Structure-function relationships of the soluble form of the antiaging protein Klotho have therapeutic implications for managing kidney disease

Abstract

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.

Abnormally decreased renal Klotho is linked to endoplasmic reticulum-associated degradation in mice

“…CKD is characterized by persistent ER stress that yields increased accumulation of misfolded or unassembled proteins (including Klotho) in the ER. Because Klotho contains eight putative N-linked glycosylation sites, it is presumably subjected to post-translational modifications before maturation [5]. As an important compensatory mechanism, ERAD is activated to alleviate protein overload and maintain normal cell function in the context of persistent ER stress.…”

“…mKlotho is a type 1 transmembrane glycoprotein with a short intracellular domain, membrane spanning domain, and large extracellular domain. sKlotho is produced by ectodomain shed of mKlotho or alternative Klotho mRNA splicing [5]. Because Klotho is mainly generated by the kidney, it is easily understood that Klotho expression is influenced by the state of kidney function [6,7].…”

New Opportunities in Glycan Engineering for Therapeutic Proteins

“…β1,4GalNAcT3 [ 51 ] and β1,4GalNAcT4 [ 52 ] account for GalNAc transfer and have a broad tissue expression coverage including fetal kidney and brain. It has been reported that adding a carboxyl-terminal 19-amino-acid α-helix stretch with several basic amino acids is sufficient to mediate GalNAc transfer to N-linked oligosaccharides [ 47 , 53 , 54 ]. Other GalNAc motifs involve three structural loops with aromatic side chains [ 50 ], as well as additional unidentified motifs [ 54 ].…”

Klotho and calciprotein particles as therapeutic targets against accelerated ageing

“…Each domain had weak homology to the family 1 glycosidases. It is still controversial whether Klotho protein has enzymatic activity as a glycosidase [42][43][44][45][46], because two conserved catalytic glutamate residues essential for the glycosidase activity and thus conserved in all the family 1 glycosidases are replaced with other amino acids in Klotho protein.…”