The fortuitously discovered antiaging membrane protein αKlotho (Klotho) is highly expressed in the kidney, and deletion of the Klotho gene in mice causes a phenotype strikingly similar to that of chronic kidney disease (CKD). Klotho functions as a co-receptor for fibroblast growth factor 23 (FGF23) signaling, whereas its shed extracellular domain, soluble Klotho (sKlotho), carrying glycosidase activity, is a humoral factor that regulates renal health. Low sKlotho in CKD is associated with disease progression, and sKlotho supplementation has emerged as a potential therapeutic strategy for managing CKD. Here, we explored the structure-function relationship and post-translational modifications of sKlotho variants to guide the future design of sKlotho-based therapeutics. Chinese hamster ovary (CHO)- and human embryonic kidney (HEK)-derived WT sKlotho proteins had varied activities in FGF23 co-receptor and β-glucuronidase assays in vitro and distinct properties in vivo Sialidase treatment of heavily sialylated CHO-sKlotho increased its co-receptor activity 3-fold, yet it remained less active than hyposialylated HEK-sKlotho. MS and glycopeptide-mapping analyses revealed that HEK-sKlotho is uniquely modified with an unusual N-glycan structure consisting of N,N'-di-N-acetyllactose diamine at multiple N-linked sites, one of which at Asn-126 was adjacent to a putative GalNAc transfer motif. Site-directed mutagenesis and structural modeling analyses directly implicated N-glycans in Klotho's protein folding and function. Moreover, the introduction of two catalytic glutamate residues conserved across glycosidases into sKlotho enhanced its glucuronidase activity but decreased its FGF23 co-receptor activity, suggesting that these two functions might be structurally divergent. These findings open up opportunities for rational engineering of pharmacologically enhanced sKlotho therapeutics for managing kidney disease.

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