THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 281, NO. 11, pp. 6955–6963, March 17, 2006 © 2006 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
Interaction of Mannan Binding Lectin with ␣2 Macroglobulin via Exposed Oligomannose Glycans A CONSERVED FEATURE OF THE THIOL ESTER PROTEIN FAMILY? *□ S
Received for publication, October 20, 2005, and in revised form, January 5, 2006 Published, JBC Papers in Press, January 5, 2006, DOI 10.1074/jbc.M511432200
James N. Arnold‡1, Russell Wallis‡§2, Antony C. Willis‡, David J. Harvey¶, Louise Royle¶, Raymond A. Dwek¶, Pauline M. Rudd¶, and Robert B. Sim‡3 From the ‡Medical Research Council Immunochemistry Unit and ¶Oxford Glycobiology Institute, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU and the §Department of Infection, Immunity and Inflammation, University of Leicester, Leicester LE1 9HH, England The serum collectin mannan-binding lectin (MBL) binds to oligomannose and GlcNAc-terminating glycans present on microorganisms. Using a commercial affinity chromatography resin containing immobilized MBL we screened human and mouse serum for endogenous MBL-binding targets. We isolated the serum protease inhibitor ␣2 macroglobulin (␣2M), a heavily glycosylated thiol ester protein (TEP) composed of four identical 180-kDa subunits, each of which has eight N-linked glycosylation sites. ␣2M has previously been reported to interact with MBL; however, the interaction was not characterized. We investigated the mechanism of formation of complexes between ␣2M and MBL and concluded that they form by the direct binding of oligomannose glycans Man5–7 occupying Asn846 on ␣2M to the lectin domains (carbohydrate recognition domains) of MBL. The oligomannose glycans are accessible for lectin binding on both active ␣2M (thiol ester intact) and proteasecleaved ␣2M (thiol ester cleaved). We demonstrate that MBL is able to interact with ␣2M in the fluid phase, but the interaction does not inhibit the binding of MBL to mannan-coated surfaces. In addition to ␣2M, two other members of the TEP family, C3 and C4, which also contain oligomannose glycans, were captured from human serum using the MBL resin. MBL binding may be a conserved feature of the TEPs, dating from their ancestral origins. We suggest that the inhibition of proteases on the surface of microorganisms by an ancestral ␣2M-like TEP may generate “arrays” of oligomannose glycans to which MBL or other lectins can bind. Binding would lead to opsonization or activation of enzyme systems such as complement.
The protease inhibitor ␣2 macroglobulin (␣2M)4 is a glycoprotein of 720 kDa, composed of four identical 180-kDa subunits, covalently
* This work was supported in part by the Glycobiology Institute endowment. The MALDI mass spectrometer for glycan analysis was purchased with a grant from the Biotechnology and Biological Sciences Research Council. 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. □ S The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S3. 1 Supported by a postgraduate studentship from the Medical Research Council. 2 Supported by Grant 077400 from the Wellcome Trust. 3 To whom correspondence should be addressed. Tel.: 44-1865-275-352; Fax: 44-1865275-729; E-mail:
[email protected]. 4 The abbreviations used are: ␣2M, ␣2 macroglobulin; MBL, mannan-binding lectin; TEP, thiol ester protein; CRD, carbohydrate recognition domain; MASP, MBL-associated serine protease; BSA, bovine serum albumin; 2AB, 2-aminobenzamide; NP-HPLC, normal phase-high-performance liquid chromatography; GU, glucose unit(s); ABS, A. ureafaciens sialidase; AMF, almond meal ␣-fucosidase; BTG, bovine testis -galactosidase; JBM, jack bean ␣-mannosidase; SPH, S. pneumonia -hexosaminidase; GuH, -Nacetylglucosaminidase; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight.
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linked in pairs by disulphide bridges. Each subunit has eight N-linked glycosylation sites located at Asn-32, Asn-47, Asn-224, Asn-373, Asn387, Asn-846, Asn-968, and Asn-1401 (1) (see Fig. 1), a total of 32 N-linked glycosylation sites per ␣2M tetramer. ␣2M is an evolutionarily ancient protease inhibitor, which circulates in human plasma at 1–2 mg/ml and is found in a wide range of species from arthropods to mammals (2). There are two regions of major functional importance, a “bait region,” which contains multiple cleavage sites for many different proteases (Fig. 1), and a second region, a reactive thiol ester (Fig. 1) also found in the complement components C3 and C4. ␣2M inhibits active proteases via a “trap mechanism” (3, 4), where cleavage of the “bait” region by the active protease triggers a conformational change in ␣2M (5) forming a cage-like structure around the protease. During the refolding, the protease or other “bystander” molecules (6) may become covalently bound via the thiol ester (7). The covalent binding, however, is not essential for protease inhibition (8). The entrapped protease is inhibited in its action on high molecular weight protein substrates, which can no longer access the cage. In contrast low molecular weight (synthetic) substrates (esters and amides) can enter the cage and still be hydrolyzed by the trapped protease (3, 5). The thiol ester can also be cleaved in ␣2M, as in C4 and C3, by spontaneous hydrolysis or can react with low molecular weight nucleophiles such as NH3. This reaction converts ␣2M to the “dead” (thiol ester cleaved) form, which, although it has not been cleaved by protease, undergoes a conformational change (9). MBL, also known as mannan/mannose-binding protein is a member of the collectin family of proteins (10), synthesized in the liver and secreted into the bloodstream. Levels of MBL in human serum vary greatly between individuals (11), from below 50 ng/ml to above 10 g/ml. MBL has a structure and function similar to that of the complement protein C1q and consists of trimers of identical polypeptide chains, each containing a carbohydrate recognition domain (CRD) and a collagen-like region. These trimers form higher order oligomers from dimers to hexamers, resembling a “bunch of tulips” in structure. MBL circulates bound to pro-enzymic MBL-associated serine proteases (MASPs) and MAp19. Three MASPs have been characterized to date: MASP-1, MASP-2 (12, 13), and MASP-3 (14). MAp19 is an alternatively spliced variant of MASP-2, which does not contain the serine protease domain (15). Binding of MBL to a target via its lectin domains leads to autocatalytic activation of the pro-MASPs. MBL can trigger the lectin pathway of complement activation (15, 16) via MASP-2, which cleaves the complement components C4 and C2, resulting in the formation of a C3 convertase C4b2a. The biological roles of MASP-1 and MASP-3 have not been firmly established, although MASP-1 has thrombin-like
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FIGURE 1. The subunit of human ␣2M. The diagram shows the full-length 180-kDa subunit of ␣2M with eight N-linked glycosylation sites at Asn32, Asn-47, Asn-224, Asn-373, Asn-387, Asn-846, Asn-968, and Asn-1401. There are two regions of functional importance, the bait region and the thiol ester. The diagram also shows the fragments and their approximate molecular weights produced upon autolytic cleavage and proteolytic cleavage.
activities, cleaving fibrinogen and activating Factor XIII, possibly implicating MASP-1 in localized coagulation (15, 17). Protease inhibition studies have shown that ␣2M inhibits activated MASP-1 with fast kinetics and reacts slowly with activated MASP-2 (17, 18). ␣2M does not inhibit C1r and C1s of the complement C1 complex: the serpin C1 inhibitor is the prime inhibitor of C1r and C1s and of MASP-2 (19, 20). MBL binds calcium dependently to sugars that have hydroxyl groups on the carbon-3 and carbon-4 orientated in the equatorial plane of the pyranose ring (21). This gives MBL affinity for mannose, fucose, and N-acetyl glucosamine (GlcNAc) (11). These sugar residues tend to be found on the surfaces of microorganisms, including bacteria, viruses, and fungi (22). MBL binds to sugar residues via the CRDs (lectin) heads. The affinity of a single CRD for carbohydrate is very weak (10⫺3 M) (23), but multiple CRD binding generates a much greater avidity. For example, interaction of three CRDs in one subunit with mannan has a Kd of 2–3 ⫻ 10⫺8 M (24). There are some human plasma proteins with which MBL does interact via oligomannose or GlcNAc terminating glycans such as has been described for specific glycoforms of IgG (IgG-G0) and IgM (25, 26). There are reports describing ␣2M co-purifying with MBL (27) and electron microscopy studies showing a complex between porcine MBL and ␣2M (28). ␣2M䡠MBL complexes have been detected in plasma (29). However, the interaction has not been characterized, and it has not been determined whether ␣2M is interacting with MBL or with the MASPs directly. We have characterized one major interaction between MBL and ␣2M as a lectin interaction. Analysis of the glycosylation of ␣2M from pooled human serum identified oligomannose glycans, occupying Asn-846. This study shows that the oligomannose glycans are accessible to MBL in both thiol ester-cleaved and thiol ester intact ␣2M conformations. The oligomannose glycans mediate Ca2⫹-dependent attachment of MBL to ␣2M.
EXPERIMENTAL PROCEDURES ␣2M Purification—The method was based on that of Salvesen and Enghild (30). Chelating Sepharose 6B (Amersham Biosciences, 5 ml) was washed with 50 ml of 0.1 M sodium phosphate buffer, pH 5.0, then with 50 ml water, and then with 5 ml of ZnSO4 (0.3 g/100 ml), and finally with 5 ml of water. Citrated human plasma (20 ml) pooled from 20 or more donors (HDS Supplies, High Wycombe, UK) was dialyzed twice against 1.2 liters of water at 4 °C. The plasma was spun at 3,000 rpm for 10 min, and the supernatant was applied to the Zn2⫹-loaded Sepharose chelate column. The column was washed with 0.1 M sodium phosphate, pH 7.0, until the A280 was below 0.01, and then washed with 0.1 M sodium phosphate, pH 6.0. The bound ␣2M was eluted with 0.1 M sodium phosphate, pH 5.0. The eluted material was then raised to pH 7.0 with 1 M sodium phosphate, pH 7.0, and 1 mM Pefabloc-SC (Penta-
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pharm Ltd., Basle, Switzerland) was added to inhibit trace proteases. The major contaminants were albumin and IgG (identified by SDSPAGE and mass spectrometry analysis). These were removed by gel filtration. The ␣2M-containing fractions were pooled and run on a Superose 6 (HR 10/30) size-exclusion column (Amersham Biosciences, repetitive loading of 1 ml of 1 mg/ml), with the running buffer 0.1 M sodium phosphate, pH 7.0. Fractions containing ␣2M were pooled and judged by SDS-PAGE to be ⬎95% pure (see supplemental Figs. S1–S3). The preparation was dialyzed into 10 mM HEPES, 140 mM NaCl, pH 7.4. Nucleophile Treatment of ␣2M—Nucleophiles such as ammonia can induce conformational change in ␣2M by cleaving the thiol ester (31, 32). The purified ␣2M (thiol ester intact) (10 mM HEPES, 140 mM NaCl, pH 7.4) was mixed 1:1 (v/v) with 0.2 M NH4HCO3, pH 8.9, and incubated for 2 h at 37 °C, and the material was dialyzed back into 10 mM HEPES, 140 mM NaCl, pH 7.4. Thrombin Cleavage of ␣2M—Thrombin cleavage was carried out at a molar ratio of 1 ␣2M subunit to 2 molecules of human thrombin (Sigma-Aldrich, T6884), based on ␣2M subunit molecular mass of 180 kDa and a thrombin molecular mass of 37 kDa. The cleavage was carried out in 10 mM HEPES, 140 mM NaCl, pH 7.4, for 2 h at room temperature, and the cleaved ␣2M was analyzed by SDS-PAGE. Cleavage was incomplete, suggesting that the thrombin was only partially active (see supplemental material). Autolytic Cleavage of ␣2M—␣2M was identified as being “live” with an intact thiol ester by analyzing autolytic cleavage. Autolytic cleavage occurs on denaturation of ␣2M and requires the thiol ester to be intact before denaturation (33). Treatment of the sample with nucleophilic agents (e.g. dithiothreitol) before denaturation cleaves the thiol ester and prevents autolytic cleavage. To prevent autolytic cleavage protein samples were resuspended in 10 l of NuPAGETM sample-reducing agent (10⫻, Invitrogen, NP0009) and incubated at 37 °C for 5 min before 10 l of NuPAGE LDS sample buffer (Invitrogen, NP0008) was added, and the samples were incubated at 95 °C for 5 min. To enhance autolytic cleavage the protein samples were resuspended in 10 l of NuPAGE LDS sample buffer (Invitrogen, NP0008) and incubated at 95 °C for 5 min, before 10 l of NuPAGETM sample reducing agent (10⫻, Invitrogen, NP0009) was added, and the mixture was incubated at 95 °C for a further 5 min. Samples were then analyzed by SDS-PAGE. MBL Purification—The purification was done by the method of Tan et al. (34), modified by Arnold et al. (26). The concentration of MBL was calculated using a mannan capture MBL enzyme-linked immunosorbent assay (35). Capture of ␣2M from Human and Mouse Serum by an Affinity Resin Containing Immobilized MBL—MBL conjugated to a 4% beaded agarose support (5 ml), is sold as a reagent for the purification of mouse IgM (ImmunoPure威 IgM purification kit, Pierce). When loaded with human or mouse serum the MBL resin bound several proteins, in addition to
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MBL Interacts with ␣2M via Oligomannose Glycans IgM, in a calcium-dependent manner (26). The running procedure was adapted from the directions of the manufacturer (www.piercenet.com) using buffers supplied by the manufacturer. The affinity column (5 ml) was pre-washed with 5 ml of ImmunoPure威 MBP column preparation buffer (Tris, NaCl, sodium azide, EDTA, pH 7.4) at room temperature and then equilibrated with 20 ml of the ImmunoPure威 IgM binding buffer (Tris, 1 M NaCl, CaCl2, and sodium azide, pH 7.4) at 4 °C. The MBL resin was used to capture proteins from serum of human and mouse. Citrated human plasma (HDS Supplies, High Wycombe, UK) was made 20 mM CaCl2 and left overnight at 4 °C to clot, and then the clot was filtered. Pooled mouse serum was obtained from Harlan Sera-Lab (Loughborough, UK). Mouse or human serum was diluted 1:1 with ImmunoPure威 IgM binding buffer, and 0.5 ml was applied to the MBL resin and incubated at 4 °C for 1 h. The column was washed with ImmunoPure威 IgM binding buffer until the A280 was ⬍0.01. The bound protein was eluted using 15 ml of ImmunoPure威 IgM elution buffer (Tris, 1 M NaCl, EDTA, and sodium azide, pH 7.4). Eluted material was run on SDS-PAGE, and protein bands were analyzed by mass spectrometry. Characterization of Binding of ␣2M to an Affinity Resin Containing Immobilized MBL—Purified ␣2M (50 g) preparations were diluted 1:1 with ImmunoPure威 IgM binding buffer, applied to the MBL resin at 4 °C, and allowed to enter the resin completely. A further 500 l of ImmunoPure威 IgM binding buffer was added and allowed to enter the resin. The column was then incubated at 4 °C for 30 min then washed with 20 ml of ImmunoPure威 IgM binding buffer. Bound ␣2M was eluted at 4 °C with 20 ml of ImmunoPure威 IgM elution buffer (Tris, 1 M NaCl, sodium azide, and EDTA, pH 7.4). The 20-ml elution and flowthrough fractions were then split 50:50, and each half was incubated with 20 l of StrataClean resin (Stratagene, Zuidoost, The Netherlands). Resin-bound protein samples were then prepared for SDS-PAGE analysis and analysis of autolytic cleavage as described above. Binding of MBL to Immobilized ␣2M—MBL binding was studied as described by Arnold et al. (35). Enzyme-linked immunosorbent assay plates (Nunc-Maxisorp) wells were coated with 100 l of 10 g/ml ␣2M (thiol ester intact) or nucleophile treated ␣2M (thiol ester cleaved). Mannan was used as a positive binding control, and BSA was used as a negative binding control, both at 10 g/ml. All were diluted in 0.1 M NaHCO3, pH 7, incubated in wells for 1 h at room temperature, then the wells blocked with 400 l of Dulbecco’s phosphate-buffered saline (8.2 mM Na2HPO4, 1.5 mM KH2PO4, 139 mM NaCl, 3 mM KCl, pH 7.4) 0.1% Tween 20 for 2 h at room temperature, washed three times with 200 l of wash buffer (10 mM HEPES, 1 M NaCl, 5 mM CaCl2, 0.1% Tween 20, pH 7.4), and incubated in triplicate for 1 h at 37 °C with 50 ng/well purified MBL diluted in 10 mM HEPES, 1 M NaCl, 5 mM CaCl2, pH 7.4. Other wells were incubated with MBL diluted with 10 mM HEPES, 1 M NaCl, 5 mM EDTA, pH 7.4 as controls for non-calcium-dependent binding. The wells were washed three times with the wash buffer and incubated for 1 h at room temperature with 100 l of 1/700 (v/v) antiMBL polyclonal antiserum (35) in wash buffer. The wells were washed and incubated 1 h, room temperature with 100 l of a 1/2000 (v/v) dilution of monoclonal anti-rabbit IgG (␥-chain specific, Clone RG-96 alkaline phosphatase conjugate, Sigma-Aldrich, A-2556) in wash buffer and washed again, 100 l of an AmpliQ, DakoCytomation (Cambridgeshire, UK) amplification kit reagent mixture was added, and the optical density was read at 492 nm after 30 min. Analytical Ultracentrifugation of MBL and ␣2M—Experiments were carried out in a Beckman Optima XL-A analytical ultracentrifuge equipped with absorbance optics using an An60Ti rotor as previously described (36). The MBL and ␣2M (thiol ester intact) were dialyzed overnight against 50 mM HEPES, 140 mM NaCl, 10 mM CaCl2, pH 7.4, at
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FIGURE 2. SDS-PAGE analysis of human and mouse serum elution from the MBLconjugated resin. Human or mouse serum (0.5 ml) was applied to the MBL-conjugated resin (Pierce) as described under “Experimental Procedures.” The column was eluted with ImmunoPure姞 IgM elution buffer (contains EDTA), and the eluted proteins were analyzed by SDS-PAGE. The ladder was MultiMark (Invitrogen). Bands were excised, and the presence of ␣2M from both the human and mouse serum elution was confirmed by MALDI-TOF mass spectrometry. Mouse ␣2M subunits were as previously identified by van Leuven et al. (2). As a control human or mouse serum (0.5 ml) was applied to a 5-ml column of Ultrogel A4 (4% beaded agarose) (Sigma-Aldrich, U0507) and eluted in the same way as above. No proteins bound to the blank resin.
4 °C. Samples (110 l of total volume) were placed into sample cells and consisted of either 8 g of MBL or 14 g of ␣2M or both, diluted in dialysis buffer. The reference cells were loaded with 125 l of dialysis buffer. Experiments were performed at 5,000 rpm at 20 °C. Equilibrium data were obtained at 280 nm in step scan mode using a separation of 0.001 cm and an average of 5 scans. Readings were taken at 6-h intervals until no difference could be detected between consecutive scans (equilibrium reached). At equilibrium, 3 scans were taken at 6-h intervals, which were averaged. Assay of MBL Binding to Mannan in the Presence of Excess ␣2M— MBL binding and detection was carried out by the enzyme-linked immunosorbent assay described above, using the wash/dilution buffer 10 mM HEPES, 140 mM NaCl, 10 mM CaCl2, pH 7.4. It was shown that 100 ng/well MBL did not saturate the mannan-coated well. Binding of MBL to the mannan was complete after 1 h but was detectable at 2-min incubation (data not shown). Serial dilutions of ␣2M and of BSA (as a negative control) from 2000-fold molar excess over MBL were incubated with 1 g/ml MBL in 10 mM HEPES, 140 mM NaCl, 10 mM CaCl2, pH 7.4 for 1 h at 37 °C (molarities calculated from 450-kDa MBL, 720kDa ␣2M, and 66-kDa BSA). Then 100 l of the mixtures was incubated on the mannan-coated wells for 2 min or 1 h at room temperature. ␣2M used was the thiol ester intact form. MBL, which had bound to the mannan, was detected as described above, developed using 100 l of 1 mg/ml p-nitrophenyl phosphate in 0.2 M Tris buffer (Sigma-Aldrich, N-2770), and the absorbance values at 405 nm were recorded after 10 min. Release of N-Linked Glycans for Analysis—Human ␣2M (25 g in 10 mM HEPES, 140 mM NaCl, 5 mM EDTA, pH 7.4) was run on SDS-PAGE according to Ku¨ster et al. (37), followed by in-gel N-linked glycan release using peptide N-glycanase F (EC 3.5.1.52, 1000 units/ml) as described by Radcliffe et al. (38).
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FIGURE 3. NP-HPLC exoglycosidase digestion profile of glycan pool from ␣2M. 2AB-labeled N-linked glycans were digested by exoglycosidases and analyzed by NP-HPLC. Structure abbreviations: all N-glycans have two core N-acetylglucosamines (GlcNAcs): Fc, core fucose linked ␣1– 6 to the inner GlcNAc: Man, number of mannose residues on core GlcNAcs: A, number of antennae on the trimannosyl core; A2, biantennary; B, bisecting GlcNAc-linked 1– 4 to inner mannose; G, number of galactose residues on antennae; S, number of sialic acids on antennae. The diagrammatic structures are represented by: black square, GlcNAc; white diamond, galactose; white diamond with a dot inside, fucose; black star, sialic acid; solid line,  linkage; dotted line, ␣ linkage; slanted line, 1– 6 linkage; solid line, 1– 4 linkage; and vertical line, 1–2 linkage. Percentage areas and GU values are shown in Table 1. The exoglycosidases used were: ABS (removes sialic acid), BTG (removes galactose), BKF (removes core fucose), and GuH (removes non-reducing terminal GlcNAc).
2AB Labeling—Released glycans were labeled by reductive amination with the fluorophore 2AB according to Bigge et al. (39), using a Ludger TagTM 2AB glycan labeling kit (Ludger Ltd., Oxford, UK). Normal Phase-HPLC—Labeled glycans were separated on NP-HPLC as described by Guile et al. (40). Glycan profiles from NP-HPLC were calibrated against a dextran ladder prepared from hydrolyzed and 2ABlabeled glucose oligomers (41). Glycans were assigned glucose units (GU) values by fitting a fifth order polynomial distribution curve to the dextran ladder (glucoses 1–15). Glycan structure/composition was assigned using GU values and confirmed using exoglycosidase digestions. Exoglycosidase Digestions—Exoglycosidases were used to confirm the structures of glycans present in the preparations, in conjunction with NP-HPLC (38). Enzyme digests were carried out using 50 mM sodium acetate buffer, pH 5.5, for 16 h at 37 °C at concentrations listed below. The following enzymes were supplied by Glyko Inc. (Upper Heyford, UK); Arthobacter ureafaciens sialidase (ABS, EC 3.2.1.18) 1–2 units/ml; almond meal ␣-fucosidase (AMF, EC 3.2.1.51), 3 milliunits/ml; bovine testis -galactosidase (BTG, EC 3.2.1.23), 1 unit/ml; jack bean ␣-mannosidase (JBM, EC 3.2.1.24), 100 milliunits/ml; Streptococcus pneumonia -hexosaminidase (SPH, EC 3.2.1.30), 120 units/ml. Europa Bioproducts (Mantes la Jolie, France) was the source of -N-
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acetylglucosaminidase (GuH, EC 3.2.1.30) (cloned from Streptococcus pneumonia, expressed in Escherichia coli), 40 units/ml. Glycan Mass Spectrometry—Non-2AB-labeled N-linked glycans were analyzed by MALDI-TOF mass spectrometry (Waters-Micromass TofSpec 2E) in reflectron mode with 2,5-dihydroxybenzoic acid as the matrix (41). Protein Mass Spectrometry—Bands were excised from SDS-PAGE gels and cut into 1- ⫻ 1-mm pieces, then destained by washing twice for 20 min at room temperature with 100 l of 50 mM NH4CO3 in 50% acetonitrile. The gel pieces were then soaked in 100 l of 70% acetonitrile for a further 20 min, the supernatant was removed, and the gel pieces were dried in a Gyro-Vac (Howe, Oxon, UK) for 30 min. The gel pieces were rehydrated with 10 l/gel piece with 20 ng/l trypsin (Sigma-Aldrich, T-6567) in 20 mM NH4CO3 and incubated for 16 h at 37 °C. The peptides were removed from the gel with 50 l of 50%(v/v) acetonitrile 0.1%(v/v) trifluoroacetic acid in water shaken for 10 min, the supernatant was extracted, and the step was repeated for a further 10 min. The supernatants were combined and dried in the SpeedVac. Mass spectrometry of the tryptic digested peptides was performed using the Ettan MALDI-ToF Pro according to the manufacturer’s instructions (Amersham Biosciences).
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MBL Interacts with ␣2M via Oligomannose Glycans TABLE 1 Normal pooled serum ␣2M N-linked glycans DS, desialylated mass detected.
1
Structures are explained, and the major structures are shown in Fig. 3. Molecular mass of unlabeled glycans, detected as [M ⫹ Na]⫹ by MALDI-TOF-MS. Sialylated structures are detected as sodium salts. All masses were within 0.2 mass units of calculated values. Sialylated structures are prone to losing sialic acid during MALDI. 3 Compositions deduced from mass values. * Not detected by NP-HPLC as is not present in native glycan pool and is only generated through exoglycosidase digests. # Free acid detected. 2
RESULTS Capture of ␣2M from Human and Mouse Serum Using Immobilized MBL—Serum was passed through an MBL-conjugated resin (Pierce) to screen for endogenous MBL-binding targets. The eluted proteins were analyzed by SDS-PAGE and mass spectrometry (Fig. 2). The MBL resin bound ␣2M from both human and mouse serum (Fig. 2). The mouse ␣2M on reducing SDS-PAGE gave a fragmentation pattern of 165- and 35-kDa fragments and intact 185-kDa subunits as previously described (2). Interestingly, non-activated serum C3 and C4 ␣-chain (113 and 95 kDa, respectively) and -chain (75 kDa), and the ␥-chain (30 kDa) of C4 were identified from human serum (Fig. 2). Characterization of the Interaction between MBL and ␣2M—Using the MBL-conjugated resin (Pierce) the interaction with ␣2M was further characterized. Each run used 50 g of purified ␣2M, and the protein eluted with EDTA was analyzed by SDS-PAGE, with and without
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autolytic cleavage (see supplemental material). The autolytic cleavage of the material, which bound the MBL resin, indicated that the thiol ester was intact, demonstrating that MBL was not solely binding thiol ester-cleaved ␣2M. All the forms of ␣2M tested (thiol ester intact, thiol ester-cleaved, and thrombin-cleaved) were bound by MBL. This assay characterized the interaction as a C-type lectin interaction and not a protein-protein interaction, because the binding was calcium-dependent (bound protein eluted with EDTA) and was also inhibitable by the addition of 10 mM mannose to the running buffer. The binding was not affected by the addition of 10 mM galactose, with which MBL does not interact. Identification of the N-Linked Glycans on ␣2M to which MBL Could Bind—The structures of 2AB-labeled glycans from ␣2M (Fig. 3 and Table 1) were assigned from GU values, shifts with enzyme digestion arrays, and MALDI-TOF mass spectrometry. Oligomannose structures Man5 through Man7, which are potential ligands of MBL, were identified. Man5 was the
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FIGURE 4. NP-HPLC profiles from ␣2M fragments. 2AB-labeled N-linked glycans from the proteolytically cleaved and autolytically cleaved fragments (Fig. 1) were analyzed by NP-HPLC. Shown (on the left) are the ␣2M peptides (a– d) analyzed and the N-linked glycosylation sites positioned on these fragments. Shown (on the right) are the NP-HPLC N-linked glycan profiles from the peptide fragments marking the oligomannose glycan peaks identified. The glycan nomenclature and structures are explained in the legend of Fig. 3. The glycan profile of fragment d shows that Asn-968 and Asn-1401 are solely occupied by complex glycans. The glycan profile from fragment c, which contains Asn-846 in addition to these two sites, includes peaks corresponding to Man5–7 and the complex glycan FcA1G1S1, indicating that these structures occupy Asn-846. It should be noted that the Man7 peak at GU 7.96 also contains A2G2S1, which co-elutes with Man7 shown in Fig. 3 and Table 1, which is also marked.
FIGURE 5. Glycan processing at Asn-846. The glycans identified to occupy the Asn-846 N-linked glycosylation site are shown. The x-axis shows the glycan processing pathway. The cellular regions to which processing is localized are labeled. The glycan processing at Asn-846 is abnormally distributed, with two predominant differently processed structures, a Man5 and an FcA1G1S1, which occupy Asn-846 at 42 and 28%, respectively (Man6 –7 and A1G1 comprise the remaining 30%). Glycan processing appears to have partially ceased through the medial Golgi. However, the tetramer of ␣2M has already fully formed by the end of the endoplasmic reticulum.
most abundant oligomannose structure and accounted for 5% of the total glycan pool. In total, oligomannose glycans comprised ⬃8% of the total glycan pool. The remaining 92% of glycan structures terminated in galac-
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tose or sialic acid. The most abundant complex glycan was FcA2G2S2 (see legend to Fig. 3 for an explanation of this nomenclature), which accounted for 27% of the total glycan pool.
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MBL Interacts with ␣2M via Oligomannose Glycans Identification of the N-Linked Glycosylation Site Occupied by Oligomannose Glycans—The N-linked glycosylation site occupied by oligomannose glycans was identified from protein fragments generated from autolytic-cleavage and thrombin-cleavage of ␣2M. Each fragment contained a different selection of the N-linked glycosylation sites (Fig. 4). Glycosylated fragments were separated using SDS-PAGE and excised for in-gel N-linked glycan release. The ␣2M protein fragment containing Asn-968 and Asn-1401 was solely occupied by complex glycans terminating in galactose and sialic acid (Fig. 4d). However, the ␣2M protein fragment containing Asn-968 and Asn-1401 as well as Asn-846 was occupied by oligomannose and complex glycans (Fig. 4c). The presence of oligomannose glycans was confirmed using JBM (mannosidase) digests (data not shown). This demonstrated that the oligomannose glycans occupied Asn-846. Comparison of fragments c and d (Fig. 4) indicated that Asn-846 was also occupied by FcA1G1S1. Differences between fragments c and d (Fig. 4) and their JBM digests show that Asn-846 is occupied by 70% Man5–7 and 30% FcA1G1S1. Extrapolating back to the whole glycan pool (Fig. 4a) with all N-linkage sites, we calculated that 12% of the total glycan structures are accounted for by these two glycans shown, which is compatible with the occupation of
FIGURE 6. Equilibrium ultracentrifugation of MBL and ␣2M. MBL and ␣2M can interact in the fluid phase as occurs in serum. MBL and ␣2M were analyzed by equilibrium sedimentation at an approximate molar ratio of 1:1 in 50 mM HEPES, 140 mM NaCl, 10 mM CaCl2, pH 7.4, at 20 °C. a, equilibrium distributions at 5000 rpm of the A280 of the observed MBL䡠␣2M mix compared with the sum (dotted line) of the separate MBL and ␣2M distributions at identical protein concentrations also shown on the graph. b, the distributions of MBL䡠␣2M mix subtracted from the sum of the distributions of individual components. The non-random distribution of points about the zero value is consistent with a weak interaction between MBL and ␣2M.
one of the eight N-linked glycosylation sites. That this Asn-846 site is occupied by both oligomannose glycans and the processed glycans suggests that there may be a non-uniform accessibility to glycan processing among the monomers in the ␣2M tetramer (Fig. 5). Because 70% of the glycans at Asn-846 are oligomannose, it suggests that on average each ␣2M tetramer contains three oligomannose glycans all at Asn-846. The fourth ␣2M monomer contains FcA1G1S1 at Asn-846. Fluid Phase Interaction between MBL and ␣2M—Analytical ultracentrifugation was used to determine whether MBL interacts with ␣2M in the fluid phase, where neither protein was immobilized. MBL and ␣2M were analyzed separately and as a mixture in a molar ratio of 1:1. Fig. 6a shows the equilibrium distribution as a function of absorbance at 280 nm of MBL and ␣2M separately and as a mixture of MBL plus ␣2M. In the absence of an interaction, the distribution of the mixture would equal the sum of the distributions of the individual components (Fig. 6a). More protein was distributed toward the bottom of the cell, indicating the presence of a MBL䡠␣2M complex in the mixture. The interaction between MBL and ␣2M in the fluid phase was relatively weak (Fig. 6b). It is worth considering that the serum concentration of ␣2M is ⬃10-fold higher than the concentration used in this experiment and that the ␣2M to MBL molar ratio is 400 – 800:1 (assuming 1000 –2000 g/ml ␣2M and 1 g/ml MBL), so a high proportion of MBL molecules may associate with ␣2M under physiological conditions. Binding of MBL to Mannan in the Presence of Molar Excess of ␣2M— The lectin interaction between MBL and the oligomannose glycans of ␣2M might modulate the binding of MBL to a mannan surface. To test this, MBL was incubated with mannan-coated microtiter plate wells for two time periods, 2 min and 1 h. MBL association with mannan was detectable but incomplete at 2 min and complete (at equilibrium) by 1 h (these times were pre-established by incubations from 1 min to 2 h, data not shown). ␣2M up to a 2000-fold molar excess over MBL did not affect the amount of MBL binding to a surface coated with mannan, again suggesting that the fluid-phase interaction between MBL and ␣2M is relatively weak compared with MBL binding to surfaces coated in arrays of “high” mannose (mannan), which can engage more CRDs of MBL to generate a high avidity interaction. MBL Binding to a Surface of Immobilized ␣2M—The binding of MBL to ␣2M (thiol ester intact) and nucleophile inactivated ␣2M (thiol ester cleaved) immobilized on microtiter plates was assessed to demonstrate that MBL can bind to immobilized arrays of ␣2M (Fig. 7). Mannan- and BSA-coated wells were used as positive and negative controls, respectively. Assays were carried out in triplicate with additional EDTA-negative controls. MBL does bind ␣2M. ␣2M (thiol ester intact) bound
FIGURE 7. MBL binding to immobilized ␣2M (thiol ester intact and cleaved). Microtiter plate wells were coated with 1 g/well thiol ester intact ␣2M or of nucleophile-treated (thiol ester cleaved) ␣2M. Mannan and BSA were coated as positive and negative controls for MBL binding. MBL was incubated at 50 ng/well for 1 h and then sequentially with anti-MBL antiserum and the monoclonal anti-rabbit IgG (␥-chain specific) alkaline phosphatase conjugate, as described by Arnold et al. (35). The assay was developed with the AmpliQ amplification kit. The bars show the mean MBL binding ⫾ 1 S.D. unit. EDTA was used in negative controls. EDTA readings were low and were subtracted from the results. The figure shows the lower end of the scale on the y-axis to clearly show MBL binding ␣2M. Much more MBL bound to the mannan-coated well (0.608 A492), at the top end of the scale.
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MBL Interacts with ␣2M via Oligomannose Glycans more MBL than the ␣2M (thiol ester cleaved). The oligomannose glycans occupying Asn-846 may become positioned at a less accessible spacing for MBL binding after the conformational change. However, MBL is able to bind to the “arrays” of oligomannose glycans presented by a surface of immobilized ␣2M (Fig. 7).
DISCUSSION Lectin Interaction between MBL and ␣2M—Our results show that MBL and ␣2M (both thiol ester intact and thiol ester-cleaved forms) interact directly by the binding of MBL lectin domains (CRDs) to oligomannose glycans on MBL. The binding has been demonstrated with immobilized MBL and fluid-phase ␣2M and vice versa. The binding is inhibited by mannose and EDTA, consistent with a C-type lectin interaction. We also demonstrated an interaction between MBL and ␣2M when both are in the fluid phase, as occurs in plasma (Fig. 6). There are multiple instances of experimental data presented in this and previous studies to suggest a predominantly lectin interaction between MBL and ␣2M. Terai et al. (27) showed a calcium-dependent MBL䡠␣2M complex in a sandwich enzyme-linked immunosorbent assay, capturing MBL from serum on a microtiter plate with an antiMBL antibody and detecting ␣2M using an anti-␣2M antibody. Terai et al. (27) also selectively eluted, using 0.2 M mannose, MBL and MASP from an anti-␣2M affinity column loaded with a preparation rich in MBL䡠MASP and ␣2M. MBL is composed of higher oligomers of a subunit consisting of trimers of CRDs. The carbohydrate binding domains of the CRDs are spaced about 45 to 53 Å apart in an MBL trimer subunit (42, 43). The dimensions of ␣2M (thiol ester intact) are 200 ⫻ 150 ⫻ 135 Å and of ␣2M (thiol ester cleaved) are 200 ⫻ 155 ⫻ 140Å (9). Considering the general structure and position of the ␣2M subunits in the tetramer (9, 44, 45) it seems unlikely that three Asn-846 oligomannose glycans could be positioned to allow a single MBL trimer arm to bind them all. It is more likely that a maximum of two Asn-846 oligomannose glycans are positioned within ⬃50 Å of each other and will bind two CRDs within one trimer. This would suggest that the Kd of the fluid-phase interaction is ⬍2–3 ⫻ 10⫺8 M (24). The lectin interaction between an MBL and fluid phase ␣2M is relatively weak and probably short-lived due to the low number of ligands (Fig. 6). This explains why large molar excesses of fluid phase ␣2M, such as is in the serum, cannot inhibit MBL binding to mannan-coated surfaces, which engage multiple CRDs simultaneously. When several MBL CRD trimer subunits bind (each with a Kd of 2.3 ⫻ 10⫺8 M (24)) a much higher avidity interaction is generated. MBL therefore does not opsonize fluid-phase ␣2M. The lectin binding of MBL to one fluid-phase ␣2M tetramer would also not trigger the lectin pathway of complement activation. On steric grounds no more than one MBL trimer unit of a single MBL molecule could interact with a single ␣2M. This would not activate the pro-MASPs as the MBL CRD trimer arms of MBL would not become clustered. However, when ␣2M is immobilized on a surface, MBL can bind to the presented “arrays” of oligomannose glycans (Fig. 7). Oligomannose Glycans Occupying Asn-846 and ␣2M Glycosylation— The presence of oligomannose glycans Man5–7 suggests that the glycans occupying Asn-846 are inaccessible for glycan processing by the endoplasmic reticulum and Golgi apparatus, because of the local three-dimensional structure of the protein. However, the terminal glycans are accessible for MBL binding in the secreted protein (Figs. 2, 6, and 7). The Asn-846 N-linked glycosylation site is situated between the “bait region” and the thiol ester in the primary amino acid sequence. In each ␣2M tetramer, three Asn-846 N-linked glycosylation sites remain oligomannose, and one Asn-846 is partially processed to a hybrid complex glycan
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FcA1G1S1 (Fig. 4) (one biantennary arm has been fully processed, and the other has not) and, therefore, must be partially accessible to glycanprocessing enzymes. In the endoplasmic reticulum ␣2M subunits form the tetramer through non-covalent binding and disulfide bridging. At this stage of glycoprotein biosynthesis all glycans have been fully processed in this compartment to Man7 structures, and they are subsequently exported to the Golgi for further processing (Fig. 5). Three of the Asn-846 glycans become inaccessible in the Golgi to further processing, and one Asn-846 glycan is partially processed (Fig. 5). Electron microscopy studies have shown that the ␣2M tetramer is asymmetrical in structure (9, 44), and thus the accessibility to the Asn-846 glycans may be different for each subunit. Complement component C5 is homologous to ␣2M but does not contain a thiol ester group and is not occupied by oligomannose glycans (46). ␣2M may undergo a conformational change, due to thiol ester formation during transit through the Golgi. This could result in the Asn-846 oligomannose glycans becoming temporarily inaccessible to processing (Fig. 5). Once the thiol ester group has formed in the mature protein the Asn-846 oligomannose glycans then become accessible for lectin binding. This mechanism may explain the presence of oligomannose glycans on C3 and C4 (46), which also contain a thiol ester. Thiol Ester Proteins and MBL—C3 and C4 are homologues of ␣2M. C4 has four N-linked glycosylation sites: Asn-207 on the -chain, Asn917, Asn-1309, and Asn-1372 on the ␣-chain. C4 is occupied by both complex and oligomannose glycans. The predominant oligomannose glycan is Man9 (46). However, the one or more N-linked glycosylation sites occupied by oligomannose glycans have not been identified. C3 has two N-linked glycosylation sites, Asn-63 on the -chain and Asn-917 on the ␣-chain, and both are occupied by unprocessed oligomannose glycans, predominantly Man6 at Asn-63 and Man8 –9 at Asn-917 (46, 47). Both unactivated C3 and C4 bound the MBL resin from human serum (Fig. 2). C3 and C4 eluted in EDTA from the MBL resin, indicative of a lectin interaction. In contrast to our results Solis et al. (48), showed MBL and conglutinin did not bind to C3 (thiol ester intact). When C3 was activated to C3(H2O) (thiol ester-cleaved), C3b, or iC3b the oligomannose glycans become exposed for conglutinin binding, but MBL did not bind to any of the C3 fragments (48). Recently the crystal structure of C3 has been published (49), and from this structure it can be seen that both oligomannose glycans at Asn-63 and Asn-917 are accessible for the CRD of MBL to bind (data not shown). Non-lectin Interactions between MBL and ␣2M—Others have observed ␣2M䡠MBL complexes (27, 28). It is not clear whether the interactions occurred directly with MBL or via the MASPs. When MBL binds a target, the pro-enzyme MASPs become active. ␣2M can inhibit the MASPs. MASP-1 reacts readily with ␣2M, whereas MASP-2 reacts slowly with ␣2M (17, 18). Because ␣2M has four thiol esters, it could be that, once it is cleaved by a MASP, ␣2M may bind covalently not only to the MASP but also to the adjacent MBL. Therefore, the MBL-␣2M interaction may be both non-covalent and covalent. Terai et al. (27) and Storgaard et al. (28) both report MBL䡠␣2M complexes. Storgaard et al. (28) eluted ␣2M䡠MBL from mannose-Sepharose using 50 mM mannose and further purified it using an anti-␣2M affinity resin, still with 50 mM mannose in the buffer. This would allow the antibody capture of ␣2M, but any lectin interaction between the MBL and ␣2M would have been dissociated. Therefore their report is likely to represent rare covalent ␣2M䡠MBL complexes arising from ␣2M inhibition of MASPs bound to MBL. Electron microscopy of the eluted material identified ␣2M䡠MBL complexes, where the ␣2M appears to be interacting with the predominantly collagen-like regions rather than the CRDs of MBL (28). Com-
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MBL Interacts with ␣2M via Oligomannose Glycans parison of the dimensions of ␣2M and MBL suggests that it may be possible for ␣2M to wrap around a collagen-like region “hugging” the MBL arm. The CRD may be too large to be entrapped. No MBL䡠MASP䡠␣2M complexes have been identified. There is support in the literature to suggest that ␣2M䡠MASP complexes dissociate from MBL once formed. Terai et al. (27) identified ␣2M in complex with MASP captured from serum using anti-MASP antibodies and detected using anti-␣2M antibodies. Interestingly, anti-MASP antibodies could apparently still bind to entrapped MASP in the ␣2M “cage.” MASPs form homodimers (50), which may be too large for both monomers to be fully captured by the same ␣2M. Petersen et al. (29) also report ␣2M䡠MASP complexes dissociating back into the fluid phase, after ␣2M has reacted with MBL䡠MASP complexes bound to mannan. They also suggest ␣2M can interact with mannan-bound MBL (possibly in a lectin interaction via unassociated CRD arms). ␣2M䡠MASP complexes are likely to be cleared from the serum via ␣2M receptors primarily found on the hepatocytes of the liver, because the receptor binding site becomes exposed upon the conformational change to ␣2M (9). Physiological Relevance—The lectin interaction between MBL and the oligomannose glycans of ␣2M and possibly the other TEPs, C3 and C4, may involve carbohydrate structures that have been conserved since the evolutionary origins of the TEPs from a common ancestor (51). The weak fluid-phase lectin interaction may represent a mechanism for the localization of ␣2M to the MBL䡠MASP complex for the inhibition of spontaneously or inappropriately activated MASP in the serum. ␣2M inhibits foreign proteases, which can be found on bacterial cell surfaces (52, 53). Dodds (54) suggested that protease inhibitor interaction with bacterial surface protease could result directly or indirectly in the assembly of an opsonic signal. We propose that activated ␣2M on the bacterial cell surface may present “arrays” of oligomannose glycans that could engage multiple CRDs of MBL, or other lectin-like receptors to generate high avidity interactions. Such interactions might opsonize or trigger enzyme cascade systems such as complement via MASP-2 (MASP-1 and MASP-3 are proteases, but it is not known what proteins they activate). This mechanism may also permit MBL to bind to microorganisms to which it cannot bind directly, using the TEPs and their oligomannose glycans as “molecular adaptors,” allowing MBL to bind to a wider variety of microorganisms.
14. 15. 16.
17. 18.
19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39.
Acknowledgments—We thank Dr. A. W. Dodds for his comments on the manuscript. J. N. A. thanks the Medical Research Council for a postgraduate studentship.
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