From the Laboratory of Molecular and Cellular
Recognition, Osaka University Graduate School of Medicine, 2-2, Yamada-oka, Suita 565-0871, Japan, the ¶ Department of Medicine
and Clinical Science, Okayama University Graduate School of Medicine
and Dentistry, 2-5-1, Shikata, Okayama 700-8558, Japan, and the
Biocenter and Department of Medical Biochemistry and Molecular
Biology, University of Oulu, P. O. Box 5000, FIN-90014 Oulu, Finland
Received for publication, December 2, 2002, and in revised form, January 27, 2003
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ABSTRACT |
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Leukocyte infiltration during inflammation is
mediated by the sequential actions of adhesion molecules and
chemokines. By using a rat ureteral obstruction model, we showed
previously that L-selectin plays an important role in leukocyte
infiltration into the kidney. Here we report the purification,
identification, and characterization of an L-selectin-binding heparan
sulfate proteoglycan (HSPG) expressed in the rat kidney. Partial amino
acid sequencing and Western blotting analyses showed that the
L-selectin-binding HSPG is collagen XVIII, a basement membrane HSPG.
The binding of L-selectin to isolated collagen XVIII was specifically
inhibited by an anti-L-selectin monoclonal antibody, EDTA, treatment of the collagen XVIII with heparitinase or heparin but not by chemically desulfated heparin. A cell binding assay showed that the
L-selectin-collagen XVIII interaction mediates cell adhesion.
Interestingly, collagen XVIII also interacted with a chemokine,
monocyte chemoattractant protein-1, and presented it to a monocytic
cell line, THP-1, which enhanced the
L-selectin was initially identified as a lymphocyte
homing receptor that plays a crucial role in lymphocyte migration into peripheral lymph nodes (1). Subsequently, this molecule was found to be
expressed on other types of leukocytes, such as neutrophils and
monocytes, and to be involved in leukocyte migration into inflamed
tissues (2-4). In agreement with these reports, L-selectin-deficient mice show severely impaired lymphocyte homing and leukocyte
infiltration to sites of inflammation (5, 6).
L-selectin-binding molecules are found not only in the high endothelial
venules of peripheral lymph nodes but also in non-vascular tissues,
such as the white matter and choroid plexus of the central nervous
system and the renal tubules (7). We have shown previously that
leukocyte infiltration into the kidney interstitium induced by ureteral
obstruction was significantly reduced by the administration of an
anti-rat L-selectin blocking
mAb1 or a sulfated
glycolipid, sulfatide, which binds to L-selectin in vitro
(8, 9). These observations prompted us to examine L-selectin-reactive
molecules that are expressed in the kidney. We first identified
sulfatide, a sulfated glycolipid expressed in the renal distal tubules,
as an L-selectin-binding molecule in the kidney (9). Subsequent
analyses revealed that, in addition to sulfatide, L-selectin-binding
chondroitin sulfate proteoglycans and heparan sulfate proteoglycans
(HSPGs) are both expressed in the kidney (10). Versican was identified
as one of the L-selectin-binding chondroitin sulfate proteoglycans (11,
12), whereas L-selectin-binding HSPGs with core protein sizes of 160 and 180 kDa (13) have remained undefined.
In the present study, we isolated these L-selectin-binding HSPGs from
the rat kidney and identified them as collagen XVIII, a basement
membrane HSPG. We also found that collagen XVIII interacts with
monocyte chemoattractant protein-1 (MCP-1) and induces the integrin
activation of monocytes, raising the possibility that collagen XVIII
plays a role in renal inflammation by bridging selectin- and
integrin-mediated cell adhesion.
Reagents--
A recombinant soluble form of rat L-selectin
(LEC-IgG) has been described previously (7). Human L-selectin-Ig and a
recombinant soluble form of human vascular cell adhesion molecule-1
(VCAM-1) were purchased from R & D Systems Inc. (Minneapolis, MN).
Completely desulfated, N-acetylated heparin
(CDSNAc-heparin), completely desulfated, N-sulfated heparin
(CDSNS-heparin), N-desulfated, N-acetylated
heparin (NDSNAc-heparin), heparitinase, heparitinase I, heparitinase
II, keratanase, and aggrecan from a rat chondrosarcoma (RC-PG) were
purchased from Seikagaku Kogyo Co. (Tokyo, Japan). Heparin (from
porcine intestine) was purchased from Sigma. The neutralizing anti-rat
L-selectin mAb HRL3 and non-neutralizing anti-rat L-selectin mAb HRL2
were prepared as described previously (14). Recombinant MCP-1 was
purchased from PeproTech (London, UK). The anti-human L-selectin
monoclonal antibody (mAb) TQ1, anti-integrin Preparation of the 1 M NaCl Fraction from the Renal
Tubular Lysates--
Renal tubules were collected from rat kidneys
(male Wistar rats, 9-weeks old) according to the method of Krisko
et al. (15). The fraction containing L-selectin-binding
molecules (1 M NaCl fraction) was prepared as described
previously (13).
Amino Acid Sequencing Analysis of the 160- and 180-kDa
Molecules--
The 1 M NaCl fraction was dialyzed against
deionized water and lyophilized. The sample was then solubilized in
buffer A (50 mM Tris-HCl, 7 M urea, 0.1% octyl
glucoside, 10 mM EDTA, 10 mM N-ethylmaleimide, 1 mM PMSF, pH 7.4) and
dialyzed against buffer B (20 mM Tris-HCl, 0.15 M NaCl, pH 7.4). After dialysis, the buffer composition was
adjusted to buffer B containing 0.046% Tween 20, 3 mM
CaCl2, 0.93 mM PMSF, 4.6 µg/ml pepstatin, 4.0 µg/ml leupeptin. To 800 µl of this solution, 63.45 µl of
CNBr-activated Sepharose 4B beads (Amersham Biosciences) coupled with 5 milliunits of heparitinase (0.0788 milliunits/µl beads) was added,
and the mixture was incubated at 37 °C for 15 h with agitation.
After brief centrifugation to remove the beads, the supernatant was
concentrated, subjected to SDS-PAGE, and blotted onto a Hybond-C
nitrocellulose membrane (Amersham Biosciences). Bands were visualized
by staining with Ponceau S, and the 160- and 180-kDa bands were excised
separately. In the next step, each membrane strip was blocked with
0.5% polyvinylpyrrolidone K30 (Wako Pure Chemicals Co., Osaka, Japan),
0.1 M acetic acid, washed with buffer C (50 mM
Tris-HCl, pH 8.0), and then incubated with 0.5 µg of trypsin
(sequencing grade from bovine pancreas; Roche Molecular Biochemicals)
in buffer C at 37 °C for 15 h. After the reaction, peptides
were fractionated on a reversed-phase C-18 column (3.9 × 150 mm;
Millipore Co., Bedford, MA) at 1 ml/min under a 40-min gradient from
0.1% (v/v) trifluoroacetic acid to 0.1% (v/v) trifluoroacetic
acid, 12% (v/v) acetonitrile, 28% (v/v) 2-propanol. The peak
fractions detected by absorbance at 220 nm were collected and dried.
The fractions (peaks 1 and 2 of the 160-kDa protein and peak 2 of the
180-kDa protein) were subjected to gas-phase microsequencing using a
model 492 protein sequencer (PerkinElmer Life Sciences).
Generation of Anti-rat Collagen XVIII Polyclonal Antibody
RES.16--
A C-terminal fragment of rat collagen XVIII that
corresponds to the mouse endostatin sequence (16) was amplified by PCR, inserted into the vector pQE-31 (Qiagen, Santa Clarita, CA), and expressed and purified as described recently (17). A polyclonal antibody RES.16 was raised against the recombinant rat endostatin using
conventional methods. Briefly, rat endostatin in complete Freund's
adjuvant (Sigma) was injected into a rabbit subcutaneously, followed by
booster injections in incomplete Freund's adjuvant at 2-week
intervals. The polyclonal antiserum was affinity-purified on a
CNBr-activated Sepharose 4B (Amersham Biosciences) column coupled with
the recombinant rat endostatin as described elsewhere (18). The
specificity of the affinity-purified RES.16 antibody was verified by
Western blotting against the recombinant rat and human endostatins and
the hepatoblastoma-derived full-length human collagen XVIII.
Immunoprecipitation and Western Blotting
Analyses--
Immunoprecipitation and Western blotting were performed
as described previously (13).
Purification of Collagen XVIII from the 1 M NaCl
Fraction--
Ten milliliters of the 1 M NaCl fraction was
mixed with 40 ml of buffer D (50 mM Tris-HCl, 8 M urea, 1 mM PMSF, pH 8.0) and 2 ml of
Ni-NTA-agarose beads (Qiagen) at 4 °C for 12 h. After incubation, the beads were packed in a column (Poly-Prep chromatography column; Bio-Rad) and washed with 10 ml of buffer D and then with 10 ml
of buffer D containing 0.3 M NaCl. Collagen XVIII was
eluted with 250 mM imidazole in buffer D, dialyzed against
PBS, and stored at Enzyme-linked Immunosorbent Assay (ELISA)--
ELISA was
performed as described previously (12).
Disaccharide Composition Analysis--
Purified collagen XVIII
was incubated overnight with a mixture of heparitinase (0.1 unit/ml),
heparitinase I (0.1 unit/ml), and heparitinase II (0.1 unit/ml) in 20 mM HEPES-NaOH, 0.14 M NaCl, 1 mM
CaCl2, 1 mM MgCl2, pH 6.8, at
37 °C. The disaccharide products were derivatized with
2-aminobenzamide (2-AB) and analyzed by high performance liquid
chromatography as described previously (19). Eluates were monitored
using a fluorescence detector with excitation and emission wavelengths
of 330 and 420 nm, respectively.
Cell Binding Assay--
Murine lymphoma EL-4 cells transfected
with the BCMGSNeo vector containing rat L-selectin cDNA (RM-1) (20)
or untransfected EL-4 cells were used for the L-selectin-mediated cell
adhesion assay as described previously (12).
Chemokine-induced cell adhesion to VCAM-1 was determined as
follows. Purified collagen XVIII (1 µg/ml) and/or VCAM-1 (0.5 µg/ml) were immobilized in the wells of 4-mm-diameter 96-well EIA/RIA
plates (Corning Glass) overnight at 4 °C. After washing, the plates
were blocked with 3% bovine serum albumin in PBS. Various concentrations of MCP-1 were added to the wells, which were incubated for 2 h, and then washed with PBS containing 1 mM
CaCl2 and 1 mM MgCl2. THP-1 cells
(provided by Dr. Katsuhiko Ishihara, Osaka University Graduate school
of Medicine) were labeled with 5 µM 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein, acetoxymethyl ester (Molecular Probes Inc., Eugene, OR), and resuspended in RPMI 1640 medium containing 2% fetal calf serum (medium A). The cells were then
added to the wells (5 × 104 cells/well) in
triplicate. After a 10-min incubation at 37 °C, the wells were
filled with medium A and sealed with parafilm. The plates were inverted
for 20 min at 37 °C to allow non-adherent cells to detach from the
bottom surface of the wells. The remaining bound cells were lysed, and
the fluorescence intensity was measured as described previously
(12).
Immunofluorescence Staining--
Immunofluorescence staining of
frozen sections of the rat kidney (male Wistar rat, 8-week-old) was
performed as described previously (11).
Identification of L-selectin-binding HSPGs in the Rat Kidney as
Collagen XVIII--
L-selectin binding HSPGs (13) with core protein
sizes of 160 and 180 kDa were purified by preparative SDS-PAGE, and
their partial amino acid sequences were analyzed. The sequences of two fragments from the 160-kDa protein and one fragment from the 180-kDa protein were determined (Fig.
1A). All the sequences
obtained showed significant homology to that of human collagen XVIII
(21-24), suggesting that both the 160- and 180-kDa proteins are rat
counterparts of human collagen XVIII.
We next performed Western blotting analysis using a polyclonal
antibody, RES.16, which had been raised against rat collagen XVIII. As
shown in Fig. 1B, 160- and 180-kDa proteins that were precipitated with LEC-IgG were specifically reactive with RES.16. The
binding of these proteins to LEC-IgG was apparently mediated by the
C-type lectin domain of L-selectin, given that the binding was blocked
by EDTA and the anti-rat L-selectin-blocking mAb HRL3 but not by the
non-blocking anti-rat L-selectin mAb HRL2. Collectively, these results
show that the L-selectin-binding HSPGs with core protein sizes of 160 and 180 kDa were both rat collagen XVIII.
Purification of Collagen XVIII from the Rat Kidney--
A
GenBankTM data base search revealed that a partial sequence
of rat collagen XVIII (GenBankTM accession number AF
189709) contains an unusual His11 segment. Because His
repeating sequences have been widely used as an affinity tag for
recombinant proteins to facilitate their binding to matrices containing
immobilized Ni2+ ions, we next tried to purify rat collagen
XVIII using nitrilotriacetic acid (NTA)-agarose coupled with
Ni2+ ions (Ni-NTA-agarose). The 1 M NaCl
fraction from DEAE-Sepharose chromatography (13) containing collagen
XVIII was applied to the Ni-NTA-agarose column, and the bound material
was then eluted with 250 mM imidazole. As shown in Fig.
2, whereas the 1 M NaCl fraction contained many proteins, the imidazole eluate contained almost
exclusively proteins of 160 and 180 kDa. These bands were reactive with
the RES.16 antibody in Western blotting
analysis.2 These results
indicate that collagen XVIII was purified to an apparent homogeneity by
Ni-NTA-agarose column chromatography.
Characterization of the Interaction between L-selectin and Collagen
XVIII--
By having obtained purified collagen XVIII, we next
examined if collagen XVIII could directly interact with L-selectin. As shown in Fig. 3A, biotinylated
collagen XVIII bound to immobilized LEC-IgG but not to control human
IgG, and the binding was inhibited by HRL3 or EDTA but not by HRL2.
Treatment of the biotinylated collagen XVIII with heparitinase but not
chondroitinase ABC or keratanase inhibited its binding to LEC-IgG (Fig.
3B). These results indicate that heparan sulfate (HS) chains
of collagen XVIII interact with the lectin domain of L-selectin.
To examine the structural features of the HS chains required for the
interaction of collagen XVIII with L-selectin, we next performed an
inhibition assay using various chemically modified glycosaminoglycans.
As shown in Fig. 4A, the
binding of biotinylated collagen XVIII to LEC-IgG was inhibited in a
dose-dependent manner by intact heparin containing both
N- and O-sulfation. The blocking ability of
intact heparin was comparable with that of chondroitin sulfate (CS) E,
which strongly interacts with L-selectin (12, 19). The binding was also
inhibited by CS B, which also interacts with L-selectin (12). On the
other hand, the binding was not inhibited by chemically modified
heparins, such as CDSNAc-heparin, CDSNS-heparin, and NDSNAc-heparin,
indicating that both N- and O-sulfation of
HS/heparin chains are required for the interaction of collagen XVIII
with L-selectin. Consistently, disaccharide composition analysis showed
that HS chains of collagen XVIII contained abundant N- and
O-sulfated disaccharide units (Fig. 4B). Peak areas of
We (12) and others (25, 26) have shown previously that HS chains can
interact with L- and P-selectin but not E-selectin or CD44. Consistent
with this, biotinylated collagen XVIII bound L- and P-selectin-IgG but
not E-selectin-IgG or CD44-IgG (Fig. 5).
In contrast, biotinylated polymeric sialyl Lewis X (sLeX),
a common ligand for selectins (27), bound the three selectins. Biotinylated versican bound L- and P-selectin-IgG and CD44-IgG, whereas
aggrecan (RC-PG) bound selectively to CD44-IgG, as described previously
(12, 28).
The L-selectin-Collagen XVIII Interaction Mediates Cell
Adhesion--
To determine whether the L-selectin-collagen XVIII
interaction could mediate cell adhesion, we next performed a
cell-binding assay using L-selectin transfectants. As shown in Fig.
6, rat L-selectin transfectants (RM-1
cells) bound to collagen XVIII-coated wells at a higher level than did
untransfected EL-4 cells, and the binding was inhibited by HRL3 or EDTA
but not by HRL2. These results indicate that the L-selectin-collagen
XVIII interaction can mediate cell adhesion.
Localization of Collagen XVIII in the Rat Kidney--
We next
examined the tissue distribution of L-selectin ligands and collagen
XVIII in the kidney (Fig. 7). Consistent
with our previous observations (7, 9-11), LEC-IgG bound abundantly to
tubules but only slightly, if at all, to the vascular bundles in the
outer medulla of the rat kidney (Fig. 7A), whereas the anti-collagen XVIII antibody RES.16 reacted with the vascular bundles
and the basement membranes of the tubules of the outer medulla (Fig.
7B). Thus, L-selectin ligands and collagen XVIII appeared to
colocalize in the basement membranes of the renal tubules of the outer
medulla. In contrast, collagen XVIII was also detected in the glomeruli
and tubules in the renal cortex, where no LEC-IgG binding was observed
(Fig. 7, C and D), suggesting that the collagen
XVIII expressed in these regions is not reactive with L-selectin.
Collagen XVIII Interacts with Chemokines to Induce
Integrin Activation in Leukocytes--
HSPGs are proposed to function
as "reservoirs" for chemokines and to present them to leukocytes
efficiently (29). Among various chemokines, MCP-1 has been shown to
play a critical role in renal inflammation (30-32). We thus examined
the possibility that collagen XVIII interacts not only with L-selectin
but also with MCP-1 and presents it to leukocytes. As shown in Fig.
8A, MCP-1 bound to immobilized
collagen XVIII in a dose-dependent manner. The binding was
inhibited by heparitinase but not by chondroitinase ABC treatment,
indicating that MCP-1 interacts with HS chains of collagen XVIII.
Binding of MCP-1 to immobilized collagen XVIII was inhibited by LEC-IgG
in a dose-dependent manner (Fig. 8B). Similarly,
binding of LEC-IgG to immobilized collagen XVIII was inhibited by MCP-1
(Fig. 8C), suggesting that MCP-1 and L-selectin interact
with the same or overlapping carbohydrate structure in the HS chains of
collagen XVIII.
We then investigated the possible functional consequences of the
collagen XVIII-MCP-1 interaction. To this end, MCP-1 was applied to
wells that had been coated with collagen XVIII alone or with a mixture
of collagen XVIII and VCAM-1. We then applied to each well a monocytic
cell line, THP-1, that expresses a receptor for MCP-1, CCR2B, and
In this study, we have purified L-selectin-binding HSPGs in the
kidney and identified them as collagen XVIII. Treatment with heparitinase, but not with any other GAG-degrading enzymes, abolished the L-selectin binding (Fig. 3B), indicating that HS chains
of collagen XVIII bind to L-selectin. Unlike sulfatide, collagen XVIII
bound to L-selectin in a divalent cation-dependent manner. This binding was inhibited by the mAb HRL3, which inhibits the binding
of L-selectin to lymph node high endothelial venules (14). These
results indicate that HS chains on collagen XVIII are recognized by the
lectin domain of L-selectin, which is known to interact with
sLeX-like sugar chains on L-selectin ligands such as CD34
and glycosylation-dependent cell adhesion molecule-1
(GlyCAM-1) (33, 34).
Collagen XVIII is a triple helical endothelial/epithelial basement
membrane protein characterized by multiple interrupted triple helical
regions and a unique C-terminal endostatin domain (21-24). Collagen
XVIII became the focus of medical interest when an 18-kDa
anti-angiogenic peptide with tumor-suppressing activity, named
endostatin, turned out to be the C-terminal part of collagen XVIII
(16). More recently, it was reported that collagen XVIII is modified
with HS chains in the human kidney (18) and in various organs in the
chick (35). Our present finding that the binding of rat collagen XVIII
to L-selectin and MCP-1 is mediated by HS chains of collagen XVIII is
consistent with these previous reports.
Amino acid sequencing and Western blotting analyses showed
that the 160- and 180-kDa proteins are both rat collagen XVIII (Fig.
1). To date, three variants of mouse collagen XVIII (36, 37) and two
variants of human collagen XVIII (38) have been reported. In the mouse
and human, collagen XVIII transcripts corresponding to the short
variant are predominantly expressed in the kidney (36, 38). The human
collagen XVIII short variant is 180 kDa after heparin lyase II and III
treatment (18). It is unclear at present whether the 160- and 180-kDa
proteins detected in the rat kidney represent forms produced by
alternative splicing or proteolytic cleavage. However, the presence of
two forms does not appear to be due to the cleaving off of the
C-terminal 18-kDa endostatin domain from the core protein, because both
proteins were reactive with the RES.16 antibody, which recognizes the
rat endostatin domain.
It appears that proteoglycans can selectively interact with
particular adhesion molecules depending on differences in their GAG
modifications. As reported previously, versican modified with CS B and
CS C (12) as well as CS E (19) binds L- and P-selectin and CD44,
whereas the rat chondrosarcoma proteoglycan aggrecan, which is modified
exclusively with CS A (28), selectively binds CD44 (Fig. 5). Collagen
XVIII modified with highly sulfated HS (Fig. 4B) bound both
L- and P-selectin but not CD44 (Fig. 5). It should be noted, however,
that only a subset of collagen XVIII, such as that expressed in the
basement membrane of distal tubules, bound L-selectin, whereas collagen
XVIII in other locations did not (Fig. 7). This result implies that
only a subset of collagen XVIII species present in the rat kidney is
modified with L-selectin-reactive HS chains. Although the precise
carbohydrate structure required for L-selectin binding has yet to be
determined, our present results suggest that both N- and
O-sulfation of heparin/HS chains are required for the
interaction with L-selectin (Fig. 4A).
Although only a weak expression of MCP-1 in tubular epithelial cells
has been reported in the normal kidney (39), a strong expression of
MCP-1 has been detected in various renal diseases. For example, strong
tubular epithelial expression of MCP-1 was found in kidney biopsies of
patients with membranous nephropathy, IgA nephropathy, and
glomerulosclerosis (39). MCP-1 expression was also found in the
glomeruli from biopsies of patients with proliferative
glomerulonephritis, Wegener's disease, and lupus nephritis (40). Our
finding that collagen XVIII interacts with MCP-1 and presents it to a
monocytic cell line to induce Under physiological conditions, L-selectin-reactive molecules are
selectively localized to the epithelial cells of distal tubules, where
leukocytes do not normally traffic. The paucity of leukocytes in the
kidney parenchyma under physiological conditions can probably be
explained by the absence of leukocyte accessibility to collagen XVIII
and other L-selectin-binding molecules in the kidney. However, in the
diseased kidney, where massive mononuclear cell infiltration can be
observed, it is possible that collagen XVIII interacts with L-selectin
on the infiltrating cells and/or presents chemokines to them, and may
accelerate the progression of inflammatory processes. Experimental
verification is now required to assess the actual involvement of
collagen XVIII in leukocyte migration under pathological conditions.
4
1 integrin-mediated binding
of the THP-1 cells to vascular cell adhesion molecule-1. Thus, collagen
XVIII may provide a link between selectin-mediated cell adhesion and
chemokine-induced cellular activation and accelerate the progression of
leukocyte infiltration in renal inflammation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
4 chain mAb
HP2/1, and anti-integrin
1 chain mAb 4B4 were purchased
from Beckman Instruments (Fullerton, CA). All the other reagents used
in this study were described previously (11-13).
80 °C until use. A portion of the purified
collagen XVIII was biotinylated as described previously (12).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Identification of the L-selectin-binding
HSPGs in the rat kidney as collagen XVIII. A,
comparison of partial amino acid sequences of the L-selectin-binding
HSPGs (peaks 160-1, 160-2, and 180-2)
with that of human collagen XVIII (hCol XVIII). Identical
residues are marked with asterisks. B, the 1 M NaCl eluate prepared from rat tubular lysates by
DEAE-Sepharose anion exchange column chromatography (13) was incubated
with human IgG1 or LEC-IgG in the presence or absence of
EDTA (20 mM), HRL2 (50 µg/ml), or HRL3 (50 µg/ml) and
then with protein A-Sepharose beads. The precipitates were treated with
heparitinase (250 milliunits/ml) at 37 °C for 5 h. The samples
were then subjected to SDS-PAGE and blotted onto a
polyvinylidene fluoride membrane (Millipore Co.). The membrane
was probed with 0.5 µg/ml normal rabbit IgG or RES.16.
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Fig. 2.
Purification of rat collagen XVIII. The
biotinylated 1 M NaCl eluate from the DEAE-Sepharose column
(13) (lane 1) and biotinylated 250 mM
imidazole eluate from the Ni-NTA-agarose column (lane 2)
were treated with heparitinase (0.1 unit/ml), subjected to SDS-PAGE,
and transferred onto a polyvinylidene fluoride membrane. The
bands were detected using ABC reagent and ECL Western blotting
detection reagents.
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Fig. 3.
Binding of collagen XVIII to L-selectin is
inhibited by HRL3, EDTA, and heparitinase treatment. A,
biotinylated collagen XVIII was added to wells coated with human IgG or
LEC-IgG (5 µg/ml) that had been incubated with 30 µg/ml HRL2 or
HRL3 or 20 mM EDTA. B, biotinylated collagen
XVIII that had been treated with 0.1 unit/ml chondroitinase ABC
(CHase ABC), heparitinase, or keratanase, or left untreated
was added to wells coated with human IgG or LEC-IgG (5 µg/ml). The
extent of binding was determined by ELISA as described previously (12).
Each bar represents the mean ± S.D. of triplicate
determinations.
DiHS-0S,
DiHS-6S,
DiHS-NS,
DiHS-di(6,N)S,
DiHS-di(U,N)S, and
DiHS-tri(U,6,N)S in Fig. 4B
were 8.1, 28.2, 19.9, 15.7, 20.4, and 7.7%, respectively.
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Fig. 4.
Both N- and
O-sulfation of HS chains are required for interaction
of collagen XVIII with L-selectin. A, binding of
collagen XVIII to L-selectin is inhibited by heparin but not by
chemically modified heparin derivatives. Biotinylated collagen
XVIII was added to wells coated with LEC-IgG (5 µg/ml) in the
presence of increasing concentrations of various GAGs. GAGs
used were heparin (open circles), CDSNAc-heparin
(closed circles), CDSNS-heparin (open
squares), NDSNAc-heparin (closed squares), CS B
(open triangles), and CS E (closed triangles).
The solid bars on the right show the binding of
biotinylated collagen XVIII to the wells coated with human
IgG1 or LEC-IgG (5 µg/ml) in the absence of GAGs.
B, disaccharide composition analysis of HS chains of
collagen XVIII. Collagen XVIII was treated with a mixture of
heparitinase, heparitinase I and heparitinase II, and the resultant
disaccharides were derivatized with 2-AB and analyzed by high
performance liquid chromatography on an amine-bound silica column as
described previously (19). The elution positions of the
2-AB-derivatized standard disaccharides, DiHS-0S (peak
1),
DiHS-6S (peak 2),
DiHS-NS (peak
3),
DiHS-di(6,N)S (peak 4),
DiHS-di(U,N)S
(peak 5), and
DiHS-tri(U,6,N)S (peak 6) are
shown.
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Fig. 5.
Collagen XVIII binds L- and P-selectin but
not E-selectin or CD44. Binding of biotinylated collagen XVIII
(Col.XVIII), biotinylated sLeX-polymeric
probe (sLeX), biotinylated versican (12), or
biotinylated aggrecan (RC-PG) to wells coated with human IgG
(bar 1), human L-selectin-IgG (bar 2), human
E-selectin-IgG (bar 3), human P-selectin-IgG (bar
4), human CD44-IgG (bar 5), or rat L-selectin-IgG
(LEC-IgG, bar 6) was determined by ELISA. Each
bar represents the mean ± S.D. of triplicate
determinations.
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Fig. 6.
Binding of rat L-selectin transfectants to
collagen XVIII.
2',7'-Bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein-labeled rat
L-selectin transfectants (RM-1) or parental cells
(EL-4) (1 × 105 cells/well) were allowed
to bind to wells coated with (black bars) or without
(striped bars) collagen XVIII (1 µg/ml) in the presence or
absence of 50 µg/ml anti-L-selectin mAbs HRL2 or HRL3 or 25 mM EDTA as described previously (12). Each bar
represents the mean ± S.D. of triplicate determinations.
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Fig. 7.
Localization of L-selectin-binding
molecules and collagen XVIII in the rat kidney. Cryosections from
a rat kidney were incubated overnight with a mixture of LEC-IgG (10 µg/ml) and RES.16 (2 µg/ml). They were subsequently incubated with
a mixture of fluorescein isothiocyanate-anti-human IgG antibody and
TRITC anti-rabbit IgG antibody. A and C, green
fluorescence (LEC-IgG staining) was detected, whereas in B
and D red fluorescence (RES.16 staining) was detected, using
an Olympus fluorescence microscope BX-FLA. A and
B, outer medulla. C and D, cortex.
T, tubule; VB, vascular bundle; G,
glomerulus.
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Fig. 8.
MCP-1 interacts with HS chains of
collagen XVIII. A, binding of MCP-1 to wells coated
with collagen XVIII. The wells of a 4-mm-diameter 96-well EIA/RIA plate
that had (open symbols) or had not (closed
circles) been coated with collagen XVIII (1 µg/ml) were either
untreated (open squares, closed circles) or treated with 0.1 units/ml heparitinase (open circles) or 0.1 units/ml
chondroitinase ABC (open triangles) at 37 °C for 2 h. After washing, MCP-1 binding was determined as described previously
(41), except that streptavidin-conjugated alkaline phosphatase (1:500,
Promega, Madison, WI) and Blue PhosTM (Kirkegaard Perry
Laboratories, Gaithersburg, MD) were used to detect the binding.
B, binding of MCP-1 (0.2 µg/ml) to wells coated with
(black bars) or lacking (striped bars) collagen
XVIII (0.2 µg/ml) in the presence or absence of LEC-IgG (3, 30, and
300 µg/ml). The binding was detected as described in A. C, binding of LEC-IgG (3 µg/ml) to wells coated with
(black bars) or lacking (striped bars) collagen
XVIII (0.2 µg/ml) in the presence or absence of MCP-1 (0.3, 3, and 30 µg/ml). The binding of LEC-IgG to collagen XVIII was detected by
ELISA (method 2) as described previously (12).
4
1 integrin. The THP-1 cells did not bind collagen XVIII in an L-selectin-dependent manner under the
conditions used in this experiment, and thus were useful to determine
the effect of chemokine presentation by collagen XVIII on
integrin-mediated cell adhesion. As shown in Fig.
9A, THP-1 cells bound to the
wells that had been coated with a mixture of collagen XVIII and VCAM-1 but not to the wells coated with collagen XVIII or VCAM-1 alone in a
manner dependent on the dose of MCP-1, indicating that MCP-1 that had
been immobilized on collagen XVIII activated the binding of THP-1 cells
to VCAM-1. This up-regulated adhesion was apparently mediated by
4
1 integrin, since the adhesion was
blocked by either the anti-
4 integrin mAb HP2/1 or the
anti-
1 integrin mAb 4B4 (Fig. 9B).
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Fig. 9.
Collagen XVIII presents MCP-1 to THP-1 cells
to activate their
4
1
integrin-mediated binding to VCAM-1. A, various
concentrations of MCP-1 were applied to wells coated with 1 µg/ml
collagen XVIII (closed circles), 0.5 µg/ml VCAM-1
(open squares), a mixture of collagen XVIII and VCAM-1
(closed squares), or to uncoated wells (open
circles). THP-1 cells were applied to the wells, and cell adhesion
was determined as described under "Experimental Procedures."
B, MCP-1 (3 µg/ml) was either applied (black
bars) or not applied (striped bars) to wells that had
been coated with PBS alone or with a mixture of collagen XVIII (1 µg/ml) and VCAM-1 (0.5 µg/ml). THP-1 cells were then applied to the
wells in the presence or absence of 5 µg/ml control mouse
IgG1, anti-human L-selectin mAb TQ1, anti-
4
integrin mAb HP2/1, or anti-
1 integrin mAb 4B4. Each
bar represents the mean ± S.D. of triplicate
determinations.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
4
1 integrin
activation in vitro (Figs. 8 and 9) indicates that collagen XVIII may bind locally produced MCP-1 and present it to infiltrating monocytes in the diseased kidney.
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ACKNOWLEDGEMENTS |
---|
We thank Yumi Takara and Bo-Gie Yang for technical assistance. We also thank Naoko Abe who helped with part of the purification work as an undergraduate student.
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FOOTNOTES |
---|
* This work was supported in part by grants-in-aid for the Center of Excellence Research and for Scientific Research on Priority Areas, Sugar Remodeling and Cellular Communications from the Ministry of Education, Science and Culture, Japan, and Taisho Pharmaceutical Co.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed: Glycobiology Program, Cancer Research Center, The Burnham Institute, La Jolla, CA. Tel.: 858-646-3100; Fax: 858-646-3193; E-mail: hkawashima@burnham.org.
Published, JBC Papers in Press, January 28, 2003, DOI 10.1074/jbc.M212244200
2 H. Kawashima, unpublished observations.
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ABBREVIATIONS |
---|
The abbreviations used are:
mAb, monoclonal
antibody;
HSPG, heparan sulfate proteoglycan;
MCP-1, monocyte
chemoattractant protein-1;
LEC-IgG, rat L-selectin-IgG chimera;
VCAM-1, vascular cell adhesion molecule-1;
CDSNAc-heparin, completely
desulfated, N-acetylated heparin;
CDSNS-heparin, completely
desulfated, N-sulfated heparin;
NDSNAc-heparin, N-desulfated, N-acetylated heparin;
RC-PG, rat
chondrosarcoma proteoglycan;
2-AB, 2-aminobenzamide;
DiHS-0S,
HexUA
1-4GlcNAc;
DiHS-6S,
HexUA
1-4GlcNAc(6-O-sulfate);
DiHS-NS,
HexUA
1-4GlcN(2-N-sulfate);
DiHS-di(6, N)S,
HexUA
1-4GlcN(2-N,6-O-disulfate);
DiHS-di(U, N)S,
HexUA(2-O-sulfate)
1-4GlcN(2-N-sulfate);
DiHS-tri(U, 6,N)S,
HexUA(2-O-sulfate)
1-4GlcN(2-N,6-O-disulfate);
ELISA, enzyme-linked immunosorbent assay;
NTA, nitrilotriacetic acid;
HS, heparan sulfate;
CS, chondroitin sulfate;
sLeX, sialyl
Lewis X;
PMSF, phenylmethylsulfonyl fluoride;
PBS, phosphate-buffered
saline;
GAG, glycosaminoglycan;
TRITC, tetramethylrhodamine
isothiocyanate.
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