Chinese Medical Journal 2004;117(5):742-747
Klotho is a serum factor related to human aging

XIAO Neng-ming 肖能明,  ZHANG Yan-ming 张焱明,  ZHENG Quan 郑 权,  GU Jun 顾 军

XIAO Neng-ming 肖能明 (Department of Biochemistry and Molecular Biology, College of Life Science, Peking University, Beijing 100871, China)

ZHANG Yan-ming 张焱明 (Department of Biochemistry and Molecular Biology, College of Life Science, Peking University, Beijing 100871, China)

ZHENG Quan 郑 权 (Department of Biochemistry and Molecular Biology, College of Life Science, Peking University, Beijing 100871, China)

GU Jun 顾 军 (Department of Biochemistry and Molecular Biology, College of Life Science, Peking University, Beijing 100871, China)

Correspondence to:Gu Jun,Department of Biochemistry and Molecular Biology, College of Life Science, Peking University, Beijing 100871, China (Tel: 86-10-62759940. Fax:86-10-62756174. E-mail:gj@pku.edu.cn)
Keywords
klotho ; aging ; polyclonal antibody
Abstract
Background Does klotho (KL) protein exist in human serum, and is there any correlation between KL protein in serum with human aging? In order to answer those questions, we identified KL protein in human serum and established the correlation between KL protein in human serum and aging.
Methods We prepared a polyclonal antibody against human KL protein that was able to recognize the C-terminal of human secreted KL protein. Western blot and enzyme-linked immunosorbent assay (ELISA) were used to identify KL protein in human serum.
Results In Western blot, the antibody specifically recognized a 60-kD KL protein in both human and mice serum. The population aged from 0 to 91 years screened by ELISA revealed that the level of serum KL declined while age increased, though each individual level was variable and that the trend of decreasing in serum KL had no difference in sex.
Conclusion Our data suggest that KL is a serum factor related to human aging.
Klotho (KL) gene defected mice exhibit a syndrome resembling human aging, including arteriosclerosis, osteoporosis, emphysema, infertility and diseases related to vascular endothelial dysfunction.[1] The KL gene has two transcripts by an alternative splicing at RNA level. One encodes a putative single-pass membrane form protein; the other encodes, in both human and mouse, a putative secreted form protein that may acts as a humoral factor.[2,3] The membrane form KL protein consists of an N-terminal signal sequence, an extracellular domain with two internal repeats named after KL1 and KL2, a single transmembrane domain, and a short intracellular domain. Each internal repeat shares homology with the β-gucosidases of both bacteria and plants and with a lactase-phlorizin hydrolase of mammals. The amino acid identity is between 20% and 40%.[1,4] The secreted form KL protein only consists of an N-terminal signal sequence and KL1 repeat, lacking KL2 repeat, the transmembrane domain and the intracellular domain.[2,3]

Human KL shares 86% identity with the mouse protein and has been localized on chromosome 13q12 spanning over 50 kb.[2] The secreted form of KL in human is much more predominant than the membrane form of KL although human and mouse both encodes membrane and secreted forms of it. To date, it has been not reported whether KL is related to human aging and age-related diseases, although association of human aging with functional variant of KL has been analyzed recently.[5] The accumulating evidences has confirmed the existence of secreted form of KL.[2,3] However, it remains unclear where it is from.

METHODS

Subjects
One hundred and twelve serum samples from healthy individuals (53 males and 59 females, aged 0 to 91 years) were obtained from Beijing Hospital.

Cloning of human klotho secreted form protein C-terminal gene
Total RNA from human kidney tissues was extracted by RNAgents(r) total RNA Isolation System (Promega, USA). The total RNA was reverse-transcribed with Oligo(dT)12-18 primer by SuperscriptTM First-Strand Synthesis System for RT-PCR (Gibco BRL, USA), and the resulting cDNA was amplified by LA-Taq DNA polymerase (Takara Shuzo, Shiga, Japan) using specific primers (sense: 5’-GTCCCCGGATCCGTGTCC ATTGCCCTAA-’; antisense: 5’-ACTAGCCTCGAG-ATCTCCAGAGCCGAAA-3’) for human Klotho cDNA. PCR condition was optimized as follows: 5-minute denaturation at 94℃; then 60-second denaturation at 94℃, 60-second annealing at 45.5℃, 90-second extension at 72℃, with 30 cycles at 10-minute extension at 72℃ at last. The PCR product was cloned into pMD18T-vector (Takara), then digested with BamH Ⅰ and Hind Ⅲ (Takara) and subcloned into the appropriate sites of the expression vector pET28a and pET30a (Novagen, Wisconsin, USA) under the control of bacteriophage T7 promoter, yielding the recombinant plasmids pET28a-KLSC and pET30a-KLSC, respec-tively.

Expression and purification of recombinant KLSC
The deduced molecular weight of 28aKLSC and 30aKLSC by computer software DNATools 5.1 version were 33.6 kDa and 35.5 kDa, respectively. The recombinant plasmids pET28a-KLSC and pET30a-KLSC were introduced into the host strain BL21 (DE3) for expression of KLSC, His-tagged at N-terminal. Cultures were grown at 37℃ in Luria-Bertain (LB) culture medium containing 50 μg/ml kanamycin until a cell density reached OD600=0.8. Isopropyl-D-thiogalacto-pyranoside (IPTG, Sigma, USA) was added to 1 mol/L LB medium. Cells were induced for 2, 4, and 6 hours, respectively. The cell pellet from 100 ml 6 hour-induced culture was resuspended in 15 ml of MCAC-0 buffer (Tris-HCl 20 mmol/L, NaCl 0.5 mol/L, glycerol 10%(v/v),PMSF 1 mmol/L, Urea 8 mol/L, pH 7.9). The resuspended cell pellet was sonicated for 4 s×50. After centrifugation, the supernatant was loaded into a column containing 2 ml of freshly prepared Ni-NTA agarose (Qiagen, Chatsworth, CA). The column was washed with 30 ml MCAC-0 buffer, followed by 15 ml MCAC-2. The protein was eluted with 1 ml MCAC-20, -40, -60, -80, -100, and -200 in turn. MCAC-2, -20, -40, -60, -80, -100, and -200 were MCAC-0 buffer added 0.02, 0.2, 0.4, 0.6, 0.8, 1, and 2 mol/L imidazole, respectively. The flow through were collected and identified by 12% SDS-PAGE, and the gel was silver dyed. The purified protein was dialyzed in phosphate buffered saline (PBS) (NaCl, 8 g; Na2HPO4·12H2O, 2.89 g; KH2PO4, 0.2 g; KCl 0.2 g in 1 L solution, pH 7.4).

Preparation of polyclonal antibody against KL protein
A recombinant partial human KL protein or KLSC containing the sequence which spans the C-terminal of human secreted form from 290 AA to 549 AA was produced by Escherichia coli (E. coli). It was used to generate antibody by injection into rabbit. For primary immunization, recombinant 30aKLSC protein was dissolved in 0.9% NaCl to 1 mg/ml and mixed with Freund’s complete adjuvant (Gibco), and then injected into three rabbits subcutaneously. Each rabbit was injected with 1 mg recombinant KLSC protein. Booster immunization was continued with antigen in 0.9% NaCl and mixed with Freund’s incomplete adjuvant (Gibco) every 10 days for 3 times, and each rabbit underwent 0.5 mg injection with recombinant KLSC protein. Rabbits were bled 7 days following the immunization and blood stood at 4℃ overnight. After removal of clot and debris by centrifugation at 3000 r/min for 10 minutes, the serum was assayed by enzyme-linked-immunosorbent assay (ELISA) and stored at -20℃. The serum was precipitated by saturated ammonium sulfate at 4℃ overnight, then centrifuged at 12000 r/min for 20 minutes. The precipitate was washed using 33% ammonium sulfate and then dissolved in PBS (5%-10% serum original volume). The dissolved rabbit IgG was dialyzed in PBS at 4℃ for 48 hours at least. The anti-KLSC IgG was purified by protein A affinity chromatograghy (Pierce Co., USA) by standard procedure.

Western blot analysis
Human serums, mouse serums, total proteins of some tissues and some cultured cells were fractionated through 10% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and then electrophoretically transferred to PVDF membranes (NEN, Brussels, Belgium) at 4℃. After transfer, the PVDF membranes were washed with Tris buffered saline (TBS) at room temperature, and then were blocked by blocking buffer (TBS containing 5% fat-free milk powder and 0.1% Tween-20) for 1 hour. After 5-minute washing with TBS/T (TBS containing 0.1% Tween-20) for three times, the PVDF membranes were incubated for 1 hour with anti-KLSC antibody diluted at 1∶1000 in 5% bovine serum albumin (BSA). After washed for three times, the PVDF membranes were incubated for 1 hour with goat anti-rabbit IgG conjugated with alkaline phosphatase (Cell Signaling Technology, Inc.) and diluted to 1∶2000. Finally, the membranes were washed for three times and developed with 5-bromo-4-chloro-3-indolyl phosphate (BCIP, Amresco, Solon, OH) and nitroblue tetrazolium chloride (NBT, BBI) as chromogenic substrates.

ELISA assay for KL protein in human serum
The antigens were coated in 96-well plates overnight at 4℃. Wells were blocked by 5% fat-free milk powder in PBS at 37℃ for 2 hours and washed three times with PBS/T (PBS containing 0.05% Tween-20). Anti-KLSC antibody was diluted 1∶20 with PBS containing 5% fat-free milk powder and added to each well. After 2-hour incubation at 37℃,wells were washed with PBS/T three times. Goat anti-rabbit IgG conjugated with alkaline phosphatase (Cell Signaling Technology) was diluted to 1∶1000 and added. After 1-hour incubation at 37℃, wells were washed again and p-nitrophenyl phosphate (Amresco) was added. The immuno-reaction was read by microplate reader (Bio-Rad, 550, USA).

Statistical analysis
Data were expressed as mean±standard deviation (SD). All analyses were made by SPSS 11.0. Student’s t test was used to analyze differences between two groups. One way analysis of variance (ANOVA) was used to test differences among groups. A P<0.05 was considered statistically significant.

RESULTS

Cloning and expression of C terminal of KLSC
Total RNA from human kidney tissues was used as templates for RT-PCR to clone the C terminal of KL secreted form gene. A 1.5 kb fragment was amplified ( Fig. 1A ). The PCR product was cloned into pMD18T-vector, then digested with BamH Ⅰ and Hind Ⅲ, and subcloned into the appropriate sites of the expression vector pET28a and pET30a. Host bacterial cells containing each of the recombinant plasmids pET28a-KLSC and pET30a-KLSC were grown and induced with IPTG for the expression of recombinant KLSC. At 4 hours, the expression level of two recombinant proteins reached their peaks ( Fig. 1B ; Lanes 3 and 7). The purification of recombinant proteins 28aKLSC and 30aKLSC was performed by Ni-NTA agarose affinity chromatograghy. The resulting proteins were assayed by SDS-PAGE and stained by silver-dye ( Fig. 1C; Lanes 5-8).

Identification of KL protein in human and mouse serum
To determine whether KL is related to human aging, we prepared a poly-clonal antibody against human secreted form of KL by bacterial expressed KL fragment which spans the C-terminal of human secreted form from 290 AA to 549 AA. The antibody only recognized the C-terminal fragment of human and mouse secreted form, not other non-related proteins ( Fig. 2A ) and proteins from several cells ( Fig. 2B ), which demonstrated that the antibody is specific for KL protein. The hypothesis that the secreted form of KL may be a humoral factor[1,3] prompted us to test if it existed in human serum. The serum samples from human and mouse were analysed using western blot assays. As shown in Fig. 2B , a 60-kD protein was detected both in human and mouse serum. It is predictable that the antibody could recognize the secreted form of KL in human and mouse since the sequences of the antigen we used are very conserved in human and mouse and not share any homology with known proteins. The result identified that KL secreted form was a humoral factor that existed in serum.

Serum level of KL protein with age
The detection of KL protein in serum by antibody provides an easy way for large screening in population to study the correlation of KL with human aging. Serums from 112 healthy individuals aged from 0 to 91 including 53 men and 59 women, have been scanned by ELISA with this antibody ( Table ). The results were analysed by SPSS 11.0 program and diagramed in Fig. 3. It showed that the level of KL protein in serum declined with aging in overall (P=0.002) ( Figs. 3A and 3C ). Further analysis revealed that the level of KL protein in serum was low before the age of 10 ( Fig. 3B ) and it reached the highest level at age between 30 and 40. It declined then with aging although it could be variable in each individual ( Figs. 3A and 3C ). The pattern of the plot has no significant difference between males and females (P=0.543) ( Fig. 3C ). However the peak appeared several years earlier in females than males ( Fig. 3B ).

DISCUSSION

We cloned and expressed the C terminal of KL secreted form protein, and then prepared a polyclonal antibody against KL protein in this study. Our results show that the Klotho protein is secreted into the blood and is a humoral factor both in human and mouse. The decline of KL protein in serum is related to human aging. The knock-out of KL in mouse leads to shortening of life in addition to other aging syndromes. The gain of wide type of KL either by protein recovery or gene delivery could rescue almost all macroscopic features observed in KL knock out mice.[1,6-8] This suggests that KL might be a susceptible factor in aging. The decline of serum KL in human may reflect the development of aging. Therefore, the protein level of KL in human serum may be used as an indicator for aging to some extent. The loss of KL in mice is also involved in aging-related diseases such as cardiovascular dysfunction, hyper-tension, diabetes, arteriosclerosis, hyperlipidemia, ischemia, osteopenia, pulmonary emphysema, chronic renal failure, cognition impairment.[9-18] The detection of human serum KL protein may become a standard for diagnosing these diseases. The low level of KL in serum at early stage after born agrees to the notion that KL may not be necessary in development.[1] The fact that KL protein level peak appears earlier in females than males may reflect the time difference in mature between sexes. The expression of KL may be controlled by many factors including hereditary background, endocrine regulation and so on, so the levels of KL in individuals at the same age should be variant. It is difficult to explain at present the significant variance in some cases, because we do not have their genetic background and the regulation of expression of the Klotho is not fully understood.We have tried to find out if there is any difference between the male and female in the trend of KL secreted protein level with aging. There is no difference between the sexes (P>0.05 ) ( Fig. 3B ).
In conclusion, our study shows that secreted form KL protein exists in both human and mouse serum and that the decline of KL protein in serum correlates with human aging. These results suggest that KL is a serum factor related to human aging. The biological function of the KL in serum needs more extensive study. However the existence of the KL in serum implicates that it may function through binding its receptor presented in the target cells to lead further cascade changes in them.


Acknowledgements: We would like to thank Dr. Yang Ze for providing us with collected samples.

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  1. National “973” Project of China,No.G2000057009;