Soluble klotho binds monosialoganglioside to regulate membrane microdomains and growth factor signaling

Abstract

Soluble klotho, the shed ectodomain of the antiaging membrane protein α-klotho, is a pleiotropic endocrine/paracrine factor with no known receptors and poorly understood mechanism of action. Soluble klotho down-regulates growth factor-driven PI3K signaling, contributing to extension of lifespan, cardioprotection, and tumor inhibition. Here we show that soluble klotho binds membrane lipid rafts. Klotho binding to rafts alters lipid organization, decreases membrane's propensity to form large ordered domains for endocytosis, and down-regulates raft-dependent PI3K/Akt signaling. We identify α2-3-sialyllactose present in the glycan of monosialogangliosides as targets of soluble klotho. α2-3-Sialyllactose is a common motif of glycans. To explain why klotho preferentially targets lipid rafts we show that clustering of gangliosides in lipid rafts is important. In vivo, raft-dependent PI3K signaling is up-regulated in klotho-deficient mouse hearts vs. wild-type hearts. Our results identify ganglioside-enriched lipid rafts to be receptors that mediate soluble klotho regulation of PI3K signaling. Targeting sialic acids may be a general mechanism for pleiotropic actions of soluble klotho.soluble klotho | lipid rafts | gangliosides | sialic acids | TRPC6 M ice homozygous for a severe hypomorphic α-klotho allele manifest multiple aging-related phenotypes and die prematurely at 2-3 mo after birth (1). α-Klotho is predominantly expressed in renal tubules, parathyroid glands, and epithelial cells of the choroids plexus, but not in myocardium. Overexpression of α-klotho extends life span in mice, indicating that it is an agingsuppression molecule (2). The full-length α-klotho protein is a single-pass membrane protein with a large extracellular domain, a membrane-spanning segment, and a short intracellular carboxyl terminus (1). Membranous α-klotho associates with FGF receptors to form coreceptors for the ligand FGF23, a bone-derived circulating hormone that plays an important role in phosphate homeostasis (3).The ectodomain of α-klotho, ∼950 aa in length, is composed of two internal repeats, KL1 and KL2, each sharing amino acid sequence homology to family-1 glycosidases (4). The ectodomain (soluble klotho, sKL) is released into the systemic circulation, urine, and cerebrospinal fluid and functions as a humoral factor (2, 5). To date, more than 10 different functions of sKL have been described (2, 6-9). sKL regulates ion transporters, antagonizes Wnt and TGF-β1signaling to suppress cellular senescence, tissue fibrosis, and cancer metastasis, and inhibits insulin and insulin-like growth factor-1 (IGF1)-driven PI3K/Akt signaling contributing to extension of lifespan in mice, cardioprotection, and inhibition of tumor cell proliferation. A fundamental gap in the understanding of sKL's mechanism of action is the lack of knowledge of potential membrane receptor(s) that mediate cellular responses to sKL.Lipid rafts are highly dynamic, cholesterol-and sphingolipidrich membrane microdomains that compartmentalize cellular processes such as ...

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

“…Coexpression of the plasmid encoding sKlotho inhibits diacylglycerol (DAG)‐stimulated TRPC6 channel function by ~50% compared to the control vector ( Fig. 5A) (16, 22). DAG increases TRPC6 channel activity by activation of channel gating as well as by stimulation of TRPC6 exocytosis (16, 22).…”

“…5A) (16, 22). DAG increases TRPC6 channel activity by activation of channel gating as well as by stimulation of TRPC6 exocytosis (16, 22). sKlotho inhibits DAG‐activated TRPC6 channel activity partially because it inhibits DAG stimulation of channel exocytosis but does not affect channel gating.…”

“…TRPC6 currents were recorded from human embryonic kidney 293 (HEK293 cells expressing recombinant TRPC6 with or without ectodomain of Klotho proteins, as previously described (16, 22). For testing recombinant KL1, cells expressing TRPC6 were treated with or without KL1 (300 pM) in serum‐containing medium overnight.…”

“…This action of sKlotho regulates cell‐surface retention of target proteins. More recently, we reported that sKlotho also targets α2–3‐sialyllactose of glycolipid gangliosides highly enriched in lipid rafts and affects multiple cellular effects mediated by lipid rafts (22). Lipid rafts are small heterogeneous, dynamic cholesterol‐ and sphingolipid‐rich membrane microdomains that serve as platforms for multiple cellular processes such as signal transduction and membrane trafficking (23).…”

“…Lipid rafts are small heterogeneous, dynamic cholesterol‐ and sphingolipid‐rich membrane microdomains that serve as platforms for multiple cellular processes such as signal transduction and membrane trafficking (23). We showed that sKlotho binds to α2–3‐sialyllactose of gangliosides GM1 and GM3 that are highly enriched in lipid rafts and inhibits raft‐dependent PI3K signaling and PI3K‐dependent exocytosis of transient receptor potential canonical type isoform 6 (TRPC6) channels, thus down‐regulating TRPC6 (22). In this study, we sought to elucidate the structural basis of sKlotho for α2–3‐sialyllactose recognition.…”