Mannan-Binding Protein Blocks the
Activation of Metalloproteases Meprin α and
β
This information is current as
of June 14, 2017.
Makoto Hirano, Bruce Yong Ma, Nana Kawasaki,
Kazumichi Okimura, Makoto Baba, Tomoaki Nakagawa,
Keiko Miwa, Nobuko Kawasaki, Shogo Oka and Toshisuke
Kawasaki
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The Journal of Immunology is published twice each month by
The American Association of Immunologists, Inc.,
1451 Rockville Pike, Suite 650, Rockville, MD 20852
Copyright © 2005 by The American Association of
Immunologists All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol 2005; 175:3177-3185; ;
doi: 10.4049/jimmunol.175.5.3177
http://www.jimmunol.org/content/175/5/3177
The Journal of Immunology
Mannan-Binding Protein Blocks the Activation of
Metalloproteases Meprin ␣ and ␤1
Makoto Hirano,2* Bruce Yong Ma,2* Nana Kawasaki,‡ Kazumichi Okimura,* Makoto Baba,*
Tomoaki Nakagawa,* Keiko Miwa,* Nobuko Kawasaki,† Shogo Oka,* and
Toshisuke Kawasaki3*§
M
annan-binding protein (MBP),4 also known as mannan-binding lectin, is a Ca2⫹-dependent (C-type) serum lectin exhibiting primary specificity for mannose,
fucose, and N-acetylglucosamine (1). MBP is an important serum
component associated with innate immunity. MBP activates complement through interaction with complement subcomponents C1r/
C1s (2, 3) or three novel C1r/C1s-like serine proteases, MBPassociated serine proteases (4 – 6). The MBP-mediated complement
activation is called the lectin pathway (7). MBP has been shown to
have complement-dependent bactericidal activity, to serve as a direct
*Department of Biological Chemistry, Graduate School of Pharmaceutical Sciences,
and †School of Health Sciences, Faculty of Medicine, Kyoto University, Kyoto, Japan; ‡Division of Biological Chemistry and Biologicals, National Institute of Health
Sciences, Tokyo, Japan; and §Research Center for Glycobiotechnology, Ritsumeikan
University, Shiga, Japan
Received for publication February 22, 2005. Accepted for publication June 3, 2005.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported in part by a Grant-in-Aid for Scientific Research on Priority Areas, A-14082203, to T.K., and by a Grant-in-Aid for Creative Scientific Research (16GS0313) to S.O. and B.Y.M. from the Japan Society for the Promotion of
Science, Ministry of Education, Culture, Sports, Science, and Technology of Japan.
2
M.H. and B.Y.M. contributed equally to this work.
3
Address correspondence and reprint requests to Dr. Toshisuke Kawasaki, Department of Biological Chemistry, Graduate School of Pharmaceutical Sciences, Kyoto
University, Kyoto 606-8501, Japan. E-mail address: [email protected]
4
Abbreviations used in this paper: MBP, mannan-binding protein; ADAM, a disintegrin and metalloproteinase; AAL, aleuria aurantia lectin; CBB, Coomassie brilliant
blue; CLD, collagen-like domain; CRD, carbohydrate recognition domain; ECM, extracellular matrix; Endo F, N-glycosidase F; Endo H, endoglycosidase H; LC, liquid
chromatography; MDCC, MBP-dependent cell-mediated cytotoxicity; MMP, matrix
metalloproteinase; MS, mass spectrometry; PFA, paraformaldehyde; PTH, parathyroid hormone; TIMP, tissue inhibitor of metalloproteinase.
Copyright © 2005 by The American Association of Immunologists, Inc.
opsonin, and to mediate the binding and uptake of bacteria that express a mannose-rich O-polysaccharide by monocytes and neutrophils
(8 –10). Furthermore, MBP can facilitate the uptake of apoptotic cells
by macrophages and immature dendritic cells (11, 12). MBP functions as a ␤-inhibitor of the influenza virus (13), and protects cells
from HIV infection by binding to gp120, a high mannose-type oligosaccharide-containing envelope glycoprotein on HIV (14). MBP may
also play an important role in other serious common diseases such as
atherosclerosis (15) and chronic pulmonary disease (16), and a MBP
deficiency could impair the normal innate immune function and increase susceptibility to infection (17).
MBP is a homo-oligomer composed of 32-kDa subunits. Each
subunit has an NH2-terminal region containing cysteines involved
in interchain disulfide bond formation, a collagen-like domain
(CLD) containing hydroxyproline and hydroxylysine residues, a
neck region, and a carbohydrate recognition domain (CRD) with
an amino acid sequence highly homologous to those of other Ctype lectins (18). Three subunits form a structural unit, and an
intact MBP molecule consists of three to six structural units. The
CRD is specific for mannooligosaccharide structures on exogenous
and endogenous ligands, whereas the CLD is believed to be responsible for interactions with other effector proteins involved in
host defense. In addition, clinical studies have demonstrated a
marked correlation between low serum levels of MBP and an immune opsonic deficiency (19). Low serum concentrations of MBP
are associated with three independent mutations in codons 52, 54,
and 57 of exon 1, resulting in amino acid changes of Arg52 to Cys,
Gly54 to Asp, and Gly57 to Glu, respectively, all of which occur in
the CLD (20, 21). These replacements appear to inhibit oligomerization of the structural unit of the molecule and consequently
0022-1767/05/$02.00
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Mannan-binding protein (MBP) is a C-type serum lectin that is known to be a host defense factor involved in innate immunity,
and recognizes mannose, fucose, and N-acetylglucosamine residues. Although some exogenous MBP ligands have been reported,
little is known about its endogenous ligands. In the present study, we found that endogenous MBP ligands are highly expressed
in the brush border epithelial cells of kidney-proximal tubules by immunohistochemistry, and both meprin ␣ and ␤ (meprins), as
novel endogenous MBP ligands, have been identified through affinity chromatography and mass spectrometry. Meprins are
membrane-bound and secreted zinc metalloproteases extensively glycosylated and highly expressed in kidney and small intestinal
epithelial cells, leukocytes, and certain cancer cells. Meprins are capable of cleaving growth factors, extracellular matrix proteins,
and biologically active peptides. Deglycosylation experiments indicated that the MBP ligands on meprins are high mannose- or
complex-type N-glycans. The interaction of MBP with meprins resulted in significant decreases in the proteolytic activity and
matrix-degrading ability of meprins. Our results suggest that core N-linked oligosaccharides on meprins are associated with the
optimal enzymatic activity and that MBP is an important regulator for modulation of the localized meprin proteolytic activity via
N-glycan binding. Because meprins are known to be some of the major matrix-degrading metalloproteases in the kidney and
intestine, MBP, which functions as a natural and effective inhibitor of meprins, may contribute, as a potential therapeutic target,
to tumor progression by facilitating the migration, intravasation, and metastasis of carcinoma cells, and to acute renal failure and
inflammatory bowel diseases. The Journal of Immunology, 2005, 175: 3177–3185.
3178
Materials and Methods
Materials
Six-week-old BALB/c mice were obtained from Japan SLC. Anti-human
MBP mAb (HYB 131-01) was purchased from Antibody Shop. Endoglycosidase H (Endo H) and N-glycosidase F (Endo F) were obtained from
Roche. Casein was obtained from Calbiochem. PTH and collagen type IV
from human placenta were purchased from Sigma-Aldrich. Tissue cryosection slides prepared from different human normal tissues were obtained
from Sigma-Aldrich. A protease inhibitor mixture and gelatin from bovine
skin were obtained from Nacalai Tesque. All chemicals for gel electrophoresis and Western blotting were obtained from Atto Bioscience, Vector
Laboratories, Bio-Rad, Pierce, and Zymed Laboratories.
Purification of MBP from human serum, and preparation of
biotin-labeled human MBP and Sepharose 4B-MBP column
Human MBP was purified from healthy donor human serum by affinity
chromatography on a Sepharose 4B-mannan column, as described previously (1). For the preparation of biotin-labeled human MBP and Sepharose
4B-MBP column, human MBP was coupled to EZ-link Sulfo-NHS-Biotin
(Pierce) and Cyanogen bromide-activated Sepharose 4B (Amersham Biosciences), respectively, according to the manufacturer’s instructions.
Immunohistochemistry
Kidneys were dissected from mice that had been perfused with PBS and
subsequently with PBS containing 4% paraformaldehyde (PFA). Each kidney was immersed in PBS containing 4% PFA overnight at 4°C, followed
by immersion in 50 mM phosphate buffer containing 10, 20, and 30%
sucrose in a stepwise manner. The kidney was then embedded in optimum
cutting temperature (OCT) compound (Tissue-Tek) and rapidly frozen on
dry ice. Cryosections (10 ␮m thick) were made with a CM3000 cryostat
(Leica Microsystems) and then mounted on poly(L-lysine)-coated glass
slides. The paraffin-embedded cryosections of human kidney (Sigma-Aldrich) were deparaffinized and hydrated with xylene and alcohol, respectively. Endogenous peroxidase activity was quenched by incubating the
sections of mouse and human kidneys with PBS containing 3% H2O2 and
1% Triton X-100. After blocking with SuperBlock blocking buffer (Pierce),
the cryosections of mouse kidney were incubated with biotinylated human
MBP, and the cryosections of human kidney were incubated with human
MBP, followed with anti-human MBP mAb (HYB 131-01) and biotinylated second Ab, respectively, in the presence of 10 mM Ca2⫹. Cryosections
were washed with TBS-Tween 20 and then incubated with a Vectastain
ABC Elite kit (Vector Laboratories). After washing with TBS-Tween 20
and TBS, immunoreactivity was visualized with a 3, 3⬘-diaminobenzidine
substrate kit (Vector Laboratories). Digital photographs were taken under
a Nikon Eclipse E600 microscope.
Isolation and identification of MBP ligands from mouse kidney
Perfused mouse kidneys were homogenized in homogenization buffer (150
mM NaCl, 20 mM Tris-HCl (pH 7.5), 0.32 M sucrose, 1 mM EDTA, and
protease inhibitor mixture). The homogenate was centrifuged at 1,000 ⫻ g
for 10 min at 4°C twice to remove cell debris and nuclei. The supernatant
was then centrifuged at 105,000 ⫻ g for 60 min at 4°C. The resulting total
membrane pellet was solubilized with lysis buffer (150 mM NaCl, 20 mM
Tris-HCl (pH 7.5), 1 mM EDTA, 1% Triton X-100, and protease inhibitor
mixture) for 60 min on a rotary shaker at 4°C, and then centrifuged at
150,000 ⫻ g for 60 min at 4°C. The supernatant was saved as the kidney
membrane proteins. CaCl2 was added to the kidney membrane proteins to
20 mM, and then the mixture was applied to a Sepharose 4B-MBP affinity
column. After washing the column with TBS buffer (pH 7.5) containing 20
mM CaCl2 and 0.1% Triton X-100, the proteins bound to the column were
eluted with TBS buffer (pH 7.5) containing 4 mM EDTA and 0.1% Triton
X-100. The eluted proteins were resolved on a 5–20% Tris-HCl gradient
gel (Atto Bioscience) and then stained with colloidal Coomassie blue
(GelCode Blue; Pierce). Bands were excised from the gel and subjected to
in-gel digestion. The released peptides from the gel were subjected to liquid chromatography/tandem mass spectrometry (LC/MS/MS) analysis using a hybrid quadrupole/time-of-flight mass spectrometer (Qstar Pulsar I;
Applied Biosystems) interfaced online with a capillary HPLC (Magic
2002; Michrom BioResources) equipped with Magic C18 column (0.2 ⫻
50 mm, 3 ␮m; Michrom BioResources). The eluents consisted of water
containing 2% CH3CN and 0.01% formic acid (pump A), and 90% CH3CN
and 0.01% formic acid (pump B), and the peptides were eluted with a linear
gradient from 5 to 65% of pump B in 20 min at a flow rate of 2 ␮l/min.
Data-dependent MS/MS acquisitions were performed on precursors with
charge states of 2 or 3 over a survey mass range of 400-2000. Proteins were
identified by searching the mass spectrometry database (MSDB) using
Mascot search engine (Matrixscience).
Deglycosylation and lectin/Western blot analysis of the purified
MBP ligands
For deglycosylation experiments, the purified MBP ligands were denatured
by boiling in the presence of 0.1% SDS and 50 mM 2-ME, and then treated
with glycosidase, Endo H, or Endo F, according to the manufacturer’s
instructions. Reactions were stopped by boiling the samples in SDS-PAGE
sample buffer. The deglycosylated and control samples were resolved on a
SDS-PAGE gel (Atto Bioscience), and then transferred to nitrocellulose
membranes, followed by immunoblot or lectin-blot detection. For visualization, a SuperSignal West Pico Chemiluminescent kit (Pierce) was used
with HRP-conjugated anti-mouse IgG Ab (Zymed Laboratories).
The inhibition of proteolytic activity of meprins toward
biologically active peptides/proteins and ECM components
by MBP
The 0.2 ␮g of purified meprins from mouse kidneys was incubated with 20
␮g of casein, 5 ␮g of PTH, 5 ␮g of collagen IV from human placenta, or
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abolish the ability to initiate complement activation without impairing the original lectin-binding specificity to oligosaccharide
ligands (22). We previously found that MBP recognizes and binds
specifically to oligosaccharide ligands expressed on the surfaces of
some human colorectal carcinomas (23). Recently, MBP was
shown to exhibit novel cytotoxic activity toward these colorectal
carcinoma cell in vivo experiments, which we proposed to term
MBP-dependent cell-mediated cytotoxicity (MDCC) (24, 25).
Several lines of evidence suggest some cellular ligands and receptors may be involved in the MDCC reaction (26, 27). Although
some exogenous MBP ligands have been reported, little is known
about its endogenous ligands.
In this study, we investigated endogenous MBP ligands that are
highly expressed in the brush border epithelial cells of kidneyproximal tubules and in some villous epithelial cells of the small
intestine by immunohistochemistry, and identified meprins (meprin ␣ and ␤), mammalian zinc metalloproteases, as novel endogenous MBP ligands in mouse kidney through affinity chromatography and mass spectrometry. Mouse meprin A (EC 3.4.24.18) is
a homo-oligomer of ␣ subunits, or a hetero-oligomer of ␣ and ␤
subunits (28). Mouse meprin B (EC 3.4.24.63) is a homo-oligomer
of ␤ subunits (29). The multidomain ␣ and ␤ meprin subunits form
a disulfide-linked dimer and higher order oligomers through noncovalent interactions (30, 31). Meprins are tissue-specific proteases that are implicated in developmental processes as well as in
normal and pathological processes in adult tissues (32, 33). They
are secreted from or localized in mammalian brush border membranes of kidney and intestine epithelial cells (34 –36). They degrade proteins of the extracellular matrix (ECM) such as collagen
type IV, gelatin, fibronectin, laminin, and nidogen, and process
biologically active peptides, including bradykinin, angiotensins,
parathyroid hormone (PTH), gastrin, the ␤-chain of insulin, growth
factors, and cytokines (37). Several proteases, including serine
proteases and metalloproteases, are implicated in tumor growth,
invasion into surrounding tissues, and metastasis. The meprins are
extensively glycosylated, containing ⬃25% carbohydrates, which
are N-linked, not O-linked oligosaccharides in meprin ␣ (30), and
both N- and O-linked ones in meprin ␤ (38). The N-linked oligosaccharides on the meprins are important for secretion and enzymatic activity, but not for apical targeting (30). In this study, we
provide convincing evidence that MBP inhibits the catalytic activation of meprins, and discuss the molecular mechanism of the
inhibition. Therefore, we suggest that the novel function of MBP
may be, at least in part, responsible for its potent antitumor and
antiangiogenic action as a natural metalloprotease inhibitor with
potential therapeutic applications.
REGULATION OF ACTIVITIES OF MEPRINS BY MBP
The Journal of Immunology
3179
5 ␮g of gelatin from bovine skin for 6 h, 30 min, 1 h, or 20 min, respectively, at 37°C in 100 mM Tris-HCl, 10 mM CaCl2, and 1 mM ZnCl2 (pH
7.5), in a total volume of 20 ␮l, before incubation with control, 1.1 or 2.2
␮g of human MBP for 1 h at room temperature. The reactions were terminated by the addition of 10 mM EDTA and boiling of the samples in
SDS-PAGE sample buffer with or without 2-ME. The samples were applied to a SDS-PAGE gel, and then stained with Coomassie brilliant blue
(CBB). For the experiment on the release by mannose of the inhibition of
the ECM-degrading ability of meprins by MBP, 20 mM mannose was
added to the reaction mixture after incubation with meprins and MBP. The
percentage of inhibition of the proteolytic activity of meprins toward biologically active peptides/proteins and ECM components by MBP was determined by densitometric scanning of substrate bands by laser
densitometry.
Results
Expression and localization of MBP ligands both in human and
mouse kidneys
Purification of MBP ligands from mouse kidney
To purify the endogenous MBP ligands expressed in mouse kidney, kidney membrane fractions were obtained, as described in
Materials and Methods. The kidney membrane proteins were applied to an affinity column of Sepharose 4B-human MBP in the
presence of 20 mM CaCl2, and the bound proteins were eluted with
4 mM EDTA. The eluted proteins were separated by SDS-PAGE
and then detected by colloidal Coomassie blue staining (Fig. 2A).
The binding with MBP was confirmed by MBP lectin blotting
(data not shown). As shown in Fig. 2A, the MBP ligands appeared
as two major bands of 83 (K5) and 91 kDa (K4) under reducing
conditions.
Identification of metalloproteases meprin ␣ and ␤ as
endogenous MBP ligands
For identification of the purified and separated MBP ligands, the
two major bands, K5 and K4, indicated by the arrows in Fig. 2A,
were excised and in-gel digested with trypsin. The digested proteins were analyzed by nano-LC/MS/MS, as described in Materials and Methods. The acquired fragmentation nano-LC/MS/MS
spectra of peptides were searched against the mouse protein database from MSDB using Mascot search engine. In this way, the K5
and K4 bands were positively identified as meprin ␣ and ␤, which
are encoded at two independent gene loci, as endogenous MBP
ligands, as shown in Fig. 2B. The peptides indicated in red within
the complete meprin ␣ and ␤ precursor sequences were found in
the fragments analyzed by mass spectrometry (Fig. 2B). Meprins,
which belong to the astacin family of metalloproteases and to the
metzincin superfamily, comprise ⬃5% of the kidney brush border
membrane proteins in mice (32). They are also expressed in in-
FIGURE 1. Immunohistochemistry to detect MBP ligands both in human and mouse kidneys. PFA-fixed cryosections of human and mouse
kidneys were stained with human MBP and counterstained with hematoxylin, as described in Materials and Methods, respectively. A, Immunohistochemical staining for human MBP ligands with human MBP. B, Immunohistochemical staining for mouse MBP ligands with human MBP. b and
c, Magnifications of the cortex and medulla in a, respectively. Blue and
green circles indicate brush border membrane staining in proximal renal
tubules and the lack of staining in renal corpuscles, respectively. Scale
bars, 50 ␮m.
testinal brush border membranes, in leukocytes, and in certain epithelial cancer cells (39, 40). The meprin subunits, ␣ and ␤, are
extensively glycosylated, and ⬃25% of the total molecular mass of
the subunits comprises carbohydrates. They associate to form homo- or hetero-oligomers via disulfide bridges (28). The meprin ␣
and ␤ subunits are 42% identical at the amino acid level and share
the same domain structure, except that meprin ␣ contains an inserted domain that is not present in meprin ␤ (41). Mouse meprin
␣ and ␤ contain 10 and 8 potential N-linked glycosylation sites,
respectively (30). In vivo, meprin ␣ is secreted as a homo-oligomer and is also found as a hetero-oligomer in the plasma membrane in association with meprin ␤, a type I integral membrane
protein; thus, any meprin oligomer containing the ␤ subunit is
localized to the cell membrane (41). The localization of MBP ligands shown in Fig. 1 corresponds to previous reports, indicating
that the ␤ homo-oligomer of meprin B and the ␣ and ␤ heterooligomer of meprin A are localized to the apical brush border of
the renal and intestinal proximal tubule epithelium.
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To investigate whether endogenous MBP ligands express in human and mouse kidneys, immunohistochemical staining of PFAfixed cryosections of kidneys from both humans and mice was
performed with human MBP, respectively. As shown in Fig. 1, the
staining patterns for endogenous MBP ligands in the human kidney cryosections with human MBP (Fig. 1A) were almost indistinguishable from that seen in the mouse kidney cryosections with
human MBP (Fig. 1B); the apical brush border membranes of kidney-proximal tubule cells of the cortex from human and mouse
stained positively for MBP ligands at higher magnification (b of
Fig. 1, A and B), whereas the renal corpuscles of the cortex and
renal medulla from both human and mouse kidney were not
stained with human MBP (b and c of Fig. 1, A and B). The results
indicate that endogenous MBP ligands are highly expressed in the
brush border epithelial cells of kidney-proximal tubules. Similarly,
obvious staining was also observed in some villous epithelial cells
of the small intestine (data not shown).
3180
REGULATION OF ACTIVITIES OF MEPRINS BY MBP
Carbohydrate analyses of meprins by glycosidase digestion and
lectin blotting
MBP binds to various types of glycoproteins with terminal mannose, fucose, and N-acetylglucosamine residues. To characterize
the oligosaccharides carried by meprins, and to investigate the interaction between MBP-CRD and the oligosaccharides of meprins,
the oligosaccharides were removed enzymatically from meprins,
and then the reactivity of the deglycosylated meprins toward MBP
was examined. Endo H removes the high mannose oligosaccharides that are found in the endoplasmic reticulum, while Endo F
removes both high mannose and complex oligosaccharides that
arise in the Golgi apparatus (42). As shown in Fig. 3, the meprins
are susceptible to both Endo H and F. Endo H cleaves at the chitobiose core of N-linked high mannose and complex oligosaccharides, leaving behind a single N-acetylglucosamine or a fucose
linked (␣-1, 6) to an N-acetylglucosamine. The Endo H-treated
meprins migrated slightly faster (lanes 3) and could be detected on
either MBP blotting or aleuria aurantia lectin (AAL) blotting,
which indicated a fucose linked (␣-1, 6) to an N-acetylglucosamine
terminal after Endo H treatment. In addition, because Endo F removes all kinds of N-linked oligosaccharides, the Endo F-treated
meprins were even smaller (lanes 2) and could not be detected on
either MBP blotting or AAL blotting. In brief, the interaction between MBP-CRD and the carbohydrates of meprins was confirmed
by the glycosidase digestion and MBP blotting described above.
The results indicate that the N-linked high mannose and complex
oligosaccharides of meprins are involved in the MBP-meprin interaction. The complex oligosaccharides that occur in the Golgi
apparatus were observed for meprin ␣ secreted from or meprin ␤
localized in brush border membranes of the kidney and intestine.
MBP inhibits the proteolytic activity of meprins
Both meprin ␣ and ␤ are known to be important metalloproteases,
abundantly expressed in the kidney and intestine, to be able to
hydrolyze a variety of biologically active peptides and proteins.
For example, meprins cleave blood pressure regulators such as
bradykinin, metabolism mediators such as PTH, and signaling
molecules such as protein kinase A (32, 43). To investigate the
effects of the proteolytic activity of meprins through the MBPmeprin interaction, casein and PTH, two well-known substrates of
meprins, were treated with purified meprins from BALB/c mice
before incubation with or without MBP. The hydrolyzed products
of casein and PTH were analyzed by SDS-PAGE under nonreducing and reducing conditions, respectively, because the m.w. of
MBP monomer is the same as that of casein (Fig. 4, A and B).
Interestingly, as shown in Fig. 4, MBP effectively blocked the
proteolytic activity of meprins in a dose-dependent manner. The
catalytic ability of meprins was decreased by ⬃85% and almost
100% for casein in the presence of 1.1 or 2.2 ␮g of MBP (Fig. 4C),
respectively, and there was almost 50% decrease for PTH in the
presence of 1.1 ␮g of MBP (Fig. 4D).
The inhibition of ECM-degrading ability of meprins by MBP
Basement membranes are organized as thin layers of a specialized
ECM that acts as a supporting scafford for epithelial and endothelial cells. Basement membranes not only provide mechanical support, but also influence cellular behavior such as the differentiation,
proliferation, and migration of various cells, including endothelial
cells. Collagen IV and gelatin are two of the major macromolecular constituents of basement membranes, and are thought to be
important in both endothelial and tumor cellular proliferation and
behavior. Recently, meprins were also shown to be crucial for the
degradion of components of the ECM, and for promotion of both
endothelial cell proliferation and migration, and tumor cell growth
and metastasis similar to matrix metalloproteases (MMPs). To examine the inhibition of the matrix-degrading ability of meprins
toward major basement membrane components by MBP, collagen
IV and gelatin were treated with purified meprins from BALB/c
mice before incubation with or without MBP, and the hydrolyzed
products were analyzed by SDS-PAGE (Fig. 5). Gelatin proved to
be the best substrate under the conditions used. After 15-min incubation with the meprins, intact gelatin was extensively hydrolyzed by the meprins in the absence of MBP (lane 2 in Fig. 5B).
Collagen IV was also degraded effectively by the meprins in the
absence of MBP, the patterns of hydrolysis being similar. The
major protein bands corresponding to 170, 150, and 90 kDa seen
for the control were hydrolyzed by the meprins, yielding bands that
migrated slightly faster than those in lane 2 in Fig. 5A. Hence, the
matrix-cleaving activity of meprins resembles that of gelatinases
(MMP-2 and -9) rather than that of collagenases (MMP-1 and -8).
However, in the presence of MBP, the matrix-degrading abilities
of the meprins were effectively inhibited (lanes 3 in Fig. 5, A and
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FIGURE 2. Purification and identification of MBP
ligands from mouse kidney. A, MBP ligands purified
by affinity chromatography. MBP ligands were purified
from mouse kidney membrane proteins on a Sepharose
4B-MBP affinity column, and fractionated on a 5–20%
reducing gradient SDS-PAGE gel, as described in Materials and Methods. A, Shows colloidal Coomassie
blue staining of this gel, and the arrows indicate the
purified MBP ligands. The m.w. markers are indicated
on the left. B, Identification of MBP ligands by mass
spectrometry. The purified MBP ligand bands were excised and digested with trypsin, and the fragments were
used for the identification of MBP ligands by mass
spectrometry, as described in Materials and Methods.
B, Shows the results of a search against the mouse protein database from National Center for Biotechnology
Information depending on the acquired fragmentation
spectra of peptides. The identified peptides are shown
in red within the complete meprin ␣ and ␤ precursor
sequences.
The Journal of Immunology
B, and bars 3 in Fig. 5, C and D). This observation suggests that
MBP may function as a potent endogenous inhibitor against meprins to degrade ECM components.
The mechanism of inhibition of metalloproteases meprin ␣ and
␤ by MBP
To further confirm that the inhibition of metalloproteases meprin ␣
and ␤ by MBP is carbohydrate dependent, 20 mM mannose was
added to the reaction mixture. As shown in lanes 4 in Figs. 4B and
5, A and B, and bars 4 in Figs. 4D and 5, C and D, mannose
effectively reverses the inhibition of the proteolytic activity of meprins by MBP in the case of both low m.w. substrate PTH and high
m.w. substrates collagen IV and gelatin. The results clearly demonstrate that MBP suppresses the metabolism mediator- and ECMdegrading ability of meprins through MBP recognition/binding of
the carbohydrates on meprins, resulting in suppression of the proteolytic activity of the meprins, and the carbohydrates of the meprins are directly involved in the MBP-meprin interaction.
Discussion
Our previous research indicated that MBP recognizes and binds
specifically to mannose, N-acetylglucosamine, or fucose-terminated oligosaccharide ligands found on the surfaces of certain human colorectal carcinomas (23). More recently, we demonstrated
that in vivo human MBP gene delivery by the recombinant vaccinia virus administered intratumorally or s.c. resulted in marked
inhibition of tumor growth and significant prolongation of the life
span of colorectal tumor SW1116-bearing mice, and the effect appeared to be a consequence of local production of MBP (24, 25).
Although the mechanism of MBP-mediated tumor growth inhibition has not yet been clearly elucidated, we proposed calling it
MDCC, supposing that some cellular ligands and receptors may be
involved in the MDCC reaction (25, 26). In the present study, we
found that endogenous MBP ligands are highly expressed in the
brush border epithelial cells of kidney-proximal tubules (Fig. 1)
and in some villous epithelial cells of the small intestine (data not
shown) by immunohistochemistry; the staining patterns with human MBP are almost indistinguishable between human and mouse
kidney cryosections (Fig. 1); and metalloproteases meprin ␣ and ␤,
as novel endogenous MBP ligands, were purified from the brush
border membranes of mouse kidneys by affinity chromatography
and identified by mass spectrometry (Fig. 2). The carbohydrate
analyses on glycosidase digestion and lectin blotting indicate that
the N-linked high mannose and complex oligosaccharides of meprins are involved in the MBP-meprin interaction (Fig. 3). Interestingly, the interaction of MBP with meprins resulted in significant decreases in the proteolytic activity (Fig. 4) and matrixdegrading ability of meprins (Fig. 5).
MBP is mainly synthesized by hepatocytes, and has been isolated from the liver and serum of several mammalian species. Only
one form of human MBP has been characterized, whereas in mice
two forms, MBP-A and MBP-C, have been described and shown
to be products of two related, but uncoupled, genes. Previous published observations demonstrated that the carbohydrate specificity
recognizing mannose, fucose, and N-acetylglucosamine residues
on glycoproteins is very similar between human and mouse MBPs,
although human MBP resembles that of mouse MBP-C more than
that of MBP-A; in addition, mouse MBP-A shows a higher affinity
for D-glucose and ␣-methyl-D-glucose than does MBP-C (44). Our
findings also indicate that the recognizing and binding for the carbohydrates on the endogenous MBP ligands, meprins, both in human and mouse kidneys with human MBP were found to be nearly
same. Furthermore, the interaction between mouse MBP and the
carbohydrates of mouse meprins was determined and quantitatively characterized by surface plasmon resonance analysis using
BIAcore X instrument (our unpublished data).
Meprin is one of the matrix-degrading metalloproteases that
comprises a closely related group of zinc metal-dependent enzymes capable of degrading one or more of the ECM components
at neutral pH. Because of their matrix-degrading ability, the metalloproteases have been suggested to play critical roles in many
biological processes, such as tissue remodeling, embryonic development, inflammation, tumor invasion, and metastasis. Several
previous reports stated that the observation of the basolateral secretion of human meprin in colorectal cancer (40, 45), and of
mouse or rat meprin in experimentally induced renal failure (46,
47) raised questions about the roles of this protease in the migration of cells across the basement membrane and in the destruction
of the ECM. Indeed, in a recent report, Lottaz et al. (45) demonstrated that altered sorting of meprin ␣ in colorectal carcinoma
cells leads to aberrant accumulation of meprin ␣ in the tumor
stroma, resulting in a proteolytic potential, which can be activated
by proteases from carcinoma cells or from cells in the tumor
stroma. In meprin ␣-positive carcinomas, the hydrolyzing activity
was 2.9-fold higher than in the corresponding normal colon mucosa, whereas in meprin ␣-negative carcinomas, this activity was
equal to that in the corresponding normal colon mucosa (45). An
important carcinoma cell-associated function is to facilitate invasion and metastasis. This process ultimately depends on degradation of the ECM. A role of meprins during this process is suggested
by the previously reported capacity of meprins to degrade ECM
components of the basement membrane, such as collagen type IV,
gelatin, fibronectin, and laminin in vitro, and therefore they may
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FIGURE 3. The carbohydrates of meprins are involved in the MBPmeprin interaction. A, The digestion of purified meprin A (␣ and ␤) with
Endo H and Endo F. The ␣ and ␤ subunits of meprin A were purified from
BALB/c mice kidney membrane proteins on a Sepharose 4B-MBP affinity
column. The purified meprins were analyzed for susceptibility to endoglycosidase treatment by enzymatic deglycosylation for 24 h at 37°C in the
absence (⫺) or presence of Endo H (H) or Endo F (F), as indicated. After
deglycosylation treatment, the control and treated enzyme proteins were
resolved on a 5–20% reducing gradient SDS-PAGE gel, and the proteins
were detected with CBB staining and lectin-blot analysis using MBP or
AAL lectin, as described in Materials and Methods. The m.w. markers are
shown on the left. B and C, Deglycosylation analysis of purified meprin A
(␣ and ␤) by MBP blotting and AAL blotting. The deglycosylated and
control samples were resolved on a 5–20% Tris-HCl gradient gel under
reducing conditions and then transferred to a nitrocellulose membrane,
followed by MBP blot (B) and AAL blot (C) detection, respectively, as
described in Materials and Methods.
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3182
REGULATION OF ACTIVITIES OF MEPRINS BY MBP
enable cells to migrate across the barrier (48). These lines of evidence strongly suggest that meprins may contribute to tumor progression by facilitating the migration, intravasation, and metastasis
of carcinoma cells.
In addition, there have been several recent reports describing
meprin exhibiting ECM-degrading activity in ischemic renal injury
(49). The renal tubular epithelium is the target in many forms of
ischemic and toxic renal injury, resulting in cell death and acute
FIGURE 5. Cleavage of ECM components by meprins, inhibition of the matrix-cleaving ability of meprins by MBP, and preincubation effect of mannose on
inhibition of the MBP-meprin interaction. A and B, The
ECM components collagen IV (A) and gelatin (B) were
incubated with the purified meprins or no enzyme in a
final volume of 20 ␮l after the meprins had been preincubated with or without 1.1 ␮g of MBP at room temperature for 1 h. The reactions were conducted at 37°C
for 1 h for collagen IV and 15 min for gelatin, respectively. The reaction was terminated by the addition of
10 mM EDTA, and samples were subjected to electrophoresis on a 5–20% reducing gradient SDS-PAGE
gel. Proteins were visualized with CBB. Representative
data for at least three independent experiments are
shown. C and D, The level of inhibition was determined by comparing the decreases in substrate concentration in the presence and absence of MBP. The relative percentage of inhibition of the proteolytic activity
of meprins toward substrates by MBP was determined
by densitometric scanning of substrate bands by laser
densitometry. The results are the averages of three independent determinations. For control lanes/bars 1,
collagen IV (A and C) or gelatin (B and D) was incubated without meprins; lanes/bars 2, meprins were
treated without MBP; lanes/bars 3, meprins were
treated with MBP; lanes/bars 4, meprins were treated
with MBP and 20 mM mannose.
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FIGURE 4. Inhibition of the proteolytic activities of
meprins by MBP. A and B, The purified meprins were
preincubated without or with 1.1 or 2.2 ␮g of MBP at
room temperature for 1 h before the addition of casein
(A) or PTH (B) as a substrate. Reactions were performed in a total volume of 20 ␮l at 37°C for 6 h for
casein and 30 min for PTH, respectively. The reaction
was terminated by the addition of 10 mM EDTA, and
samples were subjected to electrophoresis on a 15%
nonreducing SDS-PAGE gel for casein (A) and 15–
25% reducing gradient SDS-PAGE gel for PTH (B),
respectively. Proteins were visualized with CBB. Representative data for at least three independent experiments are shown. C and D, The level of inhibition was
determined by comparing the decreases in substrate
concentration in the presence and absence of MBP. The
relative percentage of inhibition of the proteolytic activity of meprins toward substrates by MBP was determined by densitometric scanning of substrate bands by
laser densitometry. The results are the averages of three
independent determinations. For control lanes/bars 1,
casein (A and C) or PTH (B and D) was incubated
without meprins; lanes/bars 2, meprins were treated
without MBP; lanes/bars 3, meprins were treated with
MBP; lanes/bars 4, meprins were treated with 2⫻
MBP (A and C) and meprins were treated with MBP
and 20 mM mannose (B and D).
The Journal of Immunology
(48). Hydroxamate-based inhibitors and TIMPs have been used
repeatedly to block tumor cell and lymphocyte migration across
basement membranes in vitro, and these observations formed the
basis for our future in vivo study. Our findings suggest that MBP
may partly function as the first potent endogenous inhibitor specific to meprins like TIMPs acting on MMPs in degree.
The present study raises more questions than it answers. Does
the inhibition of meprins by MBP attenuating the reduction in
renal function associated with ischemia-reperfusion injury and meprin-mediated cytotoxicity contribute to other types of acute renal
injury in humans? Does MBP act only on specific meprins or not
on other metalloproteases such as the MMP and ADAM families?
During the past couple of years, it has become clear that meprins,
MMPs, and ADAMs do more than degrade structural ECM proteins to promote invasion and metastasis. The use of MMP inhibitors with differing selectivities for MMP-related enzymes has
identified a novel role for metalloproteases in controlling lymphocyte transendothelial migration (53) and in blocking T cell migration across synthetic basement membranes in vitro (54). Meprin B
of the ␤ homo-oligomeric form and meprin A of the ␣ and ␤
hetero-oligomeric form are localized to apical brush border of the
renal and intestinal proximal tubule epithelium (34) (Fig. 1). Interestingly, in the kidney, MBP-A, but not MBP-C, was found to
be synthesized. Vice versa, only MBP-C biosynthesis was detected
in endothelial cells of the small intestine. In contrast, human MBP
was also detected in endothelial cells of the small intestine (our
unpublished data). The physiological function of the colocalization
of meprin and MBP is the point on which we are focusing. The
basic action of MBP, carbohydrate recognition as an animal serum
lectin, has proven sufficiently sophisticated to orchestrate various
functions. More targets and functions of MBP might still be uncovered. These could include actions that can be used more rationally and effectively for the treatment of colon cancer. Additional
studies on the actions of MBP against meprins and other certain
metalloproteases recognized by MBP are needed to assess its potential as a therapeutic target for treating colon cancer, acute renal
injury, and intestinal diseases.
Moreover, Kadowaki et al. (30) recently demonstrated that Nlinked oligosaccharides on meprin A metalloprotease are important for secretion and enzymatic activity, but not for apical targeting, by means of mutational analysis, in which potential
glycosylation sites were eliminated, and inhibitors of the biosynthesis and processing of N-linked oligosaccharides were used.
They showed that several mutants of nonglycosylation sites exhibited decreased enzymatic activity with a bradykinin analog as
the substrate, and deglycosylation of the wild-type resulted in a
75–100% loss in activity. Most recently, we characterized the
structure of MBP oligosaccharide ligands expressed on SW1116
tumor, which are shown to be a novel type of tumor-associated
carbohydrates composed of large, multiantennary N-glycans carrying highly fucosylated polylactosamine-type structure (55).
These data strongly support our observations that core N-linked
oligosaccharides on meprins are required for the catalytic property,
and that MBP is an important regulator and effector for the modulation of the localized meprin proteolytic activity via N-glycan
binding. The mechanism for inhibition of the MBP-meprin interaction may result from conformational alterations of the active site
or from effects on the subunit or domain-domain relationships that
alter the enzyme-substrate interaction.
In conclusion, the establishment of a model of the MBP-meprin
interaction is a valuable step toward elucidation of the physiological function and molecular mechanism, and provides a knowledge-based approach to novel metalloprotease inhibitor design for
therapeutic applications. It will be interesting to determine whether
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renal failure (50). Carmago et al. (47) reported that following ischemia-reperfusion renal injury, there is a rapid shift of meprin localization and intensity from the brush border to the cytoplasmic
compartment, tubular lumen, and tubular basement membranes.
Meprins are the major matrix-degrading enzymes in rat renal tubules (48), and this prompted a hunt for the role for meprin in renal
ischemia-reperfusion injury (47). They indicated that in in vivo
studies, rats exposed to ischemia-reperfusion injury were markedly
protected against acute renal failure by i.p. treatment with actinonin, a naturally occurring antibacterial agent that coincidentally
is a strong inhibitor of the astacin family of enzymes that includes
meprins (51). These observations suggested the possibility of direct cytotoxic effects of meprins on renal tubular epithelial cells,
and that the inhibition of meprins prevents ischemia-reperfusion
injury in vivo. Moreover, Trachtman et al. (46) have shown that
noncongenic mice strains with lower levels of renal tubular meprin
A expression developed less severe acute renal failure compared
with those with normal meprin A levels when exposed to ischemia-reperfusion. In this study, we obtained the first cause and
effect evidence for the inhibition of meprin metalloprotease activity by MBP as a potent endogenous inhibitor of meprins.
Most recently, Crisman et al. (43) reported that meprin ␣ and ␤
are expressed in leukocytes of mouse mesenteric lymph nodes, and
meprin ␣, but not ␤, decreased during intestinal inflammation. In
contrast, meprin ␤, but not ␣, is detected in cortical and medullary
macrophages of lymph nodes. Deletion of the meprin ␤ gene decreased the ability of leukocytes to migrate through a matrigel
compared with wild-type leukocytes. This indicated that the expression of meprins by leukocytes of the intestinal immune system
may have important implications for diseases such as inflammatory bowel diseases, which are aggravated by leukocyte infiltration
(43). Many studies describe the expression of MMPs in inflamed
tissue, and their potential roles in leukocyte extravasation (52) and
in leukocyte trafficking in the lymph node (53). The roles of meprins in leukocyte infiltration may be explained by a direct mechanism, whereby the membrane-bound meprins degrade ECM and
basement membrane to facilitate leukocyte infiltration, and the expression of meprins by macrophages involves the migration of
macrophages into the paracortex, where they commingle with T
cells (43). Based on these observations and our present findings, it
is suggested in this work that direct interaction of MBP with meprins via carbohydrate binding may be involved in the inhibitory
function of MBP toward ECM-degrading actions, and thereby may
be responsible for its protection against acute renal failure and
inflammatory bowel diseases. The potential treatment of these
diseases by targeting meprins with MBP warrants further
investigation.
The natural substrates and expression patterns of meprins in
acute renal failure, intestinal diseases, and cancerous cells implicate meprins in the regulation of growth, inflammation, cancer cell
metastasis, and matrix remodeling (39, 40, 45– 47). All components identified to date as inhibitors of meprins are either hydroxamic acid derivatives or thiol reagents such as actinonin and the
antihypertensive drug captopril. Meprins are also inhibited by the
classical inhibitors of metalloproteases, such as metal chelators
(EDTA and 1,10-phenanthroline), but not by inhibitors of serine,
cysteine, or aspartic proteinases. These compounds are rather nonspecific, because they bind not only to their target enzymes, but
also to other metalloproteases. In contrast, meprins differ from the
zinc-dependent mammalian MMPs and their close associates, a
disintegrin and metalloprotease (ADAM) and ADAM with thrombospondin repeat families, in that they are not inhibited by natural
specific tissue inhibitors of metalloproteases (TIMPs), which are
endogenous modulators of zinc-dependent mammalian MMPs
3183
3184
transendothelial migration of metastasizing tumor cells or of leukocytes at inflammatory sites is also metalloprotease dependent.
For this purpose, identification of the MBP-meprin model that
blocks tumor cell and lymphocyte transendothelial migration
across basement membranes both in vitro and in vivo is therefore
a major goal for the future.
Acknowledgments
We thank Mitsubishi Pharm for purification of the human serum MBP, and
Tomoko Honda for the secretarial assistance.
Disclosures
The authors have no financial conflict of interest.
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