Molecular Cloning and Identification of -Acyl-D-glucosamine 2-Epimerase from Porcine Kidney as a Renin-binding Protein

N-Acetylneuraminic acid (NeuAc) is an important molecule in biological recognition systems. NeuAc is known to be biosynthesized either from UDP-N-acetyl-D-glucosamine by an action of UDP-N-acetyl-D-glucosamine 2-epimerase or from N-acetyl-D-glucosamine by N-acyl-D-glucosamine 2-epimerase (GlcNAc 2-epimerase). However, the physiological function of the GlcNAc 2-epimerase in NeuAc biosynthesis has not been fully evaluated. To clarify the role of GlcNAc 2-epimerase in NeuAc biosynthesis, the enzyme and its gene were isolated from porcine kidney cortex. Escherichia coli cells transformed with the gene expressed the GlcNAc 2-epimerase having the same properties as those of the GlcNAc 2-epimerase from porcine kidney. Sequence analysis indicated that the gene was capable of synthesizing a 46.5-kDa protein (402 amino acids) with a conserved leucine zipper motif. Homology search for the cloned gene revealed that the GlcNAc 2-epimerase was identical with renin-binding protein (RnBP) in porcine kidney (Inoue, H., Fukui, K., Takahashi, S., and Miyake, Y. (1990) J. Biol. Chem. 265, 6556-6561) (identity: 99.6% in nucleotide sequence, 99.0% in amino acid sequence). That GlcNAc 2-epimerase is a RnBP was confirmed by its ability to bind porcine kidney renin and mask its protease activity. These findings provide unequivocal evidence that the enzyme GlcNAc 2-epimerase is a RnBP. N-Acetylneuraminic acid (NeuAc) is an important molecule in biological recognition systems. NeuAc is known to be biosynthesized either from UDP-N-acetyl-D-glucosamine by an action of UDP-N-acetyl-D-glucosamine 2-epimerase or from N-acetyl-D-glucosamine by N-acyl-D-glucosamine 2-epimerase (GlcNAc 2-epimerase). However, the physiological function of the GlcNAc 2-epimerase in NeuAc biosynthesis has not been fully evaluated. To clarify the role of GlcNAc 2-epimerase in NeuAc biosynthesis, the enzyme and its gene were isolated from porcine kidney cortex. Escherichia coli cells transformed with the gene expressed the GlcNAc 2-epimerase having the same properties as those of the GlcNAc 2-epimerase from porcine kidney. Sequence analysis indicated that the gene was capable of synthesizing a 46.5-kDa protein (402 amino acids) with a conserved leucine zipper motif. Homology search for the cloned gene revealed that the GlcNAc 2-epimerase was identical with renin-binding protein (RnBP) in porcine kidney (Inoue, H., Fukui, K., Takahashi, S., and Miyake, Y. (1990) J. Biol. Chem. 265, 6556-6561) (identity: 99.6% in nucleotide sequence, 99.0% in amino acid sequence). That GlcNAc 2-epimerase is a RnBP was confirmed by its ability to bind porcine kidney renin and mask its protease activity. These findings provide unequivocal evidence that the enzyme GlcNAc 2-epimerase is a RnBP. INTRODUCTIONN-Acetylneuraminic acid (NeuAc) 1The abbreviations used are: NeuAcN-acetylneuraminic acidRnBPrenin-binding proteinHPLChigh performance liquid chromatographyPAGEpolyacrylamide gel electrophoresis. is an important constituent of the carbohydrate chain of many glycoproteins and glycolipids, and has an important function in many biological recognition processes (1Shauer R. Adv. Carbohydr. Chem. Biochem. 1982; 40: 131-234Crossref PubMed Scopus (933) Google Scholar). The biosynthesis of NeuAc has been studied extensively in vivo and in vitro (2McGuire E.J. Rosenberg A. Schengrund C.L. Biological Roles of Sialic Acid. Plenum Press, New York1976: 123Crossref Google Scholar, 3Warren L. Gottschalk A. Glycoproteins. Elsevier Publishing Co., Amsterdam, London and New York1972: 1097Google Scholar). Two kinds of 2-epimerases have been assigned in the first step of the NeuAc biosynthetic pathway. One is N-acyl-D-glucosamine 2-epimerase (GlcNAc 2-epimerase) (EC.5.1.3.8) catalyzing the interconversion of N-acetylglucosamine (GlcNAc) and N-acetylmannosamine (ManNAc). The other one is UDP-N-acetyl-D-glucosamine 2-epimerase (UDP-GlcNAc 2-epimerase) (EC.5.1.3.14). The enzyme catalyzes the direct formation of ManNAc from UDP-N-acetyl-D-glucosamine (UDP-GlcNAc) (4Van Rinsum J. Van Dijk W. Hooghwinkel G.J.M. Ferwerda W. Biochem. J. 1983; 210: 21-28Crossref PubMed Scopus (25) Google Scholar). ManNAc formed by either reaction is eventually converted to cytidine 5′-monophospho-N-acetylneuraminic acid (CMP-NeuAc) through the consecutive reactions: GlcNAc or UDP-GlcNAc → ManNAc → ManNAc 6-phosphate → NeuAc 9-phosphate → NeuAc → CMP-NeuAc (5Kornfeld S. Kornfeld R. Neufeld E.F. O'Brien P.J. Proc. Natl. Acad. Sci. U. S. A. 1964; 52: 371-379Crossref PubMed Scopus (254) Google Scholar, 6Ginsburg V. Adv. Enzymol. 1964; 26: 35-88PubMed Google Scholar). CMP-NeuAc is utilized as a precursor for the synthesis of connective tissues, blood cells, and other cellular macromolecules.Of the two 2-epimerases catalyzing the formation of ManNAc, UDP-GlcNAc 2-epimerase has been considered to be essential in the NeuAc biosynthesis (4Van Rinsum J. Van Dijk W. Hooghwinkel G.J.M. Ferwerda W. Biochem. J. 1983; 210: 21-28Crossref PubMed Scopus (25) Google Scholar), and the physiological significance of the GlcNAc 2-epimerase in the formation of NeuAc is not known. However, no definitive evidence denying the participation of the GlcNAc 2-epimerase in the NeuAc biosynthesis together with UDP-GlcNAc 2-epimerase has been presented.The GlcNAc 2-epimerase has been found in porcine kidney, rat kidney, liver, spleen, brain, intestinal mucosa, thymus, pancreas, and in salivary gland (4Van Rinsum J. Van Dijk W. Hooghwinkel G.J.M. Ferwerda W. Biochem. J. 1983; 210: 21-28Crossref PubMed Scopus (25) Google Scholar, 5Kornfeld S. Kornfeld R. Neufeld E.F. O'Brien P.J. Proc. Natl. Acad. Sci. U. S. A. 1964; 52: 371-379Crossref PubMed Scopus (254) Google Scholar). Datta (7Datta A. Biochemistry. 1970; 9: 3363-3370Crossref PubMed Scopus (28) Google Scholar) and Gosh and Roseman (8Ghosh S. Roseman S. J. Biol. Chem. 1965; 240: 1531-1536Abstract Full Text PDF PubMed Google Scholar) partially purified the enzyme from porcine kidney and found that the GlcNAc 2-epimerase activity is modulated by the catalytic amount of ATP. Datta (7Datta A. Biochemistry. 1970; 9: 3363-3370Crossref PubMed Scopus (28) Google Scholar) also reported that the GlcNAc 2-epimerase possesses two distinct interaction sites, a catalytic site for substrate and an allosteric site for ATP.To clarify reaction mechanism and function of the GlcNAc 2-epimerase, we have isolated from porcine kidney the enzyme and its gene, and analyzed their properties. Surprisingly, the GlcNAc 2-epimerase was found to be identical with a renin-binding protein (RnBP) isolated from porcine kidney (9Takahashi S. Ohsawa T. Miura R. Miyake Y. J. Biochem. (Tokyo). 1983; 93: 1583-1594Crossref PubMed Scopus (29) Google Scholar, 10Inoue H. Fukui K. Takahashi S. Miyake Y. J. Biol. Chem. 1990; 265: 6556-6561Abstract Full Text PDF PubMed Google Scholar). The physiological role of RnBP has been presumed by several investigators. Leckie and McConnell (11Leckie B.J. McConnell A. Circ. Res. 1975; 36: 513-519Crossref PubMed Scopus (84) Google Scholar) suggested that RnBP is a regulator of renin because RnBP can tightly bind to renin and inhibit the renin activity. Boyd (12Boyd G.W. Circ. Res. 1974; 35: 426-438Crossref PubMed Scopus (105) Google Scholar) proposed that RnBP is a renin carrier. Murakami et al. (13Murakami K. Chino S. Hirose S. Higaki J. Biomed. Res. 1980; 1: 476-481Crossref Scopus (6) Google Scholar) showed that the binding of RnBP is highly specific to renin and does not interact with other acid proteases in the kidney. Furthermore, in tissues containing renin, RnBP was always detected (14Tada M. Takahashi S. Miyano M. Miyake Y. J. Biochem. 1992; 112: 175-182Crossref PubMed Scopus (30) Google Scholar). Takahashi et al. (15Takahashi S. Inoue H. Miyake Y. J. Biol. Chem. 1992; 267: 13007-13013Abstract Full Text PDF PubMed Google Scholar) reported that human RnBP gene is located in chromosome X, spans about 10 kilobases and consists of 11 exons separated by 10 introns. Tribioli et al. (16Tribioli C. Mancini M. Plassart E. Bione S. Rivella S. Sala C. Torri G. Toniolo D. Hum. Mol. Genet. 1994; 3: 1061-1067Crossref PubMed Scopus (43) Google Scholar) and Faranda et al. (17Faranda S. Frattini A. Vezzoni P. Gene (Amst.). 1995; 155: 237-239Crossref PubMed Scopus (11) Google Scholar) indicated that the RnBP gene is mapped in distal Xq28 chromosomal band, closely linked to a housekeeping host cell factor 1-encoding gene, and both genes are transcribed in the same direction from telomere to centromere.In this paper, we report purification and molecular cloning of GlcNAc 2-epimerase from porcine kidney, and demonstrate the renin-binding ability of GlcNAc 2-epimerase.EXPERIMENTAL PROCEDURESMaterialsPorcine kidney was obtained from the Kyoto wholesale market. Porcine kidney renin was purchased from Sigma. DEAE-cellulose DE-52 was from Whatman. Q Sepharose FF, Sephadex G-100, Superose 12 HR 10/30, and Mono Q HR 5/5 were from Pharmacia Biotech Inc. Butyl-Toyopearl 650M was from Tosoh. Hydroxyapatite and lysyl endopeptidase were from Wako Pure Chemical Industries. The ZAP-cDNA synthesis kit, Gigapack II Gold packaging extract, and the Exo/Mung deletion kit were from Stratagene. Other chemicals were of the highest grade commercially available.GlcNAc 2-Epimerase AssayGlcNAc 2-epimerase was assayed by measuring interconversion of GlcNAc and ManNAc. The reaction mixture (0.1 ml) consisted of 100 mM Tris-HCl, pH 7.4, 40 mM ManNAc, 4 mM ATP, 10 mM MgCl2, and 20 µl of enzyme. After incubation at 37°C for 30 min, the reaction was stopped by boiling for 3 min. The reaction products were treated with 1-phenyl-3-methyl-5-pyrazolone, and the derivatives were analyzed by HPLC (18Honda S. Akao E. Suzuki S. Okuda M. Kakehi K. Nakamura J. Anal. Chem. 1989; 180: 351-357Google Scholar). One unit of enzyme activity was the quantity that produced 1 µmol of GlcNAc/1 min under the assay conditions.Purification of GlcNAc 2-EpimeraseUnless otherwise noted, all operations were carried out at 0–4°C. The potassium phosphate buffer, pH 7.6 (KPB), used always contained 1.0 mM EDTA and 0.05% 2-mercaptoethanol. Centrifugation was carried out at 16,000 × g for 30 min, and dialysis was for 16 h against 20 mM KPB.GlcNAc 2-Epimerase in Porcine KidneyKidney cortex (5.6 kg) was homogenized in 12 liters of 3.0 mM KPB. The supernatant (11.7 liters) obtained after centrifugation was diluted with 11.7 liters of cold water followed by adding 705 ml of 2.0% protamine sulfate. Precipitated materials were removed, and the supernatant (22.9 liters) was treated again with 2.3 liters of 2.0% protamine sulfate. Precipitates were washed in 5 liters of 10 mM KPB, and then the extract (5.8 l) was treated for 10 min with 58 g of bentonite, which was suspended in 580 ml of 1 mM EDTA. The mixture was centrifuged, and the supernatant was dialyzed. The dialysate (6.5 liters) was put on a DEAE-cellulose DE-52 column (25 × 13 cm). After washing the column with 50 mM KPB containing 100 mM KCl, the GlcNAc 2-epimerase was eluted with 200 mM KPB containing 150 mM KCl. Ammonium sulfate (6.7 kg) was added to the eluate (12 liters) to 80% saturation. The precipitates were dissolved in 200 ml of 20 mM KPB and dialyzed. The dialysate (280 ml) was put on a hydroxyapatite column (2.6 × 9.5 cm) equilibrated with 10 mM KPB, and the enzyme was eluted with 324 ml of the same buffer. The enzyme was treated with ammonium sulfate (80%) and dialyzed. The dialysate (40 ml) was put on a Q Sepharose column (2.6 × 10 cm), and developed with a linear gradient (1 liter) of 100–400 mM KCl in 20 mM KPB. The GlcNAc 2-epimerase was eluted at 180–220 mM KCl. Active fractions were concentrated by ultrafiltration with a ultrafilter UK-10 (Toyo Roshi Kaisha) and dialyzed. The dialysate (12 ml) was put on a Mono Q column. The column was washed with 20 mM KPB containing 200 mM KCl and developed with a linear gradient (20 ml) of 200–300 mM KCl in 20 mM KPB. The GlcNAc 2-epimerase was eluted at about 240 mM KCl. Active fractions were concentrated with ammonium sulfate (80%) and used throughout this study after dialysis.GlcNAc 2-Epimerase Expressed in Escherichia coliE. coli XL1-Blue were transformed with a plasmid pEP114 (see “cDNA Library, Subcloning, and Nucleotide Sequencing” for subcloning) an aerobically grown for 16 h at 30°C in Luria-Bertani medium (7 l) supplemented with 1 mM isopropyl-β-D-thiogalactopyranoside and 0.1 mg/ml ampicillin. The washed cells (about 32 g wet weight) were suspended in 350 ml of KPB, disrupted by sonication. After adding 93 mg of protamine to 340 ml of extract, the mixture was stirred gently for 30 min and centrifuged. The precipitates were discarded, and 3.5 g of bentonite was added to 350 ml of the supernatant. After incubation for 10 min, the precipitates were discarded. The enzyme was treated with ammonium sulfate (80%) and dialyzed. The dialysate was put on a DEAE-cellulose DE-52 column (5 × 20 cm) and then the enzyme was eluted with 200 mM KPB containing 150 mM KCl. Fractions containing GlcNAc 2-epimerase were concentrated with ammonium sulfate (80%) and dialyzed. The dialysate was put on a Sephadex G-100 column (5 × 90 cm), and then the enzyme was eluted with 20 mM KPB containing 200 mM KCl. Active fractions were concentrated with ammonium sulfate (80%) and dialyzed. The dialysate was put on a Q Sepharose column (2.6 × 10 cm), and then the enzyme was eluted with a linear gradient of 100 to 400 mM KCl in 20 mM KPB. Active fractions, which were eluted between 180 and 220 mM KCl, were concentrated with ammonium sulfate (80%) and dialyzed. The dialysate was put on a Butyl-Toyopearl 650 M column (2.6 × 10 cm) and then the enzyme was eluted with a linear gradient of 30 to 0% ammonium sulfate in 20 mM KPB. The enzyme was eluted between 19.5 and 13.5% ammonium sulfate. The active fractions were concentrated with ammonium sulfate (80%), dissolved in 8 ml of 20 mM KPB and then dialyzed.Preparation of Anti-GlcNAc 2-Epimerase AntibodyA Japanese white rabbit was injected subcutaneously into foot pads on the back with 1 ml of emulsion containing 840 µg of the purified GlcNAc 2-epimerase from porcine kidney cortex and complete Friend's adjuvant. The same amount of GlcNAc 2-epimerase emulsion was injected at 2, 4, and 6 weeks after the first injection. After 8.5 weeks, bleeding was performed. Blood was allowed to clot at 37°C for 30 min and was then stored at 4°C overnight. An antiserum was separated from clots by centrifugation, added to 0.01% NaN3, and stored at 4°C. An IgG from the antiserum was purified with protein A-Sepharose (Pharmacia).Inhibition of Renin by GlcNAc 2-EpimeraseInhibitory effect on the renin activity by GlcNAc 2-epimerase was assayed according to the method of Takahashi et al. (9Takahashi S. Ohsawa T. Miura R. Miyake Y. J. Biochem. (Tokyo). 1983; 93: 1583-1594Crossref PubMed Scopus (29) Google Scholar). Porcine kidney renin (1.4 pmol) was incubated at 37°C for 30 min in the presence or absence of GlcNAc 2-epimerase in a mixture (0.1 ml) containing 100 mM sodium phosphate buffer, pH 6.5, 1 mM EDTA, 1 µM leupeptin, and 0.05% bovine serum albumin. The reaction was stopped with the addition of 0.9 ml of chilled sodium phosphate buffer, pH 6.5, containing 1 mM EDTA, 1 µM leupeptin, and 0.05% bovine serum albumin. Remaining renin activity was assayed by measuring the rate of formation of angiotensin I from porcine plasma angiotensinogen (19Takahashi S. Ohsawa T. Miura R. Miyake Y. J. Biochem. (Tokyo). 1983; 93: 265-274Crossref PubMed Scopus (47) Google Scholar).Binding of Renin with GlcNAc 2-EpimerasePorcine kidney renin (1.4 pmol) was incubated at 37°C for 30 min in the presence or absence of the GlcNAc 2-epimerase (140 pmol) in the mixture (0.1 ml) as described above. After incubation, the mixture was put on a Superose 12 HR 10/30 column equilibrated with 50 mM KPB containing 150 mM KCl and incubation products were eluted with the same buffer at 1.0 ml/min. Renin activity in each fraction (0.5 ml) was determined as described above.cDNA Library, Subcloning, and Nucleotide SequencingTotal RNAs were extracted from porcine kidney cortex in acid guanidinium thiocyanate-phenol-chloroform mixture (20Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (62983) Google Scholar), and poly(A)+ RNA was fractionated on oligo(dT)-cellulose column chromatography. The cDNA library was constructed as described by Gubler and Hoffman (21Gubler U. Hoffman B.J. Gene (Amst.). 1983; 25: 263-269Crossref PubMed Scopus (3066) Google Scholar) by using λ DNA phage (λ ZAP) vector and E. coli The cDNA library of × was by using an 2-epimerase in were using a against the protein of the GlcNAc 2-epimerase and the IgG One of the were converted to cDNA (1.4 in the between and of by in vivo in E. coli XL1-Blue host with and plasmid constructed was deletion of the plasmid was carried out by at a site of followed by with and These deletion were with DNA and the were used to E. coli One of the a deletion of about of nucleotide in cDNA and was The of cDNA were in both by the method of et al. S. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google of and 2-epimerase (0.5 from porcine kidney cortex was with 5 µg of lysyl endopeptidase in ml of 100 mM Tris-HCl, pH containing 4 M at 37°C for 12 After the were separated with a HPLC column × 150 amino of protein or were by P. Chem. Google Scholar) on an protein amino of protein were using by et al. P. J. Biochem. 1982; PubMed Scopus Google Scholar). amino and were acid at for h were analyzed with a amino acid gel was with gradient gel as described by 1970; PubMed Scopus Google Scholar). were with with other nucleotide and protein were using the Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar) with the of nucleotide and of amino acid INTRODUCTIONN-Acetylneuraminic acid (NeuAc) 1The abbreviations used are: NeuAcN-acetylneuraminic acidRnBPrenin-binding proteinHPLChigh performance liquid chromatographyPAGEpolyacrylamide gel electrophoresis. is an important constituent of the carbohydrate chain of many glycoproteins and glycolipids, and has an important function in many biological recognition processes (1Shauer R. Adv. Carbohydr. Chem. Biochem. 1982; 40: 131-234Crossref PubMed Scopus (933) Google Scholar). The biosynthesis of NeuAc has been studied extensively in vivo and in vitro (2McGuire E.J. Rosenberg A. Schengrund C.L. Biological Roles of Sialic Acid. Plenum Press, New York1976: 123Crossref Google Scholar, 3Warren L. Gottschalk A. Glycoproteins. Elsevier Publishing Co., Amsterdam, London and New York1972: 1097Google Scholar). Two kinds of 2-epimerases have been assigned in the first step of the NeuAc biosynthetic pathway. One is N-acyl-D-glucosamine 2-epimerase (GlcNAc 2-epimerase) (EC.5.1.3.8) catalyzing the interconversion of N-acetylglucosamine (GlcNAc) and N-acetylmannosamine (ManNAc). The other one is UDP-N-acetyl-D-glucosamine 2-epimerase (UDP-GlcNAc 2-epimerase) (EC.5.1.3.14). The enzyme catalyzes the direct formation of ManNAc from UDP-N-acetyl-D-glucosamine (UDP-GlcNAc) (4Van Rinsum J. Van Dijk W. Hooghwinkel G.J.M. Ferwerda W. Biochem. J. 1983; 210: 21-28Crossref PubMed Scopus (25) Google Scholar). ManNAc formed by either reaction is eventually converted to cytidine 5′-monophospho-N-acetylneuraminic acid (CMP-NeuAc) through the consecutive reactions: GlcNAc or UDP-GlcNAc → ManNAc → ManNAc 6-phosphate → NeuAc 9-phosphate → NeuAc → CMP-NeuAc (5Kornfeld S. Kornfeld R. Neufeld E.F. O'Brien P.J. Proc. Natl. Acad. Sci. U. S. A. 1964; 52: 371-379Crossref PubMed Scopus (254) Google Scholar, 6Ginsburg V. Adv. Enzymol. 1964; 26: 35-88PubMed Google Scholar). CMP-NeuAc is utilized as a precursor for the synthesis of connective tissues, blood cells, and other cellular macromolecules.Of the two 2-epimerases catalyzing the formation of ManNAc, UDP-GlcNAc 2-epimerase has been considered to be essential in the NeuAc biosynthesis (4Van Rinsum J. Van Dijk W. Hooghwinkel G.J.M. Ferwerda W. Biochem. J. 1983; 210: 21-28Crossref PubMed Scopus (25) Google Scholar), and the physiological significance of the GlcNAc 2-epimerase in the formation of NeuAc is not known. However, no definitive evidence denying the participation of the GlcNAc 2-epimerase in the NeuAc biosynthesis together with UDP-GlcNAc 2-epimerase has been presented.The GlcNAc 2-epimerase has been found in porcine kidney, rat kidney, liver, spleen, brain, intestinal mucosa, thymus, pancreas, and in salivary gland (4Van Rinsum J. Van Dijk W. Hooghwinkel G.J.M. Ferwerda W. Biochem. J. 1983; 210: 21-28Crossref PubMed Scopus (25) Google Scholar, 5Kornfeld S. Kornfeld R. Neufeld E.F. O'Brien P.J. Proc. Natl. Acad. Sci. U. S. A. 1964; 52: 371-379Crossref PubMed Scopus (254) Google Scholar). Datta (7Datta A. Biochemistry. 1970; 9: 3363-3370Crossref PubMed Scopus (28) Google Scholar) and Gosh and Roseman (8Ghosh S. Roseman S. J. Biol. Chem. 1965; 240: 1531-1536Abstract Full Text PDF PubMed Google Scholar) partially purified the enzyme from porcine kidney and found that the GlcNAc 2-epimerase activity is modulated by the catalytic amount of ATP. Datta (7Datta A. Biochemistry. 1970; 9: 3363-3370Crossref PubMed Scopus (28) Google Scholar) also reported that the GlcNAc 2-epimerase possesses two distinct interaction sites, a catalytic site for substrate and an allosteric site for ATP.To clarify reaction mechanism and function of the GlcNAc 2-epimerase, we have isolated from porcine kidney the enzyme and its gene, and analyzed their properties. Surprisingly, the GlcNAc 2-epimerase was found to be identical with a renin-binding protein (RnBP) isolated from porcine kidney (9Takahashi S. Ohsawa T. Miura R. Miyake Y. J. Biochem. (Tokyo). 1983; 93: 1583-1594Crossref PubMed Scopus (29) Google Scholar, 10Inoue H. Fukui K. Takahashi S. Miyake Y. J. Biol. Chem. 1990; 265: 6556-6561Abstract Full Text PDF PubMed Google Scholar). The physiological role of RnBP has been presumed by several investigators. Leckie and McConnell (11Leckie B.J. McConnell A. Circ. Res. 1975; 36: 513-519Crossref PubMed Scopus (84) Google Scholar) suggested that RnBP is a regulator of renin because RnBP can tightly bind to renin and inhibit the renin activity. Boyd (12Boyd G.W. Circ. Res. 1974; 35: 426-438Crossref PubMed Scopus (105) Google Scholar) proposed that RnBP is a renin carrier. Murakami et al. (13Murakami K. Chino S. Hirose S. Higaki J. Biomed. Res. 1980; 1: 476-481Crossref Scopus (6) Google Scholar) showed that the binding of RnBP is highly specific to renin and does not interact with other acid proteases in the kidney. Furthermore, in tissues containing renin, RnBP was always detected (14Tada M. Takahashi S. Miyano M. Miyake Y. J. Biochem. 1992; 112: 175-182Crossref PubMed Scopus (30) Google Scholar). Takahashi et al. (15Takahashi S. Inoue H. Miyake Y. J. Biol. Chem. 1992; 267: 13007-13013Abstract Full Text PDF PubMed Google Scholar) reported that human RnBP gene is located in chromosome X, spans about 10 kilobases and consists of 11 exons separated by 10 introns. Tribioli et al. (16Tribioli C. Mancini M. Plassart E. Bione S. Rivella S. Sala C. Torri G. Toniolo D. Hum. Mol. Genet. 1994; 3: 1061-1067Crossref PubMed Scopus (43) Google Scholar) and Faranda et al. (17Faranda S. Frattini A. Vezzoni P. Gene (Amst.). 1995; 155: 237-239Crossref PubMed Scopus (11) Google Scholar) indicated that the RnBP gene is mapped in distal Xq28 chromosomal band, closely linked to a housekeeping host cell factor 1-encoding gene, and both genes are transcribed in the same direction from telomere to centromere.In this paper, we report purification and molecular cloning of GlcNAc 2-epimerase from porcine kidney, and demonstrate the renin-binding ability of GlcNAc