Association of human aging with a functional variant of klotho

Abstract

Mice deficient in Klotho gene expression exhibit a syndrome resembling premature human aging. To determine whether variation in the human KLOTHO locus contributes to survival, we applied two newly characterized polymorphic microsatellite markers flanking the gene in a population-based association study. In a cohort chosen for its homogeneity, Bohemian Czechs, we demonstrated significant differences in selected marker allele frequencies between newborn and elderly individuals (P < 0.05). These results precipitated a search for functional variants of klotho. We identified an allele, termed KL-VS, containing six sequence variants in complete linkage disequilibrium, two of which result in amino acid substitutions F352V and C370S. Homozygous elderly individuals were underrepresented in three distinct populations: Bohemian Czechs, Baltimore Caucasians, and Baltimore African-Americans [combined odds ratio (OR) = 2.59, P < 0.0023]. In a transient transfection assay, secreted levels of klotho harboring V352 are reduced 6-fold, whereas extracellular levels of the S370 form are increased 2.9-fold. The V352/S370 double mutant exhibits an intermediate phenotype (1.6-fold increase), providing a rare example of intragenic complementation in cis by human single nucleotide polymorphisms. The remarkable conservation of F352 among homologous proteins suggests that it is functionally important. The corresponding substitution, F289V, in the closest human klotho paralog with a known substrate, cBGL1, completely eliminates its ability to cleave p-nitrophenyl-beta-D-glucoside. These results suggest that the KL-VS allele influences the trafficking and catalytic activity of klotho, and that variation in klotho function contributes to heterogeneity in the onset and severity of human age-related phenotypes.

Figure 1

Sequence variation in the KLOTHO locus. (A) Schematic of mutations detected in KLOTHO. (B) Amino acid sequence of klotho amino acid positions 346–374 and homologous proteins. Klotho has two homologous domains, here labeled “Klotho1” and “Klotho2.” The common allele of klotho is labeled “Klotho1-wt” and the rare allele is labeled “Klotho1-mt.” Alignment was performed using the CLUSTALW program (MACVECTOR 6.5.1). Amino acids found in at least 9 of 16 positions are shaded in black. Conservation of amino acid character in at least 9 of 16 positions is indicated by gray shading. The black arrow heads indicate amino acid positions at which mutations in klotho have been detected.

Figure 2

Kaplan–Meier survival curve for Czech elderly stratified by genotype. Error bars depict standard error. Individuals homozygous for V352 were too few to generate a survival curve.

Figure 3

Extracellular and intracellular levels of klotho from transiently transfected HeLa cells. (A) Western blot probed with anti-V5 or anti-NPTII antibody. Extracellular klotho is glycosylated, resulting in a 5-kDa increase in molecular mass (data not shown). (B) Bar graph depicting the extracellular to intracellular ratio of klotho protein normalized to wild-type from at least three independent assays. Error bars represent 1 SD. All ratios are significantly different from each other and wild-type (P < 0.05).

Figure 4

Enzymatic activity of wild-type and F289V substituted human cytosolic β-glucosidase (cBGL1). (A) Coomassie-stained gel demonstrating protein purification of cBGL1-GST fusions in elution fractions E2 + E3 (cBGL1-F) and E1 + E2 (cBGL1-V). No other proteins were visualized in these lanes (data not shown). (B and D) Bar graphs depicting enzymatic activity against p-nitrophenyl-β-D-glucoside. (C) Coomassie-stained gel demonstrating protein expression of cBGL1-FS and cBGL1-VS. *, No measurable enzymatic activity.