Báo cáo khoa học: De novo synthesis, uptake and proteolytic processing of lipocalin-type prostaglandin D synthase, b-trace, in the kidneys pptx

De novo synthesis, uptake and proteolytic processing of lipocalin-type prostaglandin D synthase, b-trace, in the kidneys Nanae Nagata1, Ko Fujimori1,2, Issey Okazaki1, Hiroshi Oda3, Naomi Eguchi1, Yoshio Uehara4 and Yoshihiro Urade1 Department of Molecular Behavioral Biology, Osaka Bioscience Institute, Japan Laboratory of Biodefense and Regulation, Osaka University of Pharmaceutical Sciences, Japan Central Research Institute, Maruha Nichiro Holdings, Inc., Ibaraki, Japan Health Service Center, The University of Tokyo, Japan Keywords kidney; monoclonal antibody; renal disease; urine; b-trace Correspondence Y Urade, Department of Molecular Behavioral Biology, Osaka Bioscience Institute, 6-2-4 Furuedai, Suita, Osaka 565-0874, Japan Fax: +81 6872 2841 Tel: +81 6872 4851 E-mail: uradey@obi.or.jp (Received 12 August 2009, revised 29 September 2009, accepted October 2009) doi:10.1111/j.1742-4658.2009.07426.x Lipocalin-type prostaglandin D synthase (L-PGDS) is a multifunctional protein that produces prostaglandin D2 and binds and transports various lipophilic substances after secretion into various body fluids as b-trace L-PGDS has been proposed to be a useful diagnostic marker for renal injury associated with diabetes or hypertension, because the urinary and plasma concentrations are increased in patients with these diseases However, it remains unclear whether urinary L-PGDS is synthesized de novo in the kidney or taken up from the blood circulation In crude extracts of monkey kidney and human urine, we found L-PGDS with its original N-terminal sequence starting from Ala23 after the signal sequence, and also its N-terminal-truncated products starting from Gln31 and Phe34 In situ hybridization and immunohistochemical staining with monoclonal antibody 5C11, which recognized the amino-terminal Ala23–Val28 loop of L-PGDS, revealed that both the mRNA and the intact form of L-PGDS were localized in the cells of Henle’s loop and the glomeruli of the kidney, indicating that L-PGDS is synthesized de novo in these tissues However, truncated forms of L-PGDS were found in the lysosomes of tubular cells, as visualized by immunostaining with 10A5, another monoclonal antibody, which recognized the three-turn a-helix between Arg156 and Thr173 These results suggest that L-PGDS is taken up by tubular cells and actively degraded within their lysosomes to produce the N-terminal-truncated form Structured digital abstract l MINT-7266187: L-PGDS (uniprotkb:P41222) and Cathepsin D (uniprotkb:Q4R4P0) colocalize (MI:0403) by fluorescence microscopy (MI:0416) l MINT-7266176: L-PGDS (uniprotkb:P41222) and Cathepsin B (uniprotkb:Q4R5M2) colocalize (MI:0403) by fluorescence microscopy (MI:0416) Introduction Lipocalin-type prostaglandin D synthase (L-PGDS; EC 5.3.99.2) catalyzes the isomerization of prostaglandin H2 (PGH2), a common precursor of various pro- stanoids, to produce PGD2, an endogenous regulator of sleep and pain [1–5] L-PGDS was originally purified from rat brain [6] and found to be a monomeric Abbreviations CSF, cerebrospinal fluid; DIG, digoxigenin; GST, glutathione S-transferase; KO, knockout; L-PGDS, lipocalin-type prostaglandin D synthase; MAb, monoclonal antibody; PG, prostaglandin; SPR, surface plasmon resonance 7146 FEBS Journal 276 (2009) 7146–7158 ª 2009 The Authors Journal compilation ª 2009 FEBS N Nagata et al protein with a molecular mass of approximately 26 000 [1], and was later demonstrated to be N-glycosylated at two positions, Asn51 and Asn78 [7] L-PGDS is known to be identical to b-trace [8,9], the major protein in human cerebrospinal fluid (CSF) [10] Moreover, L-PGDS is a member of the lipocalin gene family, as judged from its amino acid homology with several conserved motifs in this family [11] L-PGDS binds various lipophilic substances, such as retinoids, thyroid hormones [12], bilirubin, biliverdin [13], gangliosides [14] and amyloid b-peptide [15], with high affinities (Kd = 0.02–2 lm), similar to those of other proteins in the lipocalin gene family L-PGDS is dominantly expressed in the brain, heart and male genital organs [1], is secreted into various body fluids, such as CSF, plasma, seminal plasma and urine [5], and functions as both a PGD2-producing enzyme and an extracellular transporter of various lipophilic substances Previously, we have generated several types of mouse monoclonal antibody (MAb) against human L-PGDS and developed a sandwich ELISA with MAb-1B7 and MAb-7F5 [16] The L-PGDS level in various human body fluids, measured by the ELISA system, revealed that the urinary excretion of L-PGDS is increased in the early stage of diabetes [16] and declines with the control of the blood glucose level by hospitalization [17,18] Moreover, urinary L-PGDS excretion is increased in patients with hypertension with latent renal injury [19] Thus, L-PGDS is thought to be a useful diagnostic marker for these diseases [1,20] Urinary L-PGDS is believed to reflect the change in glomerular permeability because of its small molecular mass and anionic property However, the origin of urinary L-PGDS remains unclear Furthermore, only 10% of L-PGDS administered intravenously in canines is recovered in the urine [21], suggesting that the permeability of L-PGDS may be low and ⁄ or urinary L-PGDS is reabsorbed or metabolized after filtration through the glomerular membrane In this study, we examined the localization of L-PGDS in monkey kidney by in situ hybridization and immunohistochemical analysis with two novel MAbs against L-PGDS, and found that L-PGDS was synthesized de novo in monkey kidney, and that N-terminal-truncated forms of L-PGDS were present in monkey kidney and human urine Results Production and characterization of novel anti-human L-PGDS MAbs We purified b-trace from human CSF or the human D1)22Cys65,167Ala L-PGDS from Escherichia coli trans- Lipocalin-type prostaglandin D synthase in kidney formants In SDS-PAGE, b-trace showed a broad band at position Mr = 25 000–27 000 as a result of its glycosylation (Fig 1A, lane 1), whereas D1)22Cys65,167Ala L-PGDS showed a sharp band at position Mr = 19 000 because of a lack of glycosylation (Fig 1A, lane 2) We generated two novel anti-human L-PGDS MAbs, i.e 5C11 and 10A5, by immunizing L-PGDS knockout (KO) mice with b-trace and rats with D1)22Cys65,167Ala L-PGDS, respectively Western blot analysis showed that both MAb-5C11 (Fig 1A, lanes and 4) and MAb-10A5 (Fig 1A, lanes and 6) recognized b-trace and D1)22Cys65,167Ala L-PGDS, similar to the case of polyclonal antibody against human L-PGDS (Fig 1A, lanes and 10) By contrast, previously generated MAb1B7 (Fig 1A, lanes and 8) bound to D1)22Cys65,167Ala L-PGDS efficiently but to b-trace only slightly These results indicate that novel MAb-5C11 and MAb-10A5 recognize both highly glycosylated b-trace and non-glycosylated recombinant L-PGDS with affinities greater than those of the previously obtained MAb-1B7 [16] Epitopes recognized by MAb-5C11 and MAb-10A5 were determined by western blot analysis using glutathione S-transferase (GST)-fusion proteins containing different lengths of human L-PGDS (Fig 1B) Each GST-fusion protein was purified, separated by SDSPAGE and stained with silver (Fig 1C, top panel) MAb-5C11 bound only to the Ala23–Gln190 protein, whereas MAb-10A5 bound to all constructs, except for that carrying the Glu174–Gln190 region, indicating that epitopes for MAb-5C11 and MAb-10A5 were located in Ala23–Val28 and Arg156–Thr173, respectively In the tertiary structure of human L-PGDS (Fig 1D, E) modeled from the crystallographic (PDB codes: 2CZT and 2CZU [22]) and NMR (PDB code: 2E4J [23]) structures of mouse L-PGDS, the epitopes for MAb-5C11 and MAb-10A5 were localized to the amino-terminal loop and the three-turn a-helical region of human L-PGDS, respectively The previously generated MAb-1B7 [16] bound to the EF-loop on the side opposite the sites for MAb-5C11 and MAb-10A5 The immunoglobulin subclass and light chain type were determined to be IgG1 (j) for MAb-5C11 and IgG2a (j) for MAb-10A5 (Table 1) Surface plasmon resonance (SPR) analysis of the antigen–antibody interaction demonstrated that immobilized MAb-5C11 and MAb-10A5 bound to the soluble form of b-trace with Kd values of 91 and 2900 nm, respectively, and to the recombinant D1)22Cys65,167Ala L-PGDS with values of 167 and 57 nm, respectively The binding affinities of MAb-5C11 and MAb-10A5 were increased about 50–100-fold for the immobilized b-trace to 1.1 and 4.5 nm, respectively, and for the recombinant FEBS Journal 276 (2009) 7146–7158 ª 2009 The Authors Journal compilation ª 2009 FEBS 7147 Lipocalin-type prostaglandin D synthase in kidney N Nagata et al A MAb SYPRO orange kDa 5C11 10A5 1B7 PAb 26 19 B * Ala23 C 10 10 11 160 190 Gln190 * Ser29 Silver staining Gln35 Ser52 Asp74 Glu90 MAb-5C11 Tyr107 Val121 Gly140 Arg156 10 11 D (Amino acids) 80 120 40 Glu174 MAb-10A5 E Asn51 1B7 C D C H B Asn51 1B7 Asn78 G F E A 10A5 10A5 5C11 N 5C11 Fig Detection of human CSF b-trace and recombinant L-PGDS by MAbs against human L-PGDS and characterization of their antigenic epitopes (A) b-trace (lanes 1, 3, 5, and 9) and recombinant D1)22Cys65,167Ala human L-PGDS (lanes 2, 4, 6, and 10) were separated by SDS-PAGE and stained with SYPRO Orange (lanes and 2), followed by blotting onto a polyvinylidine difluoride membrane The blots were then reacted with human L-PGDS MAb-5C11 (lanes and 4), MAb-10A5 (lanes and 6), MAb-1B7 (lanes and 8) or polyclonal antibody (PAb, lanes and 10) for western blot analysis Molecular size markers are shown on the left (B) Schematic representation of GST-fusion proteins containing a series of amino-terminus-truncated human L-PGDS The amino-terminal amino acid residue of each mutant is indicated on the left The signal sequence of the amino-terminal 22 amino acid residues of human L-PGDS was removed in the parent mutant, D1)22Cys65,167Ala L-PGDS (line 1) Asterisks indicate the two N-glycosylation sites (C) The purified GST-fusion proteins containing the series of amino-terminus-truncated L-PGDS were separated by SDS-PAGE, followed by silver staining (top panel) or used for western blot analysis with MAb-5C11 (middle panel) or MAb-10A5 (bottom panel) Lanes 1–11 correspond to the mutants 1–11 shown in (B) (D) Positions of the antigenic epitopes for MAbs on the ribbon model of human L-PGDS, which is composed of nine b-strands (strand A, Gly40–Ala49; strand B, Cys65–Ala72; strand C, Gly76–Arg85; strand D, Gln88–Pro98; strand E, Ser104–Arg108; strand F, Tyr116–Thr123; strand G, Val128–Gly135; strand H, Phe143–Ser150; strand I, Ile177–Phe179) and a three-turn a-helix (Ala157–Ala169) The epitopes of MAb-5C11, MAb-10A5 and MAb-1B7 are shown in blue, orange and dark gray, respectively Two N-glycosylation sites (Asn51 and Asn78) are shown in green and pink, respectively (E) Positions of the antigenic epitopes on the surface model of L-PGDS drawn using PYMOL software (DeLano Scientific LLC, Palo Alto, CA, USA) The epitopes for MAb-5C11, MAb-10A5 and MAb-1B7 are shown in blue, orange and dark gray, respectively The N-glycosylation sites (Asn51) is shown in green D1)22Cys65,167Ala L-PGDS to 0.52 and 0.16 nm, respectively Thus, MAb-5C11 bound to both b-trace and the recombinant D1)22Cys65,167Ala L-PGDS with almost the same affinity, in either the soluble or immo7148 bilized state The binding affinity of MAb-5C11 for immobilized b-trace was approximately fivefold higher than the affinities of MAb-1B7 and MAb-10A5 In this model, the N-terminal and C-terminal regions of FEBS Journal 276 (2009) 7146–7158 ª 2009 The Authors Journal compilation ª 2009 FEBS N Nagata et al Lipocalin-type prostaglandin D synthase in kidney Table Characterization of the three anti-human L-PGDS MAbs used in this study Kd (nM) D1)22Cys65,167,Ala L-PGDS b-trace protein Antigen Animal Clone Subclass Soluble form Immobilized Soluble form Immobilized b-trace protein Recombinant L-PGDS Recombinant L-PGDS L-PGDS KO mouse Rat Wild-type mouse 5C11 10A5 1B7 IgG (j) IgG 2a (j) IgG (j) 91 2900 33 1.1 4.5 5.9 167 57 50 0.52 0.16 0.96 L-PGDS are exposed on the surface of the molecule However, in the soluble form, the affinities of MAb10A5 for native L-PGDS were lower than those for the recombinant protein, suggesting that the C-terminal region may be partially covered by a sugar chain in native L-PGDS, because the N-glycosylation site (Asn51) is near the C-terminal region in the surface model (Fig 1E) We used these MAbs against human L-PGDS for immunochemical and immunohistochemical analyses of monkey kidney, because the homology between human and monkey L-PGDS is very high (only five amino acid substitutions of a total of 190 amino acid residues, giving 97.4% amino acid identity; GenBank M61900 for human and DDBJ Accession No AB032480 for monkey; Fig 2A) The comparison of the amino acid sequences revealed that the sequences of the antigenic regions recognized by MAb-5C11, MAb-1B7 and MAb-10A5 were also highly conserved in both species The sequence of the antigenic epitope of MAb-5C11 (Ala23–Val28) was the same in both species; that of MAb-1B7 contained only one substitution (R108 in human and G108 in monkey) of 14 amino acid residues; and that of MAb-10A5 (Arg156– Thr173) also contained only one substitution (T164 in human and S164 in monkey) of 18 amino acid residues As a result of the highly conserved sequences between both species, all anti-human L-PGDS MAbs bound to monkey L-PGDS Western blot analysis of monkey kidney samples, partially purified by immunoaffinity columns conjugated with MAb-1B7, MAb-5C11 or MAb-10A5, revealed that all of these MAbs showed a broad single immunoreactive band at the same position (Mr = 25 000– 27 000) as that of purified human L-PGDS ⁄ b-trace from human CSF (Fig 2B) These results indicate that these MAbs selectively recognized L-PGDS and did not bind other proteins in monkey kidney Localization of L-PGDS in monkey kidney The localization of L-PGDS in monkey kidney was then determined by immunohistochemical staining with MAb-5C11, MAb-10A5 and MAb-1B7 and by in situ hybridization with antisense RNA for L-PGDS mRNA (Fig 3) The staining profile by in situ hybridization of mRNA for L-PGDS was similar to that obtained with MAb-5C11 (Fig 3A, B) By contrast, the staining profile with MAb-10A5 was similar to that with MAb-1B7 (Fig 3C, D) In the cortex and outer medulla, the L-PGDS mRNA and L-PGDS protein detected with MAb-5C11 were localized in the distal tubules, including Henle’s loop (Fig 3E, F) MAb-10A5 showed generally much weaker staining in the tubules than that obtained with MAb-1B7 (Fig 3G, H) In the glomeruli, the mRNA and L-PGDS protein detected with MAb-5C11 were localized in the epithelial cells of Bowman’s capsule and in occasional podocytes (arrowhead and arrow, respectively, in Fig 3I, J) Podocytes were identified by morphological criteria as cells located adjacent to the outer aspect of the glomerular basement membrane By contrast, MAb-10A5 showed scarce, and MAb-1B7 weak, immunoreactivity in the glomeruli (Fig 3K, L) In higher magnification analysis, positive staining in the tubules was observed in perinuclear regions by in situ hybridization and by immunostaining with MAb-5C11 (Fig 3M, N) Although punctuate structures were seen by immunostaining with MAb-10A5 (Fig 3O), diffuse cytoplasmic staining was observed with MAb-1B7 (Fig 3P) No positive signals were observed when sense RNA probe, mouse IgG or rat IgG was applied (Fig 3Q–T) Confocal laser scanning microscopy revealed that MAb-5C11-positive fluorescence was colocalized with L-PGDS mRNA in Henle’s loop (Fig 4A) and glomeruli (not shown), suggesting that L-PGDS was synthesized de novo in these regions of the kidney However, MAb-10A5-positive fluorescence overlapped that of cathepsin B, a lysosomal marker, in the tubules (Fig 4B) In higher magnification analysis, a minor immunoreactivity of cathepsin B was not colocalized with MAb-10A5 immunoreactivity, but the majority of these two immunoreactivities was colocalized to the same organella (Fig 4C) Furthermore, MAb-10A5- FEBS Journal 276 (2009) 7146–7158 ª 2009 The Authors Journal compilation ª 2009 FEBS 7149 Lipocalin-type prostaglandin D synthase in kidney N Nagata et al Fig Alignment of the amino acid sequences of monkey and human L-PGDS (A) Amino acid sequence of human L-PGDS was compared with that of monkey L-PGDS Grey tinted boxes indicate substituted amino acid residues Open boxes indicate the epitopes of MAb-5C11, MAb-1B7 and MAb-10A5 Conserved residues (*) are indicated below the sequences (B) Crude extracts of monkey kidney (1 lg protein, lane 1) and purified human CSF L-PGDS ⁄ b-trace (0.1 lg protein, lane 2) were applied for SDS-PAGE and stained with SYPRO Orange The purified human CSF L-PGDS ⁄ b-trace (0.1 lg protein, lane 3) and the partially purified samples from the crude extracts of monkey kidney (2 mg protein, lanes 4–6), obtained by immunoaffinity chromatography with MAb-1B7, MAb-5C11 or MAb-10A5, were separated by SDS-PAGE and analyzed by western blot analysis with each MAb (lanes 4, and 6, respectively) Positions of molecular size markers are shown on the left positive fluorescence overlapped that of cathepsin D, another lysosomal marker (Fig 4D), indicating that the MAb-10A5-positive punctate fluorescence was distributed to lysosomes These results, taken together, suggest that L-PGDS is re-absorbed from the urine into the tubules and proteolytically degraded within lysosomes in the tubule cells Identification of L-PGDS of various sizes in monkey kidney and human urine We applied crude extracts of monkey kidney to a MAb-1B7-conjugated immunoaffinity column and obtained purified L-PGDS Because of its N-glycosylation at positions Asn51 and Asn78 [7], purified L-PGDS showed a broad band of two different sizes, 7150 Mr = 19 000–22 000 and Mr = 25 000–27 000, the latter of which corresponded to the intact form of L-PGDS ⁄ b-trace with N-glycosylation Western blot analysis showed that MAb-1B7 and MAb-10A5 were reactive with both sizes of L-PGDS, whereas MAb5C11 selectively reacted with the intact form of L-PGDS with Mr = 25 000–27 000 (Fig 5A) After glycopeptidase F treatment, the two forms of monkey L-PGDS migrated to positions of Mr = 18 000 and Mr = 19 000, the latter of which corresponds to the non-glycosylated form of L-PGDS [7], and both of which were recognized by MAb-1B7 and MAb-10A5 By contrast, MAb-5C11 bound to non-, mono- and di-glycosylated forms of L-PGDS with Mr = 19 000, Mr = 22 000 and Mr = 27 000, respectively, but not to the small band at Mr = 18 000 (Fig 5B) FEBS Journal 276 (2009) 7146–7158 ª 2009 The Authors Journal compilation ª 2009 FEBS N Nagata et al Lipocalin-type prostaglandin D synthase in kidney 5C11 ISH B A 1B7 10A5 C D mm F E G H 50 m J I * M * N L K * O 20 m m P 20 m Q R S 50 m * m T 20 m m Fig Localization of L-PGDS-immunoreactive protein and mRNA in monkey kidney Sections of monkey kidney were used for in situ hybridization (ISH) and immunoperoxidase staining with MAb-5C11, MAb-10A5 and MAb-1B7 (A–D) Low-magnification views At high magnification, L-PGDS signals were detected in the tubules in the cortex and outer medulla (E–H) The signals were also detected in the glomeruli (I–L) Asterisks, arrowheads and arrows indicate Bowman’s space, Bowman’s capsule and cytoplasm of podocytes, respectively (M–P) High-magnification micrographs of L-PGDS signals in tubule cells (Q–S) Staining profile by in situ hybridization with sense RNA probe (Q) and immunostaining with mouse IgG (R) or rat IgG (S and T) Scale bars: (A–D) mm; (E–H, Q–S) 50 lm; (I–P, T) 20 lm; insets, lm FEBS Journal 276 (2009) 7146–7158 ª 2009 The Authors Journal compilation ª 2009 FEBS 7151 Lipocalin-type prostaglandin D synthase in kidney A Nomarski * N Nagata et al DAPI 5C11 * ISH * * Merge * 10 µm B Nomarski * DAPI 10A5 CathepsinB * * * Merge * 10 µm C Nomarski DAPI 10A5 CathepsinB Merge 10 µm D Nomarski DAPI 10A5 CathepsinD Merge 10 µm Fig Confocal laser scanning micrographs of monkey kidney (A) In situ hybridization (ISH) combined with immunohistochemistry Antisense cRNA probe for L-PGDS (DIG, red) was used in combination with anti-L-PGDS MAb-5C11 (green) Cells in Henle’s loop positive for MAb-5C11 fluorescence were also positive for L-PGDS mRNA (B) Double immunofluorescence for L-PGDS with MAb-10A5 (green) and lysosomal marker cathepsin B (red) (C, D) High-magnification micrographs of double immunofluorescence for L-PGDS with MAb-10A5 (green) and two lysosomal markers (red) cathepsin B (C) and cathepsin D (D) Asterisks indicate lumen of a tubule Scale bar, 10 lm for (A)–(D) N-terminal amino acid sequence analysis revealed that L-PGDS of the intact form in monkey kidney started from Ala23, which is the same as the aminoterminal end of human b-trace (GenBank: M61900), and that the smaller sized proteins of Mr = 18 000 began from Gln31 and Phe34, which were and 11 amino acids shorter, respectively, than the intact protein (Fig 5C) These results indicate that the kidney contained both intact L-PGDS and the N-terminaltruncated form MAb-5C11 recognized solely the intact form of L-PGDS, whereas MAb-10A5 detected both forms Furthermore, immunohistochemical staining with MAb-5C11 indicated that the intact forms of L-PGDS were localized to cells of Henle’s loop and the glomeruli of the kidney By contrast, immunostaining with MAb-10A5 indicated that the truncated forms of L-PGDS were taken up by tubular cells and degraded within their lysosomes We then analyzed human and monkey urine by western blotting with MAb-5C11 and MAb-10A5 before 7152 and after incubation with glycopeptidase F Without glycopeptidase F treatment, both MAbs showed a single immunoreactive band corresponding to the intact form of L-PGDS (Mr = 27 000, Fig 5D) By contrast, after treatment of the urine samples with glycopeptidase F, MAb-10A5, but not MAb-5C11, detected the presence of low-molecular-mass forms of L-PGDS in human urine (filled arrow) These results suggest that the N-terminal-truncated form of L-PGDS is also excreted in human urine Discussion L-PGDS (Mr = 27 000) is much smaller than serum albumin (Mr = 66 000), although these proteins share similar chemical properties to secretory proteins, such as having anionic charges at pH 7.4 Renal filtration is considered to be a major clearance pathway for low-molecular-mass proteins (Mr < 30 000 [24]) Thus, L-PGDS passes through the glomerular capillary walls FEBS Journal 276 (2009) 7146–7158 ª 2009 The Authors Journal compilation ª 2009 FEBS N Nagata et al Lipocalin-type prostaglandin D synthase in kidney Monkey kidney L-PGDS A Glycopeptidase F (–) MAb kDa 1B7 Intact form 5C11 10A5 27 kDa 28 L-PGDS Truncated form 22 19.3 16.2 B Glycopeptidase F(+) Fig Identification of novel forms of L-PGDS in monkey kidney and human urine (A) L-PGDS was purified from the homogenates of monkey kidney by MAb-1B7-conjugated affinity chromatography The purified L-PGDS proteins were separated by SDSPAGE and analyzed by western blot analysis with MAb-1B7, MAb-5C11 and MAb-10A5 Positions of molecular size markers are shown on the right (B) Purified L-PGDS was treated with glycopeptidase F and used for western blot analysis with MAb-1B7, MAb-5C11 and MAb-10A5 Truncated forms of L-PGDS were detected by MAb-1B7 and MAb-10A5, but not by MAb-5C11 Positions of molecular size marker proteins are shown on the right (C) N-terminal amino acid sequences of the three different forms of L-PGDS (D) Human and monkey urine were treated or not with glycopeptidase F (GPF), and used for western blot analysis with MAb-5C11 and MAb-10A5 N-terminal-truncated forms of L-PGDS were detected with MAb-10A5 (filled arrow) Molecular size markers are shown on the left MAb 1B7 5C11 kDa 10A5 28 27 kDa 22 Intact form L–PGDS 19.3 19 Truncated form 18 16.2 Human L–PGDS C 30 10 20 40 50 MATHHTLWMGLVLLGLLGGLQAAPEAQVSVQPNFQPDKFLGRWFSAGLAS Intact form APEAQVSVQPNF 23 QPNFQPDKFLGR 31 FQPDKFLGRWFS Truncated form 34 { D Human urine 5C11 kDa GPF – + 10A5 – + 28 – + 28 19.3 19.3 Intact form Truncated form 16.2 16.2 of the kidney more easily than does serum albumin Previously, we established a sandwich ELISA system using anti-human L-PGDS MAb-1B7 and MAb-7F5 to estimate the L-PGDS level in urine [16] Urinary L-PGDS reflects even slight changes in glomerular permeability because of its low molecular mass and anionic property Thus, urinary L-PGDS is a useful diagnostic marker for renal diseases [1,20] However, our recent study [21] demonstrated that only 10% of the intravenously administered L-PGDS in canines was recovered in urine, suggesting that the urinary L-PGDS concentration is not determined by leakage through the glomeruli only, and that a large part of the urinary L-PGDS filtered through the membranes is reabsorbed or metabolized To clarify the precise localization and metabolism of L-PGDS in the kidney, we attempted to generate novel MAbs against the epitopes distinct from those of previously obtained MAbs However, as the orthology of the amino acid sequence Monkey urine 10A5 5C11 kDa + GPF – Intact form of L-PGDS is high (70.4%) between human and mouse [25], the generation of MAbs with a variety of epitopes is difficult in wild-type mice Therefore, in this study, we used rats and L-PGDS KO mice for immunization and obtained two novel anti-human L-PGDS MAbs recognizing distinct epitopes: Ala23–Val28 for MAb-5C11 and Arg156–Thr173 for MAb-10A5 (Fig 1); both antigenic epitopes were distinct from that for the previously generated MAb-1B7, which is Tyr107–Val120 [16] These novel MAbs recognized efficiently both native and recombinant human L-PGDS proteins in western blot (Fig 1) and SPR analyses (Table 1), and detected immunohistochemically the L-PGDS immunoreactive protein in kidney tissue with high sensitivity and specificity (Figs and 4) Therefore, these novel MAbs are useful for further diagnostic analysis to clarify the localization of L-PGDS in tissues and various body fluids, such as CSF, plasma and urine FEBS Journal 276 (2009) 7146–7158 ª 2009 The Authors Journal compilation ª 2009 FEBS 7153 Lipocalin-type prostaglandin D synthase in kidney N Nagata et al In this study, we demonstrated that monkey kidney contained at least three different forms of L-PGDS, i.e an intact form (Mr = 19 000 after removing N-glycosylated groups) and two truncated forms lacking or 11 N-terminal amino acid residues (Mr = 18 000 for the non-glycosylated form; Fig 5B, C) Moreover, the L-PGDS immunoreactivity of the C-terminal epitope of MAb-10A5 was detected in lysosomes in the tubules of monkey kidney (Fig 4), suggesting that truncated L-PGDS was proteolytically degraded in the kidney We also found that the truncated forms of L-PGDS were excreted in human urine (Fig 5D) These data, taken together, suggest that a major part of urinary L-PGDS is processed by proteolytic degradation after leakage through the glomeruli As the L-PGDS level is altered in clinical samples, such as the serum, CSF and urine of patients with hypertension [19], arteriosclerosis [26], hemorrhage [27] and diabetes [17,18,28–30], L-PGDS has been proposed to be a useful diagnostic marker for these diseases [1,20] However, the previous ELISA system with anti-human L-PGDS MAb-1B7 and MAb-7F5 [16] did not detect N-terminal-truncated L-PGDS Therefore, a new ELISA system with MAb-10A5, recognizing the C-terminal a-helical region of L-PGDS, will be useful for further clinical analysis Although the localization of L-PGDS in the kidney remains controversial [19,28,29], we have clearly demonstrated by immunohistochemistry with MAb5C11 and by in situ hybridization that the L-PGDS protein is synthesized de novo in Henle’s loop, Bowman’s capsule and podocytes of the glomeruli in the kidney (Fig 3) The cellular distribution of L-PGDS in the kidney is in good agreement with the results of a previous report indicating that a substantial amount of PGH2, a substrate of L-PGDS, is released from rat glomeruli and glomerular mesangial cells [31] Although the physiological role of L-PGDS in the kidney has not yet been clarified, Shirahase et al [32] have demonstrated previously that pretreatment of rat mesenteric artery with PGD2 attenuates organ damage induced by endotoxin shock with lipopolysaccharide In addition, PGD2 and its metabolites reduce the expression of mRNA for inducible nitric oxide synthase stimulated by interleukin-1b [33] NO generation induced by the overexpression of inducible nitric oxide synthase seems to play a pathogenic role in diabetic nephropathy [34] The inhibition of cytokine-mediated NO production following an increase in PGD2 ⁄ L-PGDS in the kidney possibly attenuates the progression of kidney damage In some renal diseases, the increase in the biosynthesis of L-PGDS in the kidney may be a type of adaptation mechanism against kidney 7154 injury An L-PGDS inhibitor, AT-56, found recently [35], is useful for clarifying the pathophysiological significance of L-PGDS in Henle’s loop and the glomeruli in the kidney Materials and methods Animals Sprague–Dawley rats were purchased from Shizuoka Laboratory Animal Center (Hamamatsu, Japan) L-PGDS KO mice were generated as described previously [36] Mice and rats were maintained under specific pathogen-free conditions in isolated cages with a 12 h light ⁄ 12 h dark photoperiod in a humidity- and temperature-controlled room (55% at 24 °C) Water and food were available ad libitum The protocols used for all animal experiments in this study were approved by the Animal Research Committee of Osaka Bioscience Institute Purification of recombinant D1)22Cys65,167Ala L-PGDS and b-trace We cloned the coding region of human L-PGDS without the signal sequence at the amino-terminal portion (amino acid residues 1–22, defined translation initiation codon Met as 1) [37] into the pGEX-2T vector (GE Healthcare, Amersham, Buckinghamshire, UK) to produce a fusion protein with GST The Cys residues at positions 65 and 167 were substituted for Ala residues using a QuikChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA, USA) to minimize misfolding of the recombinant protein as a result of incorrect S–S bridging in E coli [37] The plasmid was designated as GST-D1)22Cys65,167Ala and used to transform E coli BL21 (DE3) The recombinant GST-fusion protein was produced by the addition of isopropyl-b-d-thiogalactopyranoside (final concentration, 0.6 mm) The cells were further cultured for h at 37 °C, and then harvested and disrupted by sonication The resultant cell lysates were incubated with glutathione-Sepharose 4B resin (GE Healthcare), followed by digestion of the purified GSTfusion protein with thrombin The D1)22 65,167 Cys Ala L-PGDS was further purified by gel filtration chromatography with HiLoad Superdex 75 (GE Healthcare) b-Trace was purified by MAb-1B7-conjugated immunoaffinity column chromatography [38], followed by gel filtration chromatography with a HiLoad Superdex 75 column (GE Healthcare), from the culture medium of Chinese hamster ovary cells stably expressing human L-PGDS [39], or from human CSF provided by Dr M Mase (Department of Neurosurgery, Nagoya City University Hospital, Nagoya, Japan) Proteins were analyzed by SDS-PAGE and stained with SYPRO Orange (Invitrogen, Carlsbad, CA, USA) FEBS Journal 276 (2009) 7146–7158 ª 2009 The Authors Journal compilation ª 2009 FEBS N Nagata et al Protein concentrations were measured using a BCA Protein Assay Kit (Pierce Biotechnology, Rockford, IL, USA) Preparation of MAbs for human L-PGDS Sprague–Dawley rats were subcutaneously immunized with the recombinant D1)22Cys65,167Ala human L-PGDS protein expressed in E coli Alternatively, purified b-trace from the culture medium of Chinese hamster ovary cells was used to immunize L-PGDS KO mice Splenocytes from the immunized rats or mice were fused with myeloma cells (P3U1) under standard protocols [40] Positive hybridomas were cloned by the limiting dilution method For MAb-10A5 obtained in rats, ascites was produced in male BALB ⁄ cA-nu mice by the intraperitoneal injection of hybridoma cells For MAb-5C11 generated in L-PGDS KO mice, IgG antibody was purified from the serum-free medium of hybridoma cultures The immunoglobulin isotype of MAbs was determined using a Rat Monoclonal Antibody Isotyping Kit (Serotec, Oxford, UK) or IsoStrip Mouse Monoclonal Antibody Isotyping Kit (Roche Diagnostics, Mannheim, Germany), according to the manufacturer’s instructions SPR analysis The kinetics of binding of MAbs to human L-PGDS were determined by SPR analysis using a Biacore 2000 system (GE Healthcare) D1)22Cys65,167Ala L-PGDS, b-trace or MAbs for human L-PGDS were coupled to a CM5 sensor chip (GE Healthcare) by the amine-coupling method, according to the manufacturer’s protocol The Kd values were calculated from the sensorgrams using biaevaluation 3.1 software (GE Healthcare) Western blot analysis Protein samples were dissolved in 62.5 mm Tris ⁄ Cl (pH 6.8) containing 2% (w ⁄ v) SDS, 15% (v ⁄ v) glycerol and 5% (v ⁄ v) b-mercaptoethanol, and electrophoresed in 10–20% (w ⁄ v) polyacrylamide gels, followed by silver staining with Silver Stain Reagent (Daiichi Pure Chemicals, Tokyo, Japan) or blotted onto a polyvinylidine difluoride membrane (Immobilon P; Millipore, Bedford, MA, USA) The blots were incubated with human L-PGDS MAbs After washing, the blots were incubated with anti-mouse IgG conjugated with horseradish peroxidase Immunoreactive signals were detected using the ECL Western Blotting Detection System (GE Healthcare) Mapping of antigenic epitopes recognized by MAbs The coding region of human L-PGDS was sequentially truncated from the 5¢-terminus of the coding strand by Lipocalin-type prostaglandin D synthase in kidney PCR Each amplified fragment was cloned downstream of GST in the pGEX-2T vector Expression, purification and analyses of recombinant proteins were carried out as described above N-terminal amino acid sequence analysis of L-PGDS Monkey (Macaca fascicularis) tissues were provided by Drs Y Eguchi and R Torii (Shiga University of Medical Science, Otsu, Japan) L-PGDS was purified by MAb-1B7conjugated immunoaffinity column chromatography from monkey kidney [38] The affinity-purified L-PGDS protein was separated by SDS-PAGE and transferred onto a polyvinylidine difluoride membrane The blots were stained with Coomassie brilliant blue The protein bands were excised and utilized for sequencing analysis by Edman degradation employing the HP G1005A Protein Sequencing System (Hewlett-Packard, Palo Alto, CA, USA) The blots were also used for western blot analysis as described above Immunohistochemical analysis Monkey kidney was fixed in Bouin’s fixative and embedded in paraffin The paraffin sections (thickness, lm) were mounted on glass slides, deparaffinized in xylene and rehydrated in ethanol with increasing concentrations of water The rehydrated sections were pretreated with 0.3% (v ⁄ v) H2O2 in methanol for 30 at room temperature, and then incubated with 0.3% (w ⁄ v) pepsin (Sigma, St Louis, MO, USA) in 0.01 N HCl for at room temperature Next, the sections were incubated for h at room temperature with 10% (v ⁄ v) normal goat serum, 0.1% (v ⁄ v) Triton X-100 and 0.1% (w ⁄ v) sodium azide in NaCl ⁄ Pi, for 16 h at °C with anti-human L-PGDS MAbs in NaCl ⁄ Pi containing 1% (v ⁄ v) normal goat serum and 0.1% (v ⁄ v) Triton X-100, and for h at room temperature with biotinylated antibody against mouse or rat IgG (Vector Laboratories, Burlingame, CA, USA) The immunoreactive signals were visualized as the avidin-biotinylated enzyme complex (Vectastain Elite ABC Kit; Vector Laboratories) after incubation in 50 mm Tris ⁄ Cl (pH 7.6) containing 0.001% (v ⁄ v) H2O2 and 0.02% (w ⁄ v) 3,3¢-diaminobenzidine tetrahydrochloride The sections were then counterstained with hematoxylin and observed under an ECLIPSE E600 microscope (Nikon, Tokyo, Japan) For double staining with rabbit polyclonal anti-cathepsin D IgG (Assay Designs, Ann Arbor, MI, USA) and MAb10A5, after the primary antibodies had been applied, the sections were sequentially incubated with Alexa Fluor 594conjugated antibody against rabbit IgG (Invitrogen) and biotinylated antibody against rat IgG (5 lgỈmL)1; Jackson ImmunoResearch, West Grove, PA, USA) followed by Alexa Fluor 488-conjugated streptavidin (5 lgỈmL)1; Invi- FEBS Journal 276 (2009) 7146–7158 ª 2009 The Authors Journal compilation ª 2009 FEBS 7155 Lipocalin-type prostaglandin D synthase in kidney N Nagata et al trogen) The sections were observed under an Axiovert 100M microscope connected to a Zeiss laser-scanning microscope 510META (Carl Zeiss, Jena, Germany) In situ hybridization In situ hybridization was performed using a RiboMap Kit and Discovery automatic staining module (Roche Diagnostics) The digoxigenin (DIG)-labeled RNA probe for human L-PGDS was prepared as follows The coding region of human L-PGDS was subcloned into pCR-Script Amp SK(+) vector (Stratagene) DIG-labeled cRNA probes were synthesized using T7 (antisense) or SP6 (sense) RNA polymerase according to the manufacturer’s manual (Roche Diagnostics), and further purified by a Chroma Spin Column (BD Biosciences, San Jose, CA, USA) The efficiency of DIG incorporation was determined by dot blot analysis The kidney sections were hybridized with L-PGDS RNA probe in Ribohybe hybridization solution (Roche Diagnostics) at 70 °C for h, and then incubated with horseradish peroxidase-conjugated anti-DIG IgG (DakoCytomation, Glostrup, Denmark) for 30 min, followed by 2,4-dinitrophenyl-conjugated tyramide for signal amplification Tyramide was then activated with H2O2 for 20 After heating at 85 °C for 10 min, the sections were sequentially incubated with anti-2,4-dinitrophenyl IgG for 30 min, avidin for h, biotin for h (Endogenous Biotin Blocking Kit; Roche Diagnostics) [41], biotin-conjugated anti-rabbit IgG for 10 and streptavidin-conjugated alkaline phosphatase for 16 (AmpMap with TSA; Roche Diagnostics) The chromogen reaction of alkaline phosphatase was performed with Nitro Blue tetrazolium chloride ⁄ 5-bromo-4-chloro-3¢-indolylphosphatase p-toluidine salt (BlueMap Kit; Roche Diagnostics) The sections were counterstained with Nuclear Fast Red (ISH Red Counterstain; Roche Diagnostics) For in situ hybridization combined with immunohistochemistry, after hybridization with the L-PGDS cRNA probe at 70 °C for h, the sections were incubated with anti-DIG IgG conjugated with alkaline phosphatase (1 : 1000, Roche Diagnostics) and MAb-5C11 overnight at °C The sections were then sequentially incubated for h each with avidin, biotin (Vector Laboratories), biotinylated anti-mouse IgG (5 lgỈmL)1; Jackson ImmunoResearch) and Alexa Fluor 488-conjugated streptavidin (5 lgỈmL)1; Invitrogen) The alkaline phosphatase activity was detected using a 2-hydroxy-3-naphthoic acid-2¢-phenylanilide phosphate fluorescence detection set (Roche Diagnostics), according to the manufacturer’s instructions Detection of L-PGDS in human and monkey urine Human urine samples were obtained from the Health Service Center of The University of Tokyo and human plasma samples were provided by Dr Y Eguchi (Shiga University of Medical Science, Otsu, Japan) Urine or 7156 plasma samples were pretreated with 0.5% (w ⁄ v) SDS and 0.1 m b-mercaptoethanol at 100 °C for Each sample was incubated with glycopeptidase F (Takara Bio, Kyoto, Japan) at 37 °C for 24 h in 0.1 m Tris ⁄ Cl (pH 8.6) with 1% (v ⁄ v) Nonidet P-40 The digested samples were then separated by SDS-PAGE, followed by staining with SYPRO Ruby (Invitrogen) or western blotting as described above Statement of clinical studies This study was performed in accordance with the guidelines for human studies in the respective hospitals and clinics, and was approved by the respective authoritative boards of the hospitals and departments Informed 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K, Oda H, Nakajima H, Urade Y & Endo T (2000) Comparative study of the asparagine-linked sugar chains of human lipocalin-type prostaglandin D synthase purified from urine and amniotic fluid, and. .. affinity-purified L-PGDS protein was separated by SDS-PAGE and transferred onto a polyvinylidine difluoride membrane The blots were stained with Coomassie brilliant blue The protein bands were excised and utilized... region of human L-PGDS was sequentially truncated from the 5¢-terminus of the coding strand by Lipocalin-type prostaglandin D synthase in kidney PCR Each amplified fragment was cloned downstream of