The human Klotho VS variant: focus on the processing and function of the V, S and VS isoforms
Tucker Zhou, Tracey Beth
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Klotho (KL), an anti-aging protein, attracted interest in the aging field because of the dramatic phenotype of KL deficient mice and its connection to signaling pathways implicated in aging. The KLVS variant consists of the F352V (KLV) and C370S (KLS) substitutions. It was detected in genome wide association studies (GWAS) that linked it to alterations in longevity and disease risk. The molecular mechanism(s) underlying these associations are unknown. To understand how KL increases the risk of age-related diseases, the studies in this dissertation investigated whether expression of the KLVS variant, when compared to wildtype (KLWT), displays differences in processing, protein-protein interactions and enzymatic activity. Differences in processing were evaluated by studying changes in shedding, half-life and plasma membrane localization of KL variants. The decrease in KLV shedding, as measured by the intracellular: extracellular ratio, were explained by a decreased half-life. This decreased half-life is potentially due to decreased KLV plasma membrane localization, which is attenuated by co-expression of dominant negative dynamin, suggesting a role of endocytosis in these differences. To assess whether there are changes in KLVS protein-protein interactions, differences in dimerization were measured by Blue Native gel electrophoresis and cross-linking. KLV dimerization was increased while KLS and KLVS variants decreased dimerization. Co-immunoprecipitation of tagged KL assessed whether these changes were due to alterations in homodimerization. The presence of KLVS in dimers decreased the levels of immunoprecipitated KL suggesting KLVS decreases homodimerization. Changes in heterodimerization of KLVS with fibroblast growth factor receptor (FGFR) 1c were also investigated through co-immunoprecipitation. KLVS increased heterodimerization with FGFR1c. Addition of FGF23, for which KL is a co-receptor, showed that KLVS increases FGF signaling downstream of FGFR1c. To determine differences in enzymatic activity of KLVS, 4-metylumbelliferyl-beta-D-glucuronide was used to measure alterations in glucuronidase activity. Results showed that KLVS had decreased enzymatic activity compared to KLWT. These findings are the first to show that KLVS leads to differences in function as demonstrated by decreased homodimerization and enzymatic activity and increased heterodimerization with FGFR1c. Given the association of KLVS with disease and longevity, these results suggest that these functions are integral in KL's anti-aging role in humans.