Klotho Is a Novel β-Glucuronidase Capable of Hydrolyzing Steroid β-Glucuronides

Abstract

klotho mutant mice provide a unique model to analyze mechanisms of aging because their phenotypes resemble those of human aging-associated disorders. The klotho gene encodes Klotho, a type I membrane protein that shares sequence similarity with members of the glycosidase family 1. Because Klotho lacks the glutamic acid residues that have been shown to be involved in the catalytic activity of this family of enzymes, the function of this protein was unknown. Here, we have studied the biochemical characteristics of recombinant Klotho. The purified chimeric Klotho-human IgG1 Fc protein (KLFc) was assayed with a series of 4-methylumbelliferyl (4Mu) β-glycosides as potential substrates. An enzymatic activity of Klotho was observed only with 4-methylumbelliferyl β-d-glucuronide in contrast to bovine liver β-glucuronidase, which exhibits a rather wide substrate specificity. Furthermore, the enzymatic activity of KLFc was reduced by the addition of specific inhibitors of β-glucuronidase. A number of natural β-glucuronides were screened by competitive inhibition for KLFc β-glucuronidase. We found that steroid β-glucuronides such as β-estradiol 3-β-d-glucuronide, estrone 3-β-d-glucuronide, and estriol 3β-d-glucuronide were hydrolyzed by KLFc. The artificial fluorescent substrate and the steroid conjugates share a common phenolic structure. Collectively, these data suggest that Klotho functions as a novel β-glucuronidase and that steroid β-glucuronides are potential candidates for the natural substrate(s) of Klotho. klotho mutant mice provide a unique model to analyze mechanisms of aging because their phenotypes resemble those of human aging-associated disorders. The klotho gene encodes Klotho, a type I membrane protein that shares sequence similarity with members of the glycosidase family 1. Because Klotho lacks the glutamic acid residues that have been shown to be involved in the catalytic activity of this family of enzymes, the function of this protein was unknown. Here, we have studied the biochemical characteristics of recombinant Klotho. The purified chimeric Klotho-human IgG1 Fc protein (KLFc) was assayed with a series of 4-methylumbelliferyl (4Mu) β-glycosides as potential substrates. An enzymatic activity of Klotho was observed only with 4-methylumbelliferyl β-d-glucuronide in contrast to bovine liver β-glucuronidase, which exhibits a rather wide substrate specificity. Furthermore, the enzymatic activity of KLFc was reduced by the addition of specific inhibitors of β-glucuronidase. A number of natural β-glucuronides were screened by competitive inhibition for KLFc β-glucuronidase. We found that steroid β-glucuronides such as β-estradiol 3-β-d-glucuronide, estrone 3-β-d-glucuronide, and estriol 3β-d-glucuronide were hydrolyzed by KLFc. The artificial fluorescent substrate and the steroid conjugates share a common phenolic structure. Collectively, these data suggest that Klotho functions as a novel β-glucuronidase and that steroid β-glucuronides are potential candidates for the natural substrate(s) of Klotho. klotho mutant mice (1Kuro-o M. Matsumura Y. Aizawa H. Kawaguchi H. Suga T. Utsugi T. Ohyama Y. Kurabayashi M. Kaname T. Kume E. Iwasaki H. Iida A. Shiraki-Iida T. Nishikawa S. Nagai R. Nabeshima Y.I. Nature. 1997; 390: 45-51Crossref PubMed Scopus (2811) Google Scholar, 2Nabeshima Y. Aging Res. Rev. 2002; 1: 627-638Crossref PubMed Scopus (95) Google Scholar) were originally described as a model of human aging-associated disorders because the phenotype observed in this mutant includes arteriosclerosis, ectopic calcification, emphysema, and osteoporosis. The responsible gene, klotho (1Kuro-o M. Matsumura Y. Aizawa H. Kawaguchi H. Suga T. Utsugi T. Ohyama Y. Kurabayashi M. Kaname T. Kume E. Iwasaki H. Iida A. Shiraki-Iida T. Nishikawa S. Nagai R. Nabeshima Y.I. Nature. 1997; 390: 45-51Crossref PubMed Scopus (2811) Google Scholar), encodes a type I membrane glycoprotein of 130 kDa. Expression of this gene is seen in renal tubule cells, parathyroid secretory cells, and epithelial cells of the choroid plexus. The extracellular domain of Klotho contains two internal repeats (termed mKL1 and mKL2) that display significant similarity to family 1 β-glycosidases (3Henrissat B. Biochem. J. 1991; 280: 309-316Crossref PubMed Scopus (2624) Google Scholar, 4Henrissat B. Bairoch A. Biochem. J. 1993; 293: 781-788Crossref PubMed Scopus (1771) Google Scholar, 5Henrissat B. Bairoch A. Biochem. J. 1996; 316: 695-696Crossref PubMed Scopus (1187) Google Scholar). Glycosidases are the enzymes that hydrolyze the glycosidic bond between two carbohydrates or between carbohydrate and non-carbohydrate moieties (6Davies G. Henrissat B. Structure. 1995; 3: 853-859Abstract Full Text Full Text PDF PubMed Scopus (1612) Google Scholar, 7Sinnott M. Chem. Rev. 1990; 90: 149-154Crossref Scopus (1492) Google Scholar). Glycosidases have been classified into about 90 families on the basis of amino acid similarities ((3Henrissat B. Biochem. J. 1991; 280: 309-316Crossref PubMed Scopus (2624) Google Scholar, 4Henrissat B. Bairoch A. Biochem. J. 1993; 293: 781-788Crossref PubMed Scopus (1771) Google Scholar, 5Henrissat B. Bairoch A. Biochem. J. 1996; 316: 695-696Crossref PubMed Scopus (1187) Google Scholar) carbohydrate-active enzymes (CAZy) data base at afmb.cnrs-mrs.fr/~cazy/CAZY/index.html), and they participate in essential steps of synthesis and degradation of oligosaccharides and polysaccharides (8Herscovics A. Biochim. Biophys. Acta. 1999; 1473: 96-107Crossref PubMed Scopus (240) Google Scholar), which are involved in pathogen defense systems (9Wittstock U. Halkier B.A. Trends Plant Sci. 2002; 7: 263-270Abstract Full Text Full Text PDF PubMed Scopus (457) Google Scholar, 10Rask L. Andreasson E. Ekbom B. Eriksson S. Pontoppidan B. Meijer J. Plant Mol. Biol. 2000; 42: 93-113Crossref PubMed Scopus (531) Google Scholar), detoxification (11Gopalan V. Pastuszyn A. Galey Jr., W.R. Glew R.H. J. Biol. Chem. 1992; 267: 14027-14032Abstract Full Text PDF PubMed Google Scholar, 12LaMarco K.L. Glew R.H. Biochem. J. 1986; 237: 469-476Crossref PubMed Scopus (26) Google Scholar), control of signal transduction (13Wells L. Vosseller K. Hart G.W. Science. 2001; 291: 2376-2378Crossref PubMed Scopus (805) Google Scholar), and modification of hormone (14Brzobohaty B. Moore I. Kristoffersen P. Bako L. Campos N. Schell J. Palme K. Science. 1993; 262: 1051-1054Crossref PubMed Scopus (298) Google Scholar) among others. In humans, inheritable deficiencies of glycosidases are known to induce a variety of impairments such as lysosomal storage diseases (15Neufeld E.F. Annu. Rev. Biochem. 1991; 60: 257-280Crossref PubMed Scopus (484) Google Scholar), Gaucher’s and Krabbe’s diseases, and lactose intolerance (16Semenza G. Auricchio S. Scriver C.R. Beaudet A.L. Sly W.S. Valle D. The Metabolic Basis of Inherited Deseases. 6th Ed. McGraw-Hill information Services Company, New York1989: 2975-2997Google Scholar, 17Buller H.A. Grand R.J. Annu. Rev. Med. 1990; 41: 141-148Crossref PubMed Scopus (63) Google Scholar, 18Montgomery R.K. Buller H.A. Rings E.H. Grand R.J. FASEB J. 1991; 5: 2824-2832Crossref PubMed Scopus (71) Google Scholar). Glycosidase family 1 groups together enzymes of varying substrate specificity (3Henrissat B. Biochem. J. 1991; 280: 309-316Crossref PubMed Scopus (2624) Google Scholar, 4Henrissat B. Bairoch A. Biochem. J. 1993; 293: 781-788Crossref PubMed Scopus (1771) Google Scholar, 5Henrissat B. Bairoch A. Biochem. J. 1996; 316: 695-696Crossref PubMed Scopus (1187) Google Scholar), such as β-glucosidase, β-galactosidase, 6-phospho-β-glucosidase, 6-phospho-β-galactosidase, myrosinase, and lactase-phlorizin hydrolase (LPH). 1The abbreviations used are: LPH, lactase-phlorizin hydrolase; CHO, Chinese hamster ovary; Fc, crystallizable fragment; GlcU, glucuronide; HPLC, high performance liquid chromatography; IdoUA, iduronic acid; KL, Klotho; 4Mu, 4-methylumbelliferyl. These enzymes are widely distributed throughout bacteria, archaea, and eukaryotes. Family 1 glycosidases operate with a molecular mechanism leading to overall retention of the anomeric configuration and involving the formation and breakdown of a covalent glycosyl enzyme intermediate. Two Glu residues are directly involved in this catalytic mechanism, one acting as a nucleophile and the other as an acid/base (19Rye C.S. Withers S.G. Curr. Opin. Chem. Biol. 2000; 4: 573-580Crossref PubMed Scopus (436) Google Scholar, 20McCarter J.D. Withers S.G. Curr. Opin. Struct. Biol. 1994; 4: 885-892Crossref PubMed Scopus (811) Google Scholar). Intriguingly, unlike other members of β-glycosidase family 1, Klotho does not possess these two catalytic glutamic acids. In the KL1 region, the nucleophilic Glu residue is present, but the acid/base Glu residue is replaced by an Asn residue. Inversely, in the KL2 region, the acid/base Glu residue is present, but the nucleophile is replaced by Ala and Ser residues in mouse and human Klotho, respectively (1Kuro-o M. Matsumura Y. Aizawa H. Kawaguchi H. Suga T. Utsugi T. Ohyama Y. Kurabayashi M. Kaname T. Kume E. Iwasaki H. Iida A. Shiraki-Iida T. Nishikawa S. Nagai R. Nabeshima Y.I. Nature. 1997; 390: 45-51Crossref PubMed Scopus (2811) Google Scholar). Together with Klotho, other proteins form a distinct subfamily in glycosidase family 1. First, β-Klotho (βKL) (21Ito S. Kinoshita S. Shiraishi N. Nakagawa S. Sekine S. Fujimori T. Nabeshima Y.I. Mech. Dev. 2000; 98: 115-119Crossref PubMed Scopus (262) Google Scholar), a type I membrane protein with 41.2% similarity to Klotho, contains two internal repeats highly homologous to those of family 1 glycosidases. Like in Klotho, the first repeat lacks the catalytic acid/base, whereas the second repeat lacks the nucleophile. Another protein, Klotho-LPH-related (KLPH) (22Ito S. Fujimori T. Hayashizaki Y. Nabeshima Y. Biochim. Biophys. Acta. 2002; 1576: 341-345Crossref PubMed Scopus (83) Google Scholar), which is also a type I membrane protein, has a single extracellular domain with 37.5% similarity to the KL1 domain of the mouse Klotho proteins, and this domain lacks the acid/base catalytic residue. LPH (23Naim H.Y. Histol. Histopathol. 2001; 16: 553-561PubMed Google Scholar), a major glycosidase contributing to the metabolism of dietary lactose in mammals, is a membranebound enzyme that acts extracellularly. The mature form of this enzyme is composed of two internal repeats (LPH3 and LPH4, each belonging to glycosidase family 1) and a transmembrane domain with a short cytoplasmic domain at the carboxyl terminus. The overall structure of the mature form of LPH resembles that of Klotho, and each of the Klotho internal repeats exhibits 36–42% similarity to those of LPH, respectively. Unlike Klotho, however, each of the LPH repeats possesses the two catalytic glutamic acids of glycosidase family 1. In contrast, it should be noted that myrosinase (9Wittstock U. Halkier B.A. Trends Plant Sci. 2002; 7: 263-270Abstract Full Text Full Text PDF PubMed Scopus (457) Google Scholar, 10Rask L. Andreasson E. Ekbom B. Eriksson S. Pontoppidan B. Meijer J. Plant Mol. Biol. 2000; 42: 93-113Crossref PubMed Scopus (531) Google Scholar), a plant homolog with 28–32% similarity to Klotho, has S-glycosidase enzymatic activity, even though it lacks the glutamic acid that serves as a general acid/general base catalyst. For myrosinase, it has been shown that the acid/base residue is not necessary in the first step of catalysis because of the excellent leaving group ability of the aglycone of the natural substrates (glucosinolates). For the second step, Burmeister and colleagues (24Burmeister W.P. Cottaz S. Rollin P. Vasella A. Henrissat B. J. Biol. Chem. 2000; 275: 39385-39393Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar) have shown by high resolution x-ray crystallography that ascorbate is a cofactor for myrosinase and substitutes for the function of the catalytic base. The deficiency of the Klotho proteins leads to impaired calcium homeostasis and enhancement of the expression of the 1α-hydroxylase gene, the rate-limiting enzyme in 1,25-(OH)2-vitamin D synthesis, and results in persistently high serum levels of 1,25-(OH)2-vitamin D in kl–/–mice (25Yoshida T. Fujimori T. Nabeshima Y. Endocrinology. 2002; 143: 683-689Crossref PubMed Scopus (169) Google Scholar, 26Tsujikawa H. Kurotaki Y. Fujimori K. Nabeshima Y. Mol. Endocrinology. PubMed Scopus Google Scholar). in the D participate in of the phenotypes in kl–/–mice levels of 1,25-(OH)2-vitamin D and calcium H. Kurotaki Y. Fujimori K. Nabeshima Y. Mol. Endocrinology. PubMed Scopus Google Scholar). of of the phenotypes is observed Klotho protein serum of calcium and 1,25-(OH)2-vitamin D are to levels H. Kurotaki Y. Fujimori K. Nabeshima Y. Mol. Endocrinology. PubMed Scopus Google Scholar). is that Klotho is in the of D to Klotho calcium one of the is that Klotho acts as an enzyme because it significant similarity to members of β-glycosidase family 1. this is one that the substrate(s) is to an form by the enzymatic of Klotho, and Klotho be the of the We have the biochemical characteristics of Klotho to enzymatic An 4-methylumbelliferyl was found to be hydrolyzed by Klotho at a but in a specific with other family We screened natural substrates with and found that steroid β-glucuronides were hydrolyzed by Klotho. we that Klotho functions as an enzyme in a unique that of β-glucuronidase. Expression a protein that of the extracellular domain of mouse Klotho acids with human Fc at carboxyl terminus. The expression was as and were to the of mouse klotho the and an artificial and enzyme into the and were These were with the of klotho and into this was with and and with the of the human IgG1 Fc gene G. S. G. B. Biol. 1990; PubMed Scopus Google Scholar), into expression to LPH and protein used as a was were to LPH by with and at the was with of human Fc gene and into the expression to cells were in essential bovine and the chimeric proteins, of were into cells For were in the of and In the of were in the of 1 of cells KLFc were in essential bovine serum and were of and at for at to were and of protein or a of the was into with and with with the addition of at was by a were with and to a with protein an of at at were with 1 of and and with 1 of The with KLFc or were used for enzymatic protein was by and were in a and The was with Y. E. Kinoshita S. A. A. K. K. Iida A. H. N. A. A. Fujimori T. M. N. K. Nabeshima Y. Biochem. Biophys. Res. 2000; 267: PubMed Scopus Google Scholar) by with were by to x-ray with were at acid for at were with acid for and were by a of the potential substrates for the general The 4-methylumbelliferyl (4Mu) or and of KLFc. The was in a of were at with a at an of and an of were on the basis of B. Biochim. Biophys. Acta. PubMed Scopus Google Scholar). were used as a control the addition of of purified human IgG1 and β-glucuronidase bovine liver and were used for a series of between and and between and were were by or to the to the of we other fluorescent substrate β-glucuronides to β-glucuronidase activity of KLFc. and were and were with the as The inhibitors of β-glucuronidase was assayed acid and acid to the of to the and were at in and of KLFc For between and were each 1 of was and by the addition of of were as of the of natural steroid β-estradiol estrone estriol estriol and estriol 3β-d-glucuronide of and and and and were by competitive inhibition For the inhibition each was to KLFc and the of each substrate was with KLFc proteins the for and were into and were into and were into and were into respectively. analyze the of the were on a with a were used in at a of at were on the The of 1 was as acid and in were for acid and for and acid and for The at or was we the catalytic for of were at substrate were of analyze the of amino acid was an that of the was the Expression and of recombinant Klotho, we an expression mouse klotho and human The the extracellular domain of Klotho was to the human Fc For the control we used mouse LPH, a type 1 membrane protein with two internal repeats homologous to family 1 The expression for was to that for KLFc. These expression were into cells to these were and to The proteins were at molecular of (KLFc) and respectively protein were by with the of the and to the of purified proteins, KLFc and each as by were by and the was with to proteins The KLFc protein was as a single that this protein be purified The protein also be purified The are of bovine serum bovine A bovine serum was used as a for were with to the of KLFc and The results with those by We used the purified proteins for the of the and of purified were or proteins were purified with protein A were by proteins, the were with 1, of human IgG1 as a bovine serum used as a for of and of the enzymatic activity of the KLFc protein, a of was These substrates are not but their fluorescent which was a as a function of to the of hydrolyzed of were KLFc with and hydrolyzed this A. A. B. T. and in the of other potential substrates were to the levels observed with IgG1 that KLFc does not hydrolyze these the other β-glucosidase, and β-glucuronidase were on a of substrates as shown in I. I that the specific activity of Klotho is about that of β-glucuronidase bovine liver and of that we other and that proteins these to results in a A. A. B. T. and Furthermore, and the other fluorescent were and found to be hydrolyzed the the β-glucuronidase activity of KLFc of the enzymatic activity of recombinant KLFc in a of for the KLFc protein was with a series of The of and were as shown in and substrate in a of that substrate was at for of KLFc on the of these the was for each of substrate and are in The is shown in The and were this and are shown in and that the of KLFc protein on of KLFc protein as a β-glucuronidase as the in a and of the of on the enzyme activity of KLFc was used between and and was used between and to the between to by The activity of KLFc for the of was seen at that this is the for KLFc. Because on for the activity of has been H. H. G. S. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, J. Biol. Chem. 1990; Full Text PDF PubMed Google Scholar), we the of and on the enzymatic activity of Klotho. of these to have an on the enzymatic activity of to the KLFc protein, we the of known inhibitors for β-glucuronidase on enzymatic activity liver β-glucuronidase was used as a to the between known acid and which are known competitive inhibitors for bovine liver β-glucuronidase, were to shown in A and KLFc enzymatic activity was reduced by these These were also observed on β-glucuronidase, but at a was for KLFc. These results that the enzymatic of KLFc were with known inhibitors of β-glucuronidase. for Klotho for Klotho potential we screened natural conjugates S. A. J. Biochem. Biol. 2000; PubMed Scopus Google Scholar), steroid Chem. Biol. 2000; PubMed Scopus Google Scholar, T. M. R. K. 1999; PubMed Scopus Google Scholar), Biochem. J. 1996; PubMed Scopus Google Scholar), J. G. 2001; PubMed Scopus Google Scholar), and Sly W.S. G. 2001; PubMed Scopus Google Scholar). We first these the by the KLFc was to KLFc and of or and GlcU, and on the of by KLFc the of We and be hydrolyzed with KLFc. was with KLFc The were on a and with at or a KLFc hydrolyzed and with catalytic and to those for and was hydrolyzed by whereas and were not hydrolyzed at of KLFc protein as in a In the recombinant Klotho was found to a but specific activity as a β-glucuronidase. We in of Klotho for enzyme because of expression levels in and We a unique of protein cells and the of a chimeric protein to Fc of the human IgG1 a Klotho protein that was and in and be purified protein A a significant β-glucuronidase activity be in systems fluorescent and were also hydrolyzed with KLFc. of β-glucuronidase, acid and were also found to the enzymatic activity of Klotho. recombinant Klotho be as a β-glucuronidase by substrate specificity and the data which in families on amino acid Klotho in glycosidase family 1. family groups together enzymes, and but to β-glucuronidase members have been Klotho the first enzyme with β-glucuronidase activity in this such as structure be necessary to the basis of this specificity because known to the glycosidase family (3Henrissat B. Biochem. J. 1991; 280: 309-316Crossref PubMed Scopus (2624) Google Scholar, 4Henrissat B. Bairoch A. Biochem. J. 1993; 293: 781-788Crossref PubMed Scopus (1771) Google Scholar, 5Henrissat B. Bairoch A. Biochem. J. 1996; 316: 695-696Crossref PubMed Scopus (1187) Google Scholar). on the J.D. Res. 1994; PubMed Scopus Google Scholar) similarity between Klotho and the of a novel substrate specificity such as β-glucuronidase in family 1 glycosidases is not the between the substrates in this family are and the of glycosidases of substrate specificity in the family a common (3Henrissat B. Biochem. J. 1991; 280: 309-316Crossref PubMed Scopus (2624) Google Scholar, 4Henrissat B. Bairoch A. Biochem. J. 1993; 293: 781-788Crossref PubMed Scopus (1771) Google Scholar, 5Henrissat B. Bairoch A. Biochem. J. 1996; 316: 695-696Crossref PubMed Scopus (1187) Google Scholar, B. G. Curr. Opin. Struct. Biol. 1997; 7: PubMed Scopus Google Scholar). In and as noted the glycosidase families on sequence and similarity does not with the of glycosidases on their substrate specificity (3Henrissat B. Biochem. J. 1991; 280: 309-316Crossref PubMed Scopus (2624) Google Scholar, 4Henrissat B. Bairoch A. Biochem. J. 1993; 293: 781-788Crossref PubMed Scopus (1771) Google Scholar, 5Henrissat B. Bairoch A. Biochem. J. 1996; 316: 695-696Crossref PubMed Scopus (1187) Google Scholar, B. G. Curr. Opin. Struct. Biol. 1997; 7: PubMed Scopus Google Scholar). Furthermore, on sequence together with structure and mechanism have shown that glycosidase families be together in a which includes families 1, and B. G. Curr. Opin. Struct. Biol. 1997; 7: PubMed Scopus Google Scholar, data this groups a wide of substrate In novel members to but not to their glycosidase have M. E. Henrissat B. Trends Plant Sci. Full Text Full Text PDF PubMed Scopus Google Scholar). A is hydrolase A. 42: PubMed Scopus Google Scholar), which sequence similarity to glycosidase family 1, of the known catalytic the catalytic residues of this enzyme are and have their but they a on a non-carbohydrate substrate A. 42: PubMed Scopus Google Scholar). Klotho catalytic an catalytic is not be to mechanism, of a cofactor that a catalytic domain but are in the of a A of is by the structure of Klotho, in which of the catalytic is in the first and the other is in the second In an to these we to the of of the catalytic residue in each repeat of Klotho to which of the two repeats contains the the proteins were in the cells, and it was to analyze the enzymatic of the Klotho We also the of the cofactor of myrosinase, but was on the enzymatic activity of the of a other cofactor for Klotho be The enzymatic activity of Klotho activity at a to the for bovine liver β-glucuronidase. the enzymatic activity of β-glucuronidase was by that of Klotho was In the enzymatic activity of Klotho was reduced by known inhibitors of on these we that Klotho is a β-glucuronidase. We screened for natural β-glucuronides that be hydrolyzed by Klotho. We found that steroid β-glucuronides the β-glucuronidase activity of Klotho. Furthermore, we observed that Klotho hydrolyze steroid β-glucuronides such as and and share a common phenolic aglycone with the to the of the A in these that Klotho is specific for the is necessary to the natural substrate of Klotho, β-glucuronides with a phenolic aglycone are but are to in the steroid of these These be substrates for Klotho, which into functions for the of to Furthermore, such as be and for the and of Klotho which are and the phenotypes of The of Klotho were The of Klotho is that of purified human and serum respectively B. Biochem. J. PubMed Scopus Google Scholar), that the of Klotho for this artificial substrate is that of known In we the and of natural substrates and and with those of The of and were of the of as those of These results suggest that Klotho has an enzymatic activity that of other glycosidases. is that the observed activity of Klotho is for in we the that Klotho in with groups such as the of substrates are necessary for the of the activity of Klotho. We also the ability of Klotho to substrates acid to the that Klotho function as a be that the of Klotho is to be an enzyme rather a the the and the of by Klotho is a of is to that Klotho to that are for the of calcium homeostasis and of impairments observed in klotho the substrate of Klotho is to be it is that the of Klotho be by the addition of the form of the of the phenotypes of klotho mice the the molecular mechanisms Klotho function is the first to that Klotho as an enzyme in and information in the for natural substrate(s) and in and of Klotho proteins in of a and in aging-associated disorders. We R. for on the M. for human IgG1 and for and We also K. and K. for