The klotho gene encodes a putative type I membrane protein, which consists of an N-terminal signal sequence, an extracellular domain with two internal repeats (KL1 and KL2), a single transmembrane domain, and a short intracellular domain (1). The two internal repeats share homology with members of the family 1 glycosidases and exhibit 20–40% sequence identity to aGeBs -glucosidases from bacteria and plants as well as mammalian lactase glycosylceramidase (1, 2).

Analysis of cDNA revealed that the klotho gene also expresses a secreted form, lacking KL2, transmembrane, and intracellular domains, due to alternative RNA splicing. Thus, klotho may also act as a humoral factor (1, 3). Further evidence for this hypoth- esis includes the observation that klotho mRNA expression was not detectable in many organs in which severe changes occur in klotho mice (1). Similarly, transgenic expression of klotho limited

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phosphotransferase II (Upstate Biotechnology, Lake Placid, NY) were carried out as specified by the manufacturers.

Plasmid Construction. The human KLOTHO expression vector (pSecKlwt-V5) was constructed by amplifying a 1,684-bp fragment from Human Placenta Marathon Ready cDNA (CLONTECH) with primers E1-cDNA and SecNoStop, by using the Advantage-GC cDNA PCR Kit (CLONTECH). The PCR   5 /I product was cloned into pcDNA3.1 V5 His-TOPO (Invitrogen) and verified by direct sequencing. The substitutions F352V and C370S were introduced with the QuickChange XL Site-Directed Mutagenesis Kit (Stratagene).

Protein Purification and Enzyme Assay. Total protein was isolated wit from logarithmically growing E. coli strain BL21-SI (GIBCO BRL) harboring the GST-tagged wild-type or mutant cBGL1 expression constructs. Following salt induction according to the manufacturer’s protocol, 100 ml of bacterial culture was pellet- ted and resuspended in 10 ml PBS supplemented with 1 Com- plete Mini protease inhibitor mixture tablet (Roche Molecular eratpl Biochemicals), and 10 l 1 M DTT. Cells were disrupted by re) pul sonication and further solubilized by the addition of 500 l of 20% Triton X-100 and gentle mixing for 30 min at 4°C. Samples FAL:xX were then centrifuged at 12,000 g for 10 min at 4°C, and the GST-tagged protein was purified from the supernatant with a HiTrap affinity column (Amersham Pharmacia). Elution frac- tions of 1 ml were collected. Purification was monitored by 4S/P SDS PAGE [NuPage 4–12% Bis-Tris (Invitrogen)] followed by   visualization with Bio-Safe Coomassie (Bio-Rad). -Glucosi- dase enzyme assays were performed as described (14).

The human cytosolic -glucosidase (cBGL1) expression vec- tor (pCBGDF) was constructed by amplifying a 1,474-bp frag- ment from Human Liver Quick Clone cDNA (CLONTECH)   _ with primers Glu5 and Glu3 , using Pfu turbo polymerase (Stratagene). The PCR product was cloned into the N-terminal GST-fusion expression vector pDEST15 by using the GATE-   WAY cloning system (GIBCO BRL), and verified by direct sequencing. The substitutions F289V and Q307S were intro- duced as described above. All primer sequences are available on request.

Statistics. Individual allele distributions were analyzed using the fu hahy sek/~a GAS package Version 2.3 (http: users.ox.ac.uk ayoung), and overall marker comparisons were made using the CLUMP pro- gram (15), which employs a Monte Carlo-based method for determining P. Individual genotype distributions were analyzed using Pearson’s (cid:3)2 test (http:(cid:2)(cid:2) home.clara.net(cid:2) sisa(cid:2) binomi- al.htm) and the CLUMP program was used to determine overall significance. Survival advantage or disadvantage was calculated as an odds ratio (OR) with a 95% confidence interval (CI) Ave://b   homa/b (http: home.clara.net sisa binomial.htm). Mantel–Haenszel log rank analysis with 1-sided P of Kaplan–Meier survival curves was performed using PRISM, Version 3.0 for Windows (Graph- Pad, San Diego). The combined population analysis was calcu- lated using the Mantel–Haenszel estimate of joint OR and significance (16).

Marker Genotyping. Multiple dinucleotide repeat sequences were identified by visual inspection of the genomic sequence for KLOTHO and flanking regions (GenBank accession nos. Z92540 and Z92541). Marker 1 (sense, 5(cid:2) -CATACGCAGAC-  CCAGAGCC; antisense, 5 -GCTTCTCATTACGAAAGTA- CAGCTGGG, italic sequence is a tail to force reaction to  completion) is 11 kb 3 of exon 5, and Marker 2 (sense, wwe5/- ns 5 -CTTACCAGCATGTGAGCTATC; antisense, 5 -TTTTA- an5! CAGGTGATCTGAGACTC) is 66 kb 5 of exon 1. Size frac- tionation of PCR products derived from multiple unrelated individuals was used to demonstrate polymorphic variation (data not shown). Markers were amplified using AmpliTaq Gold (Perkin–Elmer). Sense primers were fluorescently labeled, and product size was determined using a Perkin–Elmer (Abd) 373a DNA sequencer.

Results