosides and lipid rafts are membrane receptors for sKlotho and that the KL1 domain is sufficient for the tested biologic activities. These findings can help guide the design of a simpler Klotho mimetic.-Wright, J. D., An, S.-W., Xie, J., Yoon, J., Nischan, N., Kohler, J. J., Oliver, N., Lim, C., Huang, C.-L. Modeled structural basis for the recognition of α2-3-sialyllactose by soluble Klotho.

Soluble klotho regulates TRPC6 calcium signaling via lipid rafts, independent of the FGFR‐FGF23 pathway

“…The aKL1 and aKL2 domain each adopts a triosephosphate isomerase barrel fold, comprising a central core formed by b-strands stabilized by a-helices on the surface. a2-3-Sialyllactose is predicted to bind in this central core (9), in accord with its binding in b-strand cores seen in crystal structures of a2-3sialyllactose-bound proteins such as hemagglutininneuraminidase (PDB entries 5b2d, 1z4x) (11,12) and…”

“…sKlotho consists of 2 homologous domains, KL1 and KL2, at residues 57-506 (aKL1) and residues 515-953 (aKL2), respectively. Using homology modeling and molecular docking, we had elucidated residues within aKL1 domain for a2-3-sialyllactose recognition and confirmed the predictions by mutagenesis and functional studies (9). These studies reveal that binding to a2-3sialyllactose of gangliosides in lipid rafts and inhibition of growth factor-stimulated PI3K-dependent TRPC6 channel exocytosis underlies the mechanism of cardioprotection by sKlotho.…”

A Controlled Increase in Dietary Phosphate Elevates BP in Healthy Human Subjects

“…38 Recombinant Klotho administration to FGF-23 knockout mice is reported to exhibit a phosphaturic effect, 45 albeit with n=4, raising the possibility that proximal tubule effects of klotho are not confined to its role as coreceptor with membrane FGFR1c for circulating FGF-23 agonism. 46 The present results with elevated Klotho and FGF-23 levels leave open the question as to whether both of the reported proximal tubule effects of the Klotho response to hyperphosphatemia [i.e., phosphaturia and decreased 1,25(OH) 2 D] are dependent on or independent of increased FGF-23 in humans. The elevations in serum and urinary Klotho levels in response to hyperphosphatemia in humans reinforce the mounting evidence for Klotho's key role as a homeostatic phosphatonin and, potentially, for homeostatic beneficial effects in multiple organs.…”

FGF23 expression is stimulated in transgenic α-Klotho longevity mouse model

“…Additional studies found that sKL inhibits TNF-α, IGF-1, Wnt, and TGF-β signaling (1,(14)(15)(16)(17)(18). In addition, sKL is proposed to bind to gangliosides containing α-2-3-sialyllactose in lipid rafts to inhibit PI3K signaling (19), as well as regulate calcium-permeable transient receptor potential canonical type isoform 6 (TRPC6) channels (20).…”