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
Trang 1lipocalin-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
1 Department of Molecular Behavioral Biology, Osaka Bioscience Institute, Japan
2 Laboratory of Biodefense and Regulation, Osaka University of Pharmaceutical Sciences, Japan
3 Central Research Institute, Maruha Nichiro Holdings, Inc., Ibaraki, Japan
4 Health Service Center, The University of Tokyo, Japan
Introduction
Lipocalin-type prostaglandin D synthase (L-PGDS;
EC 5.3.99.2) catalyzes the isomerization of
prostaglan-din 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 puri-fied from rat brain [6] and found to be a monomeric
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 6 6872 2841
Tel: +81 6 6872 4851
E-mail: uradey@obi.or.jp
(Received 12 August 2009, revised 29
September 2009, accepted 6 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 How-ever, 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-ter-minal 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 local-ized 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 recog-nized 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 ) colocal-ize ( MI:0403 ) by fluorescence microscopy ( MI:0416 )
l MINT-7266176 : L-PGDS (uniprotkb: P41222 ) and Cathepsin B (uniprotkb: Q4R5M2 ) colocal-ize ( MI:0403 ) by fluorescence microscopy ( MI:0416 )
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.
Trang 2protein with a molecular mass of approximately
26 000 [1], and was later demonstrated to be
N-gly-cosylated 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],
gan-gliosides [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
domi-nantly 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
func-tions as both a PGD2-producing enzyme and an
extra-cellular 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
mar-ker 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
How-ever, the origin of urinary L-PGDS remains unclear
Furthermore, only 10% of L-PGDS administered
intra-venously in canines is recovered in the urine [21],
sug-gesting 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-formants In SDS-PAGE, b-trace showed a broad band
at position Mr= 25 000–27 000 as a result of its gly-cosylation (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 3 and 4) and MAb-10A5 (Fig 1A, lanes 5 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 9 and 10) By contrast, previously generated MAb-1B7 (Fig 1A, lanes 7 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-gly-cosylated 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 gluta-thione S-transferase (GST)-fusion proteins containing different lengths of human L-PGDS (Fig 1B) Each GST-fusion protein was purified, separated by SDS-PAGE 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, respec-tively 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 previ-ously 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 Kdvalues 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
Trang 3D1)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
immo-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
1B7
5C11 10A5 PAb
MAb
A
B
C
19 26 kDa
1
SYPRO orange
Silver staining
MAb-10A5
MAb-5C11
(Amino acids)
Gln190
* * Ala23
1 Ser29 2 Gln35 3 Ser52 4
Asp74 5
Glu90 6
Tyr107 7
Val121 8
Gly140 9
Arg156 10
Glu174 11
1 2 3 4 5 6 7 8 9 10 11
1 40 80 120 160 190
10A5
5C11
1B7
Asn51
2
1 3
A
H B G F
D C
E
N
C
10A5
5C11
1B7 Asn51
Asn78
Fig 1 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, 7 and 9) and recombinant D1)22Cys65,167Ala human L-PGDS (lanes 2, 4, 6, 8 and 10) were separated by SDS-PAGE and stained with SYPRO Orange (lanes 1 and 2), followed by blotting onto a polyvinylidine difluoride membrane The blots were then reacted with human L-PGDS MAb-5C11 (lanes 3 and 4), MAb-10A5 (lanes 5 and 6), MAb-1B7 (lanes 7 and 8) or polyclonal antibody (PAb, lanes 9 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)22Cys 65,167 Ala 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.
Trang 4L-PGDS are exposed on the surface of the molecule.
However, in the soluble form, the affinities of
MAb-10A5 for native L-PGDS were lower than those for
the recombinant protein, suggesting that the
C-termi-nal 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
substitu-tion (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
resi-dues 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,
par-tially purified by immunoaffinity columns conjugated
with MAb-1B7, MAb-5C11 or MAb-10A5, revealed
that all of these MAbs showed a broad single
immuno-reactive 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 hybrid-ization 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 glomeru-lar 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 struc-tures 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 glome-ruli (not shown), suggesting that L-PGDS was synthe-sized 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-Table 1 Characterization of the three anti-human L-PGDS MAbs used in this study.
K d (n M ) b-trace protein D1)22Cys 65,167, Ala L-PGDS
Soluble form Immobilized Soluble form Immobilized
Trang 5positive 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-glycosyla-tion at posiN-glycosyla-tions Asn51 and Asn78 [7], purified
L-PGDS showed a broad band of two different sizes,
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 MAb-5C11 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)
Fig 2 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, 5 and 6, respectively) Positions of molecular size markers are shown on the left.
Trang 6* *
*
*
G
K
L
E
C B
A
J I
1B7
1 mm
50 m
20 m 5 m
5 m
20 m
50 m
T
5 m
20 m
D
Fig 3 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) 1 mm; (E–H, Q–S) 50 lm; (I–P, T) 20 lm; insets, 5 lm.
Trang 7N-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
amino-terminal end of human b-trace (GenBank: M61900),
and that the smaller sized proteins of Mr= 18 000
began from Gln31 and Phe34, which were 8 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-terminal-truncated 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
wes-tern blotting with MAb-5C11 and MAb-10A5 before
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
A
B
D
C
*
*
*
*
DAPI
DAPI
Nomarski
*
*
Nomarski
Nomarski Nomarski
10 µm
10 µm
DAPI
10 µm
10 µm
Fig 4 Confocal laser scanning micrographs of monkey kidney (A) In situ hybridization (ISH) combined with immunohistochemistry Anti-sense 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 lyso-somal 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).
Trang 8of 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
local-ization 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
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 immu-nization 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 immunohistochemi-cally the L-PGDS immunoreactive protein in kidney tissue with high sensitivity and specificity (Figs 3 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
MAb
Glycopeptidase F (–)
Monkey kidney L-PGDS
Human urine Monkey urine
Glycopeptidase F(+)
1B7 5C11 10A5
APEAQVSVQPNF
34 31
FQPDKFLGRWFS
MATHHTLWMGLVLLGLLGGLQAAPEAQVSVQPNFQPDKFLGRWFSAGLAS
Human L–PGDS
L-PGDS
27 kDa 22
19.3 kDa
30
28
16.2
19.3
kDa 28
16.2
C B
A
MAb 1B7 5C11 10A5
L–PGDS
27 kDa 22 19 18
{
Truncated form Intact form
Truncated form
Truncated form
Intact form
Intact form
Truncated form
D
Intact form
10A5 5C11
19.3 28
16.2
kDa
19.3 28
16.2
kDa
Intact form
10A5 5C11
Fig 5 Identification of novel forms of
L-PGDS in monkey kidney and human urine.
(A) L-PGDS was purified from the
homo-genates of monkey kidney by
MAb-1B7-con-jugated affinity chromatography The purified
L-PGDS proteins were separated by
SDS-PAGE 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-trun-cated forms of L-PGDS were detected with
MAb-10A5 (filled arrow) Molecular size
markers are shown on the left.
Trang 9In 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-gly-cosylated groups) and two truncated forms lacking 8
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
degra-dation 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
use-ful 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-ter-minal 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
MAb-5C11 and by in situ hybridization that the L-PGDS
protein is synthesized de novo in Henle’s loop,
Bow-man’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
kid-ney has not yet been clarified, Shirahase et al [32] have
demonstrated previously that pretreatment of rat
mes-enteric 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
progres-sion 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
injury An L-PGDS inhibitor, AT-56, found recently [35], is useful for clarifying the pathophysiological sig-nificance of L-PGDS in Henle’s loop and the glomeruli
in the kidney
Materials and methods
Animals
Sprague–Dawley rats were purchased from Shizuoka Labo-ratory Animal Center (Hamamatsu, Japan) L-PGDS KO mice were generated as described previously [36] Mice and rats were maintained under specific pathogen-free condi-tions in isolated cages with a 12 h light⁄ 12 h dark photo-period in a humidity- and temperature-controlled room (55% at 24C) 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, Amer-sham, 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-Direc-ted 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-thioga-lactopyranoside (final concentration, 0.6 mm) The cells were further cultured for 6 h at 37C, 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
Cys65,167Ala L-PGDS was further purified by gel filtra-tion chromatography with HiLoad Superdex 75 (GE Healthcare)
b-Trace was purified by MAb-1B7-conjugated immunoaf-finity column chromatography [38], followed by gel filtra-tion chromatography with a HiLoad Superdex 75 column (GE Healthcare), from the culture medium of Chinese ham-ster 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)
Trang 10Protein 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
immu-nized 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
Isotyp-ing Kit (Serotec, Oxford, UK) or IsoStrip Mouse
Monoclo-nal 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
mem-brane (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
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-1B7-conjugated immunoaffinity column chromatography from monkey kidney [38] The affinity-purified L-PGDS protein was separated by SDS-PAGE and transferred onto a poly-vinylidine 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, 5 lm) were mounted on glass slides, deparaffinized in xylene and rehy-drated in ethanol with increasing concentrations of water The rehydrated sections were pretreated with 0.3% (v⁄ v)
H2O2 in methanol for 30 min at room temperature, and then incubated with 0.3% (w⁄ v) pepsin (Sigma, St Louis,
MO, USA) in 0.01 N HCl for 5 min at room temperature Next, the sections were incubated for 1 h at room tempera-ture 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 4C with anti-human L-PGDS MAbs in NaCl ⁄ Pi con-taining 1% (v⁄ v) normal goat serum and 0.1% (v ⁄ v) Triton X-100, and for 1 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 incu-bation in 50 mm Tris⁄ Cl (pH 7.6) containing 0.001% (v ⁄ v)
H2O2 and 0.02% (w⁄ v) 3,3¢-diaminobenzidine tetrahydro-chloride The sections were then counterstained with hema-toxylin 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 MAb-10A5, after the primary antibodies had been applied, the sections were sequentially incubated with Alexa Fluor 594-conjugated antibody against rabbit IgG (Invitrogen) and biotinylated antibody against rat IgG (5 lgÆmL)1; Jackson ImmunoResearch, West Grove, PA, USA) followed by Alexa Fluor 488-conjugated streptavidin (5 lgÆmL)1;