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 α and β (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.

Mannan-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, MBP-associated serine proteases (4, 5, 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 opsonin, and to mediate the binding and uptake of bacteria that express a mannose-rich O-polysaccharide by monocytes and neutrophils (8, 9, 10). Furthermore, MBP can facilitate the uptake of apoptotic cells by macrophages and immature dendritic cells (11, 12). MBP functions as a β-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 C-type 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 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 kidney-proximal tubules and in some villous epithelial cells of the small intestine by immunohistochemistry, and identified meprins (meprin α and β), 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 α subunits, or a hetero-oligomer of α and β subunits (28). Mouse meprin B (EC 3.4.24.63) is a homo-oligomer of β subunits (29). The multidomain α and β 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, 35, 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 β-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 α (30), and both N- and O-linked ones in meprin β (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.

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.

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.

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 μm 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.

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 μm; 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 μl/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).

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 0.2 μg of purified meprins from mouse kidneys was incubated with 20 μg of casein, 5 μg of PTH, 5 μg of collagen IV from human placenta, or 5 μg 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 μl, before incubation with control, 1.1 or 2.2 μg 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.

To investigate whether endogenous MBP ligands express in human and mouse kidneys, immunohistochemical staining of PFA-fixed 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. 1,A) were almost indistinguishable from that seen in the mouse kidney cryosections with human MBP (Fig. 1,B); 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).

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 μm.

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 μm.

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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. 2,A). The binding with MBP was confirmed by MBP lectin blotting (data not shown). As shown in Fig. 2 A, the MBP ligands appeared as two major bands of 83 (K5) and 91 kDa (K4) under reducing conditions.

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 α and β precursor sequences.

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 α and β precursor sequences.

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For identification of the purified and separated MBP ligands, the two major bands, K5 and K4, indicated by the arrows in Fig. 2,A, 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 α and β, which are encoded at two independent gene loci, as endogenous MBP ligands, as shown in Fig. 2,B. The peptides indicated in red within the complete meprin α and β precursor sequences were found in the fragments analyzed by mass spectrometry (Fig. 2,B). 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 intestinal brush border membranes, in leukocytes, and in certain epithelial cancer cells (39, 40). The meprin subunits, α and β, 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 α and β subunits are 42% identical at the amino acid level and share the same domain structure, except that meprin α contains an inserted domain that is not present in meprin β (41). Mouse meprin α and β contain 10 and 8 potential N-linked glycosylation sites, respectively (30). In vivo, meprin α is secreted as a homo-oligomer and is also found as a hetero-oligomer in the plasma membrane in association with meprin β, a type I integral membrane protein; thus, any meprin oligomer containing the β subunit is localized to the cell membrane (41). The localization of MBP ligands shown in Fig. 1 corresponds to previous reports, indicating that the β homo-oligomer of meprin B and the α and β hetero-oligomer of meprin A are localized to the apical brush border of the renal and intestinal proximal tubule epithelium.

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 (α-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 (α-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 α secreted from or meprin β localized in brush border membranes of the kidney and intestine.

FIGURE 3.

The carbohydrates of meprins are involved in the MBP-meprin interaction. A, The digestion of purified meprin A (α and β) with Endo H and Endo F. The α and β 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 (α and β) 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.

FIGURE 3.

The carbohydrates of meprins are involved in the MBP-meprin interaction. A, The digestion of purified meprin A (α and β) with Endo H and Endo F. The α and β 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 (α and β) 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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Both meprin α and β 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 MBP-meprin 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 μg of MBP (Fig. 4,C), respectively, and there was almost 50% decrease for PTH in the presence of 1.1 μg of MBP (Fig. 4 D).

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 μg 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 μl 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).

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 μg 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 μl 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).

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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. 5,B). 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. 5,A. 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 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.

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 μl after the meprins had been preincubated with or without 1.1 μg 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.

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 μl after the meprins had been preincubated with or without 1.1 μg 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.

Close modal

To further confirm that the inhibition of metalloproteases meprin α and β by MBP is carbohydrate dependent, 20 mM mannose was added to the reaction mixture. As shown in lanes 4 in Figs. 4,B and 5, A and B, and bars 4 in Figs. 4,D 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 ECM-degrading 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.

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 α and β, 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 matrix-degrading 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 α-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 α in colorectal carcinoma cells leads to aberrant accumulation of meprin α 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 α-positive carcinomas, the hydrolyzing activity was 2.9-fold higher than in the corresponding normal colon mucosa, whereas in meprin α-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 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 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 α and β are expressed in leukocytes of mouse mesenteric lymph nodes, and meprin α, but not β, decreased during intestinal inflammation. In contrast, meprin β, but not α, is detected in cortical and medullary macrophages of lymph nodes. Deletion of the meprin β 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, 46, 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 (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 β homo-oligomeric form and meprin A of the α and β 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 N-linked 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 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.

We thank Mitsubishi Pharm for purification of the human serum MBP, and Tomoko Honda for the secretarial assistance.

The authors have no financial conflict of interest.

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.

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.

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