Identification of Cleavage Sites Leading to the Shed Form of the Anti-Aging Protein Klotho

Abstract

Membrane protein shedding is a critical step in many normal and pathological processes. The anti-aging protein klotho (KL), mainly expressed in kidney and brain, is secreted into the serum and CSF, respectively. KL is proteolytically released, or shed, from the cell surface by ADAM10 and ADAM17, which are the α-secretases that also cleave the amyloid precursor protein and other proteins. The transmembrane KL is a coreceptor with the FGF receptor for FGF23, whereas the shed form acts as a circulating hormone. However, the precise cleavage sites in KL are unknown. KL contains two major cleavage sites: one close to the juxtamembrane region and another between the KL1 and KL2 domains. We identified the cleavage site involved in KL release by mutating potential sheddase(s) recognition sequences and examining the production of the KL extracellular fragments in transfected COS-7 cells. Deletion of amino acids T958 and L959 results in a 50–60% reduction in KL shedding, and an additional P954E mutation results in further reduction of KL shedding by 70–80%. Deletion of amino acids 954–962 resulted in a 94% reduction in KL shedding. This mutant also had moderately decreased cell surface expression, yet had overall similar subcellular localization as that of WT KL, as demonstrated by immunofluorescence. Cleavage-resistant mutants could function as a FGFR coreceptor for FGF23, but they lost activity as a soluble form of KL in proliferation and transcriptional reporter assays. Cleavage between the KL1 and KL2 domains is dependent on juxtamembrane cleavage. Our results shed light onto mechanisms underlying KL release from the cell membrane and provide a target for potential pharmacologic interventions aimed at regulating KL secretion.

Renal Production, Uptake, and Handling of Circulating αKlotho

“…[50][51][52] Studies on cultured cell and kidney slices indicated that a-secretase (ADAM 10/ 17) and b-secretase modulate aKlotho ectodomain shedding and becoming soluble aKlotho protein, 18,19 by acting on two cleavage sites: one close to the juxtamembrane region (aa 950 approximately981) and another between the KL1 and KL2 domains. 53 To examine if secretases modulate circulating aKlotho in vivo, we injected a-secretase inhibitor doxycline hyclate and/or b-secretase inhibitor III into the intraperitoneal cavity of normal mice for 2 days and determined serum aKlotho in 48 hours. Serum aKlotho levels were decreased with either one or both a-and b-secretase inhibitors ( Figure 1E) supporting the notion that aKlotho release depended on secretases.…”

Klotho/FGF23 Axis in Chronic Kidney Disease and Cardiovascular Disease

“…ing of 2 repeat sequences (Kl1 and Kl2) can be shed by secretases to yield full fragment and Kl1 fragment of extracellular domain, respectively, and released into the circulation as cleaved Klotho [5,6] . Another form of Klotho protein encompassing the Kl1 fragment is generated by alternative transcript splicing and is called secreted Klotho [1,7] .…”

Human alternative Klotho mRNA is a nonsense-mediated mRNA decay target inefficiently spliced in renal disease

“…Secondly, in the absence of secreted Klotho, the proteins commonly designated as circulating or soluble Klotho are likely derived from cleaved membrane-bound Klotho only, and proteolytic cleavage would gain relevance as the process generating circulating Klotho (13,14,(35)(36)(37). An experiment performed by Hu et al is in line with cleaved Klotho as the sole contributor to circulating Klotho; Hu et al show that inhibition of both α-and β-secretases for 48 hours in mice virtually depletes circulating Klotho (38).…”