active centers of many glycosidases (2, 27), it has been proposed that other residues, perhaps N239 and S872 in human klotho, may serve as proton donor and nucleophile, respectively, or support alternative hydrolysis pathways. Interestingly, a novel Bk mouse protein termed klotho also lacks the corresponding glutamates, perhaps defining a distinct subset within the family 1 glycosidase superfamily (28). Thus, homozygotes for the KL-VS allele could have a severe deficiency of extracellular klotho function despite increased secretion of the protein. This scenario would recapitulate the situation in the hypomorphic mouse model of premature aging. Clearly, more details regard- ing klotho biology are needed to derive the precise pathogenetic mechanism.

proteins that may be required for klotho export, such as ER chaperones.

Surprisingly, extracellular levels of klotho harboring S370 are increased 2.9-fold. This substitution has the ability to comple- ment the V352 phenotype in cis, as demonstrated by the V352(cid:2) S370 double mutant which exhibits an intermediate phe- notype (1.6-fold increase). In screening a total of over 300 individuals taken from the geographically and(cid:2) or ethnically distinct cohorts used in this study, we have not found any individuals who harbor only V352 or S370. Although individuals harboring either of these mutations alone may exist at a low frequency in the population, it is also possible that neither genotype is viable. This hypothesis requires that secretion is essential to klotho function. In this scenario the V352 phenotype would correspond to that of a severe hypomorph, similar to the klotho mouse, and would be negatively selected. Likewise, if the homozygous disadvantage exhibited by KL-VS allele-containing individuals is due, at least in part, to increased extracellular klotho levels, individuals harboring only S370 would experience an even greater negative selection. Because the mutation iden- tified at C370 results in a serine residue, which is present in this position in mice (1) and rats (25), it is possible that this substitution contributes to species-specific differences in age- related phenotypes. Additional examples exist, including  -synuclein, in which protein coding changes that result in amino acids present in other organisms can lead to phenotypic variation in humans, including disease (26).

Multiple models of aging invoke accelerated or excessive posttranslational modification of proteins including glycation. Resultant advanced glycation end-products (AGEs) elicit a wide range of responses that have been proposed to contribute to many age related phenotypes, including atherosclerosis, Alzhei- mer’s disease, diabetic complications, and microvascular changes (29). It is possible that the proposed glycosidase activity of klotho retards the accumulation of AGEs. Alternatively, klotho may contribute to or regulate signaling cascades with downstream targets that influence aging. Based on observations in klotho- deficient mice, these targets may include factors involved in NO production and maintenance of proper endothelial function (4, 5). These hypotheses remain to be tested in humans or mouse models of normal or accelerated aging.

The remarkable evolutionary conservation of the F352 amino acid among homologous proteins suggests that it is likely to be important to klotho function. Indeed, the substitution of valine for phenylalanine at the corresponding residue in the closest human klotho paralog with a known substrate, cBGL1, com- ee-B-1 pletely eliminates the ability to cleave p-nitrophenyl- -D- glucoside. The introduction of serine at the position correspond- ing to klotho aa 370 failed to restore enzymatic activity. These results suggest that the KL-VS allele may exhibit both altered secretion and a loss of catalytic activity. Whereas both KL1 and KL2 lack one of two conserved glutamate residues found in

We thank Charlotte Ferencz for access to epidemiologic data from the Baltimore–Washington Infant Study (supported by NHLBI R37- HL25629) and Christopher Loffredo for access to samples. We thank Aravinda Chakravarti for useful suggestions. This work was supported by National Institutes of Health Grants AR21135 (to H.C.D.) and AR44702 (to I.M.); Grants IGA MZCR (6497-3, 6411-3, 6462-3, 6250-3, 6464-3, 00000064203) and MSMT CR (ME457, LN00AO79, 111300003) from the Czech Republic (to M.M., Jr., A.K., and M.M., Sr.); and U.S. Department of Energy Contract No. DE-AC03-76SF00098 (to I.S.M.). The Women’s Health and Aging Study is supported by NOI-AG-1-2112 (to L.F.). H.C.D. is an investigator in the Howard Hughes Medical Institute.

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