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.