Identification of a Metabolizing Enzyme in Human Kidney by Proteomic Correlation Profiling
2013; Elsevier BV; Volume: 12; Issue: 8 Linguagem: Inglês
10.1074/mcp.m112.023853
ISSN1535-9484
AutoresHidetaka Sakurai, Kazuishi Kubota, Shin‐ichi Inaba, Kaoru Takanaka, Akira Shinagawa,
Tópico(s)Lipid metabolism and biosynthesis
ResumoMolecular identification of endogenous enzymes and biologically active substances from complex biological sources remains a challenging task, and although traditional biochemical purification is sometimes regarded as outdated, it remains one of the most powerful methodologies for this purpose. While biochemical purification usually requires large amounts of starting material and many separation steps, we developed an advanced method named "proteomic correlation profiling" in our previous study. In proteomic correlation profiling, we first fractionated biological material by column chromatography, and then calculated each protein's correlation coefficient between the enzyme activity profile and protein abundance profile determined by proteomics technology toward fractions. Thereafter, we could choose possible candidates for the enzyme among proteins with a high correlation value by domain predictions using informatics tools. Ultimately, this streamlined procedure requires fewer purification steps and reduces starting materials dramatically due to low required purity compared with conventional approaches. To demonstrate the generality of this approach, we have now applied an improved workflow of proteomic correlation profiling to a drug metabolizing enzyme and successfully identified alkaline phosphatase, tissue-nonspecific isozyme (ALPL) as a phosphatase of CS-0777 phosphate (CS-0777-P), a selective sphingosine 1-phosphate receptor 1 modulator with potential benefits in the treatment of autoimmune diseases including multiple sclerosis, from human kidney extract. We identified ALPL as a candidate protein only by the 200-fold purification and only from 1 g of human kidney. The identification of ALPL as CS-0777-P phosphatase was strongly supported by a recombinant protein, and contribution of the enzyme in human kidney extract was validated by immunodepletion and a specific inhibitor. This approach can be applied to any kind of enzyme class and biologically active substance; therefore, we believe that we have provided a fast and practical option by combination of traditional biochemistry and state-of-the-art proteomic technology. Molecular identification of endogenous enzymes and biologically active substances from complex biological sources remains a challenging task, and although traditional biochemical purification is sometimes regarded as outdated, it remains one of the most powerful methodologies for this purpose. While biochemical purification usually requires large amounts of starting material and many separation steps, we developed an advanced method named "proteomic correlation profiling" in our previous study. In proteomic correlation profiling, we first fractionated biological material by column chromatography, and then calculated each protein's correlation coefficient between the enzyme activity profile and protein abundance profile determined by proteomics technology toward fractions. Thereafter, we could choose possible candidates for the enzyme among proteins with a high correlation value by domain predictions using informatics tools. Ultimately, this streamlined procedure requires fewer purification steps and reduces starting materials dramatically due to low required purity compared with conventional approaches. To demonstrate the generality of this approach, we have now applied an improved workflow of proteomic correlation profiling to a drug metabolizing enzyme and successfully identified alkaline phosphatase, tissue-nonspecific isozyme (ALPL) as a phosphatase of CS-0777 phosphate (CS-0777-P), a selective sphingosine 1-phosphate receptor 1 modulator with potential benefits in the treatment of autoimmune diseases including multiple sclerosis, from human kidney extract. We identified ALPL as a candidate protein only by the 200-fold purification and only from 1 g of human kidney. The identification of ALPL as CS-0777-P phosphatase was strongly supported by a recombinant protein, and contribution of the enzyme in human kidney extract was validated by immunodepletion and a specific inhibitor. This approach can be applied to any kind of enzyme class and biologically active substance; therefore, we believe that we have provided a fast and practical option by combination of traditional biochemistry and state-of-the-art proteomic technology. Molecular identification for an enzyme reaction or biologically active substance in an organism is challenging, although molecular biological methodologies such as expression cloning (1Yamauchi T. Kamon J. Ito Y. Tsuchida A. Yokomizo T. Kita S. Sugiyama T. Miyagishi M. Hara K. Tsunoda M. Murakami K. Ohteki T. Uchida S. Takekawa S. Waki H. Tsuno N.H. Shibata Y. Terauchi Y. Froguel P. Tobe K. Koyasu S. Taira K. Kitamura T. Shimizu T. Nagai R. Kadowaki T. Cloning of adiponectin receptors that mediate antidiabetic metabolic effects.Nature. 2003; 423: 762-769Crossref PubMed Scopus (2645) Google Scholar), recombinant protein panel (2Lin H. Lee E. Hestir K. Leo C. Huang M. Bosch E. Halenbeck R. Wu G. Zhou A. Behrens D. Hollenbaugh D. Linnemann T. Qin M. Wong J. Chu K. Doberstein S.K. Williams L.T. Discovery of a cytokine and its receptor by functional screening of the extracellular proteome.Science. 2008; 320: 807-811Crossref PubMed Scopus (556) Google Scholar) and RNAi screening (3Perocchi F. Gohil V.M. Girgis H.S. Bao X.R. McCombs J.E. Palmer A.E. Mootha V.K. MICU1 encodes a mitochondrial EF hand protein required for Ca(2+) uptake.Nature. 2010; 467: 291-296Crossref PubMed Scopus (661) Google Scholar) have been introduced recently as alternative approaches. Conventional biochemical purification has provided a number of successes and thus still remains a powerful, though labor-intensive strategy. In the traditional protein purification, it had been necessary to purify an individual protein nearly to homogeneity at a microgram amount so that the purified protein could be analyzed by N-terminal amino acid sequencing. Protein identification by mass spectrometry subsequently revolutionized this technology by enabling identification of proteins at much lower abundances: individual proteins could then be associated with specific activities as soon as a band in SDS-PAGE could be observed, even when the purified protein was far from homogeneity (4Kubota K. Sakikawa C. Katsumata M. Nakamura T. Wakabayashi K. PDGF BB purified from osteoclasts acts as osteoblastogenesis inhibitory factor (OBIF).J. Biomol. Tech. 2002; 13: 62-71PubMed Google Scholar, 5Kubota K. Nakahara K. Ohtsuka T. Yoshida S. Kawaguchi J. Fujita Y. Ozeki Y. Hara A. Yoshimura C. Furukawa H. Haruyama H. Ichikawa K. Yamashita M. Matsuoka T. Iijima Y. Identification of 2′-phosphodiesterase, which plays a role in the 2–5A system regulated by interferon.J. Biol. Chem. 2004; 279: 37832-37841Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 6Yonesu K. Kubota K. Tamura M. Inaba S. Honda T. Yahara C. Watanabe N. Matsuoka T. Nara F. Purification and identification of activating enzymes of CS-0777, a selective sphingosine 1-phosphate receptor 1 modulator, in erythrocytes.J. Biol. Chem. 2011; 286: 24765-24775Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). Although this streamlined the workflow by reducing the required starting materials as well as the separation steps for protein purification, a faster and more generalized approach from smaller starting material has still been desired because some proteins are physiochemically difficult for example in solubilization and stability. To solve these problems, we devised a proteomic correlation profiling methodology (7Kubota K. Anjum R. Yu Y. Kunz R.C. Andersen J.N. Kraus M. Keilhack H. Nagashima K. Krauss S. Paweletz C. Hendrickson R.C. Feldman A.S. Wu C.L. Rush J. Villén J. Gygi S.P. Sensitive multiplexed analysis of kinase activities and activity-based kinase identification.Nat. Biotechnol. 2009; 27: 933-940Crossref PubMed Scopus (89) Google Scholar). The basic concept of proteomic correlation profiling was originally developed by Andersen et al. (8Andersen J.S. Wilkinson C.J. Mayor T. Mortensen P. Nigg E.A. Mann M. Proteomic characterization of the human centrosome by protein correlation profiling.Nature. 2003; 426: 570-574Crossref PubMed Scopus (1051) Google Scholar). They quantitatively profiled hundreds of proteins across several centrifugation fractions by mass spectrometry and identified centrosomal proteins by calculating the correlation of these protein expression profiles with already known centrosomal proteins. In the following study, Foster et al. applied this strategy to map more than 1400 proteins to ten subcellular locations (9Foster L.J. de Hoog C.L. Zhang Y. Xie X. Mootha V.K. Mann M. A mammalian organelle map by protein correlation profiling.Cell. 2006; 125: 187-199Abstract Full Text Full Text PDF PubMed Scopus (469) Google Scholar). Although these studies used centrifugation as a separation method and a known marker profile as a standard for correlation, we extended this concept to use chromatography as a separation method and kinase activity as a basis for comparison; our approach successfully identified a kinase responsible for phosphorylation of peptide substrates just after one step chromatography, and was termed proteomic correlation profiling (7Kubota K. Anjum R. Yu Y. Kunz R.C. Andersen J.N. Kraus M. Keilhack H. Nagashima K. Krauss S. Paweletz C. Hendrickson R.C. Feldman A.S. Wu C.L. Rush J. Villén J. Gygi S.P. Sensitive multiplexed analysis of kinase activities and activity-based kinase identification.Nat. Biotechnol. 2009; 27: 933-940Crossref PubMed Scopus (89) Google Scholar). Independently, Kuromitsu et al. reported identification of an active substance in the serum response element-dependent luciferase assay from interstitial cystitis urine after three-step chromatography by a similar concept (10Kuromitsu S. Yokota H. Hiramoto M. Yuri M. Naitou M. Nakamura N. Kawabata S. Kobori M. Katoh M. Furuchi K. Mita H. Yamada T. Combination of MS protein identification and bioassay of chromatographic fractions to identify biologically active substances from complex protein sources.Mol. Cell. Proteomics. 2009; 8: 1318-1323Abstract Full Text Full Text PDF PubMed Scopus (2) Google Scholar). In theory, this general proteomic correlation profiling strategy can be adapted to any kind of separation method and activity profile but no other example has been reported thus far, therefore, actual examples where the method can be applied to other enzyme classes are required to prove its generality. Multiple sclerosis is the most common autoimmune disorder of the central nerve system in which the fatty myelin sheaths around the axons of the brain and spinal cord are damaged, leading to demyelination and scarring (11Compston A. Coles A. Multiple sclerosis.Lancet. 2008; 372: 1502-1517Abstract Full Text Full Text PDF PubMed Scopus (3614) Google Scholar, 12Brinkmann V. Billich A. Baumruker T. Heining P. Schmouder R. Francis G. Aradhye S. Burtin P. Fingolimod (FTY720): discovery and development of an oral drug to treat multiple sclerosis.Nat. Rev. Drug Discov. 2010; 9: 883-897Crossref PubMed Scopus (961) Google Scholar). Until recently, the standard treatments for multiple sclerosis such as interferon beta, glatiramer acetate, mitoxantrone, and natalizumab would often cause severe adverse events (13Ransohoff R.M. Natalizumab for Multiple Sclerosis.N. Engl. J. Med. 2007; 356: 2622-2629Crossref PubMed Scopus (212) Google Scholar, 14Galetta S.L. Markowitz C. 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Sphingosine 1-phosphate receptor 1 (S1P1) 1The abbreviations used are:S1P1sphingosine 1-phosphate receptor 1ALPLalkaline phosphatase, tissue-nonspecific isozymeALPIalkaline phosphatase, intestinalALPPalkaline phosphatase, placentalALPPLalkaline phosphatase, placental-like 2DMn-Dodecyl-β-D-maltopyranosideLPP1lipid phosphate phosphohydrolase 1LPP2lipid phosphate phosphohydrolase 2LPP3lipid phosphate phosphohydrolase 3SPP1sphingosine-1-phosphate phosphatase 1SPP2sphingosine-1-phosphate phosphatase 2FN3Kfructosamine 3-kinaseFN3K-RPfructosamine 3-kinase-related proteinHEPES4-(2-hydroxyethyl)-1-piperazineethanesulfonic acidLC-MS/MSliquid chromatography equipped with tandem mass spectrometrySDS-PAGEsodium dodecyl sulfate-polyacrylamide gel electrophoresisS1Psphingosine 1-phosphateS1P3sphingosine 1-phosphate receptor 3SPHK1sphingosine kinase 1SPHK2sphingosine kinase 2. 1The abbreviations used are:S1P1sphingosine 1-phosphate receptor 1ALPLalkaline phosphatase, tissue-nonspecific isozymeALPIalkaline phosphatase, intestinalALPPalkaline phosphatase, placentalALPPLalkaline phosphatase, placental-like 2DMn-Dodecyl-β-D-maltopyranosideLPP1lipid phosphate phosphohydrolase 1LPP2lipid phosphate phosphohydrolase 2LPP3lipid phosphate phosphohydrolase 3SPP1sphingosine-1-phosphate phosphatase 1SPP2sphingosine-1-phosphate phosphatase 2FN3Kfructosamine 3-kinaseFN3K-RPfructosamine 3-kinase-related proteinHEPES4-(2-hydroxyethyl)-1-piperazineethanesulfonic acidLC-MS/MSliquid chromatography equipped with tandem mass spectrometrySDS-PAGEsodium dodecyl sulfate-polyacrylamide gel electrophoresisS1Psphingosine 1-phosphateS1P3sphingosine 1-phosphate receptor 3SPHK1sphingosine kinase 1SPHK2sphingosine kinase 2. modulators are emerging as a new class of drugs with potential therapeutic application in multiple sclerosis (15Marsolais D. Rosen H. Chemical modulators of sphingosine-1-phosphate receptors as barrier-oriented therapeutic molecules.Nat. Rev. Drug Discov. 2009; 8: 297-307Crossref PubMed Scopus (146) Google Scholar), and fingolimod is a nonselective sphingosine 1-phosphate (S1P) receptor modulator (16Horga. A. Castilló. J. Montalban. X. Fingolimod for relapsing multiple sclerosis: an update.Expert Opin. Pharmacother. 2010; 11: 1183-1196Crossref PubMed Scopus (16) Google Scholar, 17Fujita T. Hirose R. Yoneta M. Sasaki S. Inoue K. Kiuchi M. Hirase S. Chiba K. Sakamoto H. Arita M. Potent Immunosuppressants, 2-Alkyl-2-aminopropane-1,3-diols.J. Med. Chem. 1996; 39: 4451-4459Crossref PubMed Scopus (129) Google Scholar, 18Brinkmann V. Pinschewer D.D. Feng L. Chen S. FTY720: altered lymphocyte traffic results in allograft protection.Transplantation. 2001; 72: 764-769Crossref PubMed Scopus (156) Google Scholar, 21Brinkmann V. Davis M.D. Heise C.E. Albert R. Cottens S. Hof R. Bruns C. Prieschl E. Baumruker T. Hiestand P. Foster C.A. Zollinger M. Lynch K.R. The immune modulator FTY720 targets sphingosine 1-phosphate receptors.J. Biol. Chem. 2002; 277: 21453-21457Abstract Full Text Full Text PDF PubMed Scopus (1314) Google Scholar, 22Mandala S. Hajdu R. Bergstrom J. Quackenbush E. Xie J. Milligan J. Thornton R. Shei G.J. Card D Keohane C. Rosenbach M. Hale J. Lynch C.L. Rupprecht K. Parsons W. Rosen H. Alteration of lymphocyte trafficking by sphingosine-1-phosphate receptor agonists.Science. 2002; 296: 346-349Crossref PubMed Scopus (1434) Google Scholar). Given its structural similarity to sphingosine, fingolimod is phosphorylated in vivo by sphingosine kinase, in particular sphingosine kinase 2 (SPHK2) (19Kharel Y. Lee S. Snyder A.H. Sheasley-O'Neill S.L. Morris M.A. Setiady Y. Zhu R. Zigler M.A. Burcin T.L. Ley K. Tung K.S. Engelhard V.H. Macdonald T.L. Pearson-White S. Lynch K.R. Sphingosine kinase 2 is required for modulation of lymphocyte traffic by FTY720.J. Biol. 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Alteration of lymphocyte trafficking by sphingosine-1-phosphate receptor agonists.Science. 2002; 296: 346-349Crossref PubMed Scopus (1434) Google Scholar). By this mechanism, fingolimod-P induces internalization of S1P1 on lymphocytes, blocking the ability of the receptor to support lymphocyte egress and recirculation through secondary lymphoid organs. This suppresses immune responses and is presumably the main immunomodulatory mode of action of fingolimod. sphingosine 1-phosphate receptor 1 alkaline phosphatase, tissue-nonspecific isozyme alkaline phosphatase, intestinal alkaline phosphatase, placental alkaline phosphatase, placental-like 2 n-Dodecyl-β-D-maltopyranoside lipid phosphate phosphohydrolase 1 lipid phosphate phosphohydrolase 2 lipid phosphate phosphohydrolase 3 sphingosine-1-phosphate phosphatase 1 sphingosine-1-phosphate phosphatase 2 fructosamine 3-kinase fructosamine 3-kinase-related protein 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid liquid chromatography equipped with tandem mass spectrometry sodium dodecyl sulfate-polyacrylamide gel electrophoresis sphingosine 1-phosphate sphingosine 1-phosphate receptor 3 sphingosine kinase 1 sphingosine kinase 2. sphingosine 1-phosphate receptor 1 alkaline phosphatase, tissue-nonspecific isozyme alkaline phosphatase, intestinal alkaline phosphatase, placental alkaline phosphatase, placental-like 2 n-Dodecyl-β-D-maltopyranoside lipid phosphate phosphohydrolase 1 lipid phosphate phosphohydrolase 2 lipid phosphate phosphohydrolase 3 sphingosine-1-phosphate phosphatase 1 sphingosine-1-phosphate phosphatase 2 fructosamine 3-kinase fructosamine 3-kinase-related protein 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid liquid chromatography equipped with tandem mass spectrometry sodium dodecyl sulfate-polyacrylamide gel electrophoresis sphingosine 1-phosphate sphingosine 1-phosphate receptor 3 sphingosine kinase 1 sphingosine kinase 2. CS-0777 (Fig. 1) is a novel selective S1P1 modulator (23Nishi T. Miyazaki S. Takemoto T. Suzuki K. Iio Y. Nakajima K. Ohnuki T. Kawase Y. Nara F. Inaba S. Izumi T. Yuita H. Oshima K. Doi H. Inoue R. Tomisato W. Kagari T. Shimozato T. Discovery of CS-0777: A potent, selective, and orally active S1P1agonist.ACS Med Chem Lett. 2011; 2: 368-372Crossref PubMed Scopus (50) Google Scholar). Although the immunomodulatory effects are supposed to be mainly mediated by S1P1, some lines of evidence suggest that the agonist activity on S1P receptor 3 (S1P3) could cause acute toxicity and cardiovascular deregulation, including bradycardia in rodents (24Sanna M.G. Liao J. Jo E. Alfonso C. Ahn M.Y. Peterson M.S. Webb B. Lefebvre S. Chun J. Gray N. Rosen H. Sphingosine 1-phosphate (S1P) receptor subtypes S1P1 and S1P3, respectively, regulate lymphocyte recirculation and heart rate.J. Biol. Chem. 2004; 279: 13839-13848Abstract Full Text Full Text PDF PubMed Scopus (538) Google Scholar, 25Forrest M. Sun S.Y. Hajdu R. Bergstrom J. Card D. Doherty G. Hale J. Keohane C. Meyers C. Milligan J. Mills S. Nomura N. Rosen H. Rosenbach M. Shei G.J. Singer II Tian M. West S. White V. Xie J. Proia R.L. Mandala S. Immune cell regulation and cardiovascular effects of sphingosine 1-phosphate receptor agonists in rodents are mediated via distinct receptor subtypes.J. Pharmacol. Exp. Ther. 2004; 309: 758-768Crossref PubMed Scopus (297) Google Scholar). Thus, CS-0777 was designed to have more selectivity on S1P1 over S1P3 in contrast to fingolimod-P which has potent agonistic activity for S1P3, S1P4, and S1P5 in vitro (22Mandala S. Hajdu R. Bergstrom J. Quackenbush E. Xie J. Milligan J. Thornton R. Shei G.J. Card D Keohane C. Rosenbach M. Hale J. Lynch C.L. Rupprecht K. Parsons W. Rosen H. Alteration of lymphocyte trafficking by sphingosine-1-phosphate receptor agonists.Science. 2002; 296: 346-349Crossref PubMed Scopus (1434) Google Scholar). Like fingolimod, CS-0777 is also a prodrug phosphorylated in vivo, and the phosphorylated CS-0777 (CS-0777-P, Fig. 1) agonizes S1P1 with more than 300-fold selectivity relative to S1P3 whereas CS-0777-P has weaker effects on S1P5 and no activity on S1P2 (23Nishi T. Miyazaki S. Takemoto T. Suzuki K. Iio Y. Nakajima K. Ohnuki T. Kawase Y. Nara F. Inaba S. Izumi T. Yuita H. Oshima K. Doi H. Inoue R. Tomisato W. Kagari T. Shimozato T. Discovery of CS-0777: A potent, selective, and orally active S1P1agonist.ACS Med Chem Lett. 2011; 2: 368-372Crossref PubMed Scopus (50) Google Scholar). CS-0777 showed immunosuppressive activity in mouse and rat models of experimental autoimmune encephalitis, animal models for multiple sclerosis. In healthy volunteers, single oral doses of CS-0777 caused marked, dose-dependent decreases in numbers of circulating lymphocytes, including marked and reversible decreases in circulating T and B cells (26Moberly J.B. Rohatagi S. Zahir H. Hsu C. Noveck R.J. Truitt K.E. Pharmacological modulation of peripheral T and B lymphocytes by a selective sphingosine 1-phosphate receptor-1 modulator.J. Clin. Pharmacol. 2011; 52: 996-1006Crossref PubMed Scopus (14) Google Scholar, 27Deleted in proofGoogle Scholar). Furthermore, in multiple sclerosis patients, single oral doses of CS-0777 caused dose-dependent decreases in circulating lymphocytes, with a slightly greater suppression of CD4+ versus CD8+ T cells. Therefore, CS-0777 would alter immune responses solely through activation of S1P1 without S1P3 modulation in humans, which could circumvent a bradycardia adverse effect, although the relationships associating selectivity of S1P1 to S1P3 with bradycardia in humans are not fully understood (12Brinkmann V. Billich A. Baumruker T. Heining P. Schmouder R. Francis G. Aradhye S. Burtin P. Fingolimod (FTY720): discovery and development of an oral drug to treat multiple sclerosis.Nat. Rev. Drug Discov. 2010; 9: 883-897Crossref PubMed Scopus (961) Google Scholar). Orally administrated CS-0777 is phosphorylated and rapidly reaches equilibrium with CS-0777-P as in the case of fingolimod (22Mandala S. Hajdu R. Bergstrom J. Quackenbush E. Xie J. Milligan J. Thornton R. Shei G.J. Card D Keohane C. Rosenbach M. Hale J. Lynch C.L. Rupprecht K. Parsons W. Rosen H. Alteration of lymphocyte trafficking by sphingosine-1-phosphate receptor agonists.Science. 2002; 296: 346-349Crossref PubMed Scopus (1434) Google Scholar), suggesting that the high kinase activity in blood is balanced by phosphatases. Therefore, identification of a phosphatase, the inactivating enzyme of an active metabolite, as well as identification of a kinase, the activating enzyme of a prodrug, are critical to fully understand the mechanism of action at the molecular level for both CS-0777 and fingolimod. Sphingosine kinase 2 (SPHK2) was identified as the major kinase of fingolimod (21Brinkmann V. Davis M.D. Heise C.E. Albert R. Cottens S. Hof R. Bruns C. Prieschl E. Baumruker T. Hiestand P. Foster C.A. Zollinger M. Lynch K.R. The immune modulator FTY720 targets sphingosine 1-phosphate receptors.J. Biol. Chem. 2002; 277: 21453-21457Abstract Full Text Full Text PDF PubMed Scopus (1314) Google Scholar, 28Billich A. Bornancin F. Dévay P. Mechtcheriakova D. Urtz N. Baumruker T. Phosphorylation of the immunomodulatory drug FTY720 by sphingosine kinases.J. Biol. Chem. 2003; 278: 47408-47415Abstract Full Text Full Text PDF PubMed Scopus (394) Google Scholar, 29Paugh S.W. Payne S.G. Barbour S.E. Milstien S. Spiegel S. The immunosuppressant FTY720 is phosphorylated by sphingosine kinase type 2.FEBS Lett. 2003; 554: 189-193Crossref PubMed Scopus (269) Google Scholar) and lipid phosphate phosphatase 3 (LPP3) was reported to be a phosphatase for fingolimod-P dephosphorylation (30Mechtcheriakova D. Wlachos A. Sobanov J. Bornancin F. Zlabinger G. Baumruker T. Billich A. FTY720-phosphate is dephosphorylated by lipid phosphate phosphatase 3.FEBS Lett. 2007; 581: 3063-3068Crossref PubMed Scopus (48) Google Scholar), although contribution of LPP3 in vivo has not been fully studied. In our previous work, we have identified CS-0777 kinases in human blood as fructosamine 3-kinase-related protein (FN3K-RP) and fructosamine 3-kinase (FN3K) (6Yonesu K. Kubota K. Tamura M. Inaba S. Honda T. Yahara C. Watanabe N. Matsuoka T. Nara F. Purification and identification of activating enzymes of CS-0777, a selective sphingosine 1-phosphate receptor 1 modulator, in erythrocytes.J. Biol. Chem. 2011; 286: 24765-24775Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar), whereas the phosphatase of CS-0777-P had not been identified thus far. In this study, we have successfully identified alkaline phosphatase, tissue-nonspecific isozyme (ALPL) as the major CS-0777-P phosphatase candidate in the human kidney by proteomic correlation profiling. According to available information, this is the first report applying proteomic correlation profiling to enzyme classes other than kinases; similarly, we believe this to be first application of proteomic correlation profiling to human tissue extract, which therefore has opened up wide usage of proteomic correlation profiling for all types of enzyme identification. All chemical compounds were synthesized in Daiichi Sankyo Co., Ltd. Chemical structures are illustrated in Fig. 1. Human frozen tissue blocks (kidney, liver, and lung) were collected by National Disease Research Interchange, and provided by the Human Animal Bridging Research Organization. The human small intestines were provided by Xenotech. Ethical approval was obtained from the Research Ethics Committee at Daiichi Sankyo Co., Ltd. All purification procedures were conducted at 4 °C. The block of human kidney was minced and homogenized in 9 volumes of homogenate buffer, 0.25 m sucrose containing 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), pH 7.0, and protease inhibitor mixture (Complete Tablet, Roche Applied Science) using a polytron homogenizer (Kinematica). The homogenate was sonicated by a Bioruptor (COSMO BIO) and filtered by a few sheets of gauze. The tissue homogenate was centrifuged with 9000 × g for 60 min at 4 °C and the supernatant was used as S9 fraction. The homogenate was centrifuged at 100,000 × g for 60 min, and the precipitate equivalent to 1.1 g of human kidney was dissolved in 10 ml of homogenate buffer containing 1% n-Dodecyl-β-d-maltopyranoside (DM), and then dialyzed against 1 L of anion exchange chromatography (AEX) buffer (50 mm Tris-HCl, pH 9.0, containing 0.1% DM, 5 mm CaCl2, 5 mm MgCl2, and 1 mm dithiotreitol). The dialyzed sample was filtered and loaded onto a multimodal anion exchange column (HiTrap Capto Adhere 5 ml, GE Healthcare). The column was equilibrated with AEX buffer over 30 min at a flow rate of 5 ml/min and the bound proteins were eluted with a linear gradient of 0–500 mm NaCl in AEX buffer for 10 min. The active fractions (10 ml) were dialyzed against 1 L of AEX buffer. The dialyzed sample was loaded onto an anion exchange column (Mono Q 5/50 GL, GE Healthcare). The column was equilibrated with AEX buffer over 30 min at a flow rate of 1 ml/min. The bound proteins were eluted with a linear gradient of 0–250 mm NaCl in AEX buffer for 20 min. The active fraction (1 ml) from the Mono Q column was loaded onto a gel filtration column (GFC, Superdex 200 5/50 GL, GE Healthcare). The column was equilibrated with 20 mm HEPES (pH 7.0) containing 0.1% DM, 500 mm NaCl, and 1 mm dithiotreitol (DTT) over 12 h. The separation was performed by an isocratic elution with the same buffer over 30 min at a flow rate of 40 μl/min. Five hundred fmol of bovine serum albumin (BSA) was added as the internal standard to the fractions from the gel filtration chromatography, and then the proteins were precipitated by methanol chloroform precipitation to remove the detergent and salts (31Wessel D. Flügge U.I. A method for the quantitaive recovery of protein in diluted solution in the presence of detergents and lipid.Anal. Biochem. 1984; 138: 141-143Crossref PubMed Scopus (3167) Google Scholar). Briefly, four volumes of methanol, one volume of chloroform, and three volumes of water were serially added and mixed vigorously to the fractions. After centrifugation, the aqueous phase was removed and four volumes of methanol were added. The precipitated proteins were collected by centrifugation and dried completely with a centrifuge evaporator. The dried proteins were dissolved with 8 m urea, 50 mm Tris-HCl, pH 8.0, 10 mm EDTA, pH 8.0, and 0.005% DM, and 10 mm DTT. The proteins were reduced at 37 °C for 20 min, followed by alkylation by incubation at 25 °C for 20 min in the dark with 20 mm iodoacetamide. The proteins were digested with 500 ng of trypsin (Modified trypsin, Promega, Madison, WI) at 37 °C for 12 h. The reaction was stopped by acidification with 5% formic acid to a pH lower than 2.5. Samples were desalted and concentrated by using slightly modified Stage Tips (32Rappsilber J. Ishihama Y. Mann M. Stop and go extractio
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