Localization of the Serine Protease-binding Sites in the Collagen-like Domain of Mannose-binding Protein
2004; Elsevier BV; Volume: 279; Issue: 14 Linguagem: Inglês
10.1074/jbc.m400171200
ISSN1083-351X
AutoresRussell Wallis, Jonathan Shaw, Joost C.M. Uitdehaag, Ce-Belle Chen, Dawn Torgersen, Kurt Drickamer,
Tópico(s)Monoclonal and Polyclonal Antibodies Research
ResumoMutations in the collagen-like domain of serum mannose-binding protein (MBP) interfere with the ability of the protein to initiate complement fixation through the MBP-associated serine proteases (MASPs). The resulting deficiency in the innate immune response leads to susceptibility to infections. Studies have been undertaken to define the region of MBP that interacts with MASPs and to determine how the naturally occurring mutations affect this interaction. Truncated and modified MBPs and synthetic peptides that represent segments of the collagen-like domain of MBP have been used to demonstrate that MASPs bind on the C-terminal side of the hinge region formed by an interruption in the Gly-X-Y repeat pattern of the collagen-like domain. The binding sites for MASP-2 and for MASP-1 and -3 overlap but are not identical. The two most common naturally occurring mutations in MBP result in substitution of acidic amino acids for glycine residues in Gly-X-Y triplets on the N-terminal side of the hinge. Circular dichroism analysis and differential scanning calorimetry demonstrate that the triple helical structure of the collagen-like domain is largely intact in the mutant proteins, but it is more easily unfolded than in wild-type MBP. Thus, the effect of the mutations is to destabilize the collagen-like domain, indirectly disrupting the binding sites for MASPs. In addition, at least one of the mutations has a further effect on the ability of MBP to activate MASPs. Mutations in the collagen-like domain of serum mannose-binding protein (MBP) interfere with the ability of the protein to initiate complement fixation through the MBP-associated serine proteases (MASPs). The resulting deficiency in the innate immune response leads to susceptibility to infections. Studies have been undertaken to define the region of MBP that interacts with MASPs and to determine how the naturally occurring mutations affect this interaction. Truncated and modified MBPs and synthetic peptides that represent segments of the collagen-like domain of MBP have been used to demonstrate that MASPs bind on the C-terminal side of the hinge region formed by an interruption in the Gly-X-Y repeat pattern of the collagen-like domain. The binding sites for MASP-2 and for MASP-1 and -3 overlap but are not identical. The two most common naturally occurring mutations in MBP result in substitution of acidic amino acids for glycine residues in Gly-X-Y triplets on the N-terminal side of the hinge. Circular dichroism analysis and differential scanning calorimetry demonstrate that the triple helical structure of the collagen-like domain is largely intact in the mutant proteins, but it is more easily unfolded than in wild-type MBP. Thus, the effect of the mutations is to destabilize the collagen-like domain, indirectly disrupting the binding sites for MASPs. In addition, at least one of the mutations has a further effect on the ability of MBP to activate MASPs. Serum mannose-binding protein (MBP) 1The abbreviations used are: MBP, mannose-binding protein; MASP, MBP-associated serine protease; CRD, carbohydrate-recognition domain; CUB domain, domain found in complement subcomponents C1r/C1s, Uegf, and bone morphogenetic protein-1; EGF, epidermal growth factor; Hyp, hydroxyproline. interacts with carbohydrates on the surface of microorganisms and thus forms the pathogen recognition component of the lectin pathway of complement activation (1Gadjeva M. Thiel S. Jensenius J.C. Curr. Opin. Immunol. 2001; 13: 74-78Google Scholar, 2Jack D.L. Klein N.J. Turner M.W. Immunol. Rev. 2001; 180: 86-99Google Scholar). MBP, which is also referred to as mannose-binding lectin, binds to surface arrays containing repeated mannose or N-acetylglucosamine residues. It circulates as a complex with one or more MBP-associated serine proteases (MASPs) that autoactivate when the complex binds to an appropriate surface. The surface recognition function of MBP is mediated by clusters of three C-type carbohydrate-recognition domains (CRDs) held together by coiled-coils of α-helices (Fig. 1A). The N-terminal portion of the MBP polypeptide consists of a collagen-like domain composed of Gly-X-Y triplets with a single interruption that forms a bend in the domain (3Wallis R. Immunobiology. 2002; 205: 433-445Google Scholar). A short N-terminal domain contains several cysteine residues that form interchain disulfide bonds. Serum MBPs assemble into larger forms containing 2–4 trimeric subunits in rodents and as many as six subunits in humans. All three oligomeric forms of rat serum MBP, designated MBP-A, can fix complement, although the larger oligomers have higher specific activity. Many species express a second form of MBP. In rats, the second form, MBP-C, is found in the liver. MBP-C does not form higher oligomers beyond the simple subunit that contains three polypeptides. Analysis of chimeras between rat MBP-A and MBP-C suggests that the collagen-like domains contain the MASP-binding sites (4Wallis R. Drickamer K. J. Biol. Chem. 1999; 274: 3580-3589Google Scholar). Ficolins contain N-terminal collagen-like domains and C-terminal fibrinogen-like domains that bind carbohydrate and also activate complement through MASPs (5Matsushita M. Endo Y. Fujita T. J. Immunol. 2000; 164: 2281-2284Google Scholar). Ficolins are believed to play a role in innate immunity, although their physiological ligands are unknown. Three MASPs in humans, rats, and other mammals share a common domain organization (6Dahl M.D. Thiel S. Matsushita M. Fujita T. Willis A.C. Christensen T. Vorup-Jensen T. Jensenius J.C. Immunity. 2001; 15: 127-135Google Scholar, 7Thiel S. Vorup-Jensen T. Stover C.M. Schwaeble W. Laursen S.B. Poulsen K. Willis A.C. Eggleton P. Hansen S. Holmskov U. Reid K.B.M. Jensenius J.C. Nature. 1997; 386: 506-510Google Scholar, 8Matsushita M. Fujita T. J. Exp. Med. 1992; 176: 1497-1502Google Scholar). Dimerization and binding to MBP is mediated by an N-terminal segment consisting of two CUB domains separated by an epidermal growth factor-like (EGF) domain (9Wallis R. Dodd R.B. J. Biol. Chem. 2000; 275: 30962-30969Google Scholar). MBP dimers form 1:1 complexes with MASPs, whereas MBP trimers and tetramers bind up to two MASP dimers (10Chen C.-B. Wallis R. J. Biol. Chem. 2001; 276: 25894-25902Google Scholar). The crystal structure of the N-terminal portion of MASP-2 reveals an elongated molecule in which protomers interact in an antiparallel arrangement through interactions involving the first CUB domain and the EGF domain (11Feinberg H. Uitdehaag J.C.M. Davies J.M. Wallis R. Drickamer K. Weis W.I. EMBO J. 2003; 22: 2348-2359Google Scholar). The remainder of each MASP polypeptide comprises two complement-consensus repeat modules and a serine protease domain. MBP·MASP-2 complexes are sufficient to trigger complement activation by cleaving C4 and C2 to form C3 convertase, which leads to release of the anaphylatoxin C3a and deposition of C3b on the cell surface (10Chen C.-B. Wallis R. J. Biol. Chem. 2001; 276: 25894-25902Google Scholar, 12Vorup-Jensen T. Petersen S.V. Hansen A.G. Poulsen K. Schwaeble W. Sim R.B. Reid K.B.M. Davis S.J. Thiel S. Jensenius J.C. J. Immunol. 2000; 165: 2093-2100Google Scholar). C3b targets the cell for phagocytosis or lysis by the later complement components. MASP-1 and MASP-3 are alternatively spliced products from a common gene (6Dahl M.D. Thiel S. Matsushita M. Fujita T. Willis A.C. Christensen T. Vorup-Jensen T. Jensenius J.C. Immunity. 2001; 15: 127-135Google Scholar). They are identical apart from the protease domain and a short segment that links it to the complement consensus repeat-2 module. MASP-1 may form part of the complement cascade by cleaving components C2 and C3 upon activation (13Rossi V. Cseh S. Bally I. Thielens N.M. Jensenius J.C. Arlaud G.J. J. Biol. Chem. 2001; 276: 40880-40887Google Scholar). The function of MASP-3 is not known. Genes encoding three different variant forms of human MBP are found at frequencies of up to 30% in some human populations (14Turner M.W. Immunol. Today. 1996; 17: 532-540Google Scholar). Individuals who are either heterozygous or homozygous for these variant alleles are immunocompromised. The immunodeficiency associated with MBP is particularly evident in children between the ages of 6 months and 2 years, because these individuals are susceptible to recurrent, severe infections. The Arg32 → Cys substitution results in almost complete failure to form higher oligomers. Inefficient complement fixation follows from the inability of single MBP subunits to bind and activate MASPs (3Wallis R. Immunobiology. 2002; 205: 433-445Google Scholar). Proteins containing the Gly34 → Asp and Gly37 → Glu mutations form higher oligomers that fail to fix complement effectively. In previous studies, substitutions equivalent to the human mutations have been introduced into rat MBP-A, and truncated forms of MASPs have been shown to bind less efficiently to these variant proteins (9Wallis R. Dodd R.B. J. Biol. Chem. 2000; 275: 30962-30969Google Scholar, 15Wallis R. Cheng J.Y.T. J. Immunol. 1999; 163: 4953-4959Google Scholar). In the work reported here, recombinant fragments of MBP and synthetic peptides have been used to localize the binding site for MASPs within the C-terminal portion of the collagen-like domain of MBP-A. The binding motif is found in other MBPs as well as in ficolins, suggesting a general binding mechanism for the activating components of the lectin pathway of complement activation. Substitution of glycine residues on the N-terminal side of the hinge with aspartate or glutamate destabilizes the collagen-like domain and thus indirectly decreases the binding of MASPs. However, these studies and binding experiments with full-length MASPs indicate that at least the Gly37 → Glu form of MBP is specifically defective in the ability to activate MASPs. Mutagenesis and Expression Systems—Wild-type and mutant rat MBP-A as well as fragments of MASP-1 and MASP-2, comprising the N-terminal CUB and EGF domains with a C-terminal histidine tag, and catalytically inactive rat MASPs were expressed in Chinese hamster ovary cells as previously described (4Wallis R. Drickamer K. J. Biol. Chem. 1999; 274: 3580-3589Google Scholar, 9Wallis R. Dodd R.B. J. Biol. Chem. 2000; 275: 30962-30969Google Scholar, 10Chen C.-B. Wallis R. J. Biol. Chem. 2001; 276: 25894-25902Google Scholar, 15Wallis R. Cheng J.Y.T. J. Immunol. 1999; 163: 4953-4959Google Scholar). The cDNA for MBP-C was modified to insert a factor Xa site by replacement of restriction fragments with synthetic double-stranded oligonucleotides (Applied Biosystems). Truncated forms of MBP-A were created by ligating restriction fragments from the cDNA to a cDNA encoding the dog prepro-insulin signal sequence. The resulting constructs were inserted into the plasmid pED (16Kaufman R.J. Davies M.V. Wasley L.C. Michnick D. Nucleic Acids Res. 1991; 19: 4485-4490Google Scholar). Proteins were produced in Chinese hamster ovary cells as described previously for full-size MBPs and were purified by affinity chromatography on mannose-Sepharose columns (4Wallis R. Drickamer K. J. Biol. Chem. 1999; 274: 3580-3589Google Scholar, 17Wallis R. Drickamer K. Biochem. J. 1997; 325: 391-400Google Scholar). Single subunits of the Cys6 → Ser mutant were separated from trace amounts of larger oligomers by anion exchange chromatography on a MonoQ column (HR 5/5; Amersham Biosciences) as described (4Wallis R. Drickamer K. J. Biol. Chem. 1999; 274: 3580-3589Google Scholar). Radiolabeled MBP-A was produced by incubating confluent cells in methionine-free minimum essential medium α, supplemented with 10% dialyzed fetal calf serum, 0.5 μm methotrexate, and 0.1 mCi/ml [35S]methionine/[35S]cysteine mix (Promix cell labeling mix from Amersham Biosciences) for 16 h. Protein was purified as for unlabeled proteins. Purification of Collagen-like Domain—Purified MBP-C containing a factor Xa site (2 mg) in 2 ml of loading buffer (0.15 m NaCl, 25 mm Tris-Cl, pH 7.8, and 25 mm CaCl2) was treated for 3 h at 37 °C with 10 μg of factor Xa (New England BioLabs). The reaction was stopped with phenylmethylsulfonyl fluoride at a final concentration of 0.3 mg/ml by dilution from a stock solution of 30 mg/ml in ethanol. The digest was passed over a 2-ml column of mannose-Sepharose (18Fornstedt N. Porath J. FEBS Lett. 1975; 57: 187-191Google Scholar) to remove the CRD, and the column was rinsed with loading buffer. Fractions of 1 ml were collected throughout, and the collagen-like domain was identified by gel electrophoresis of aliquots from the fractions. The wash-through pool consisting of fractions 2–4 was injected in 1-ml aliquots onto a C4 reverse phase high performance liquid chromatography column. The column was equilibrated with 20% acetonitrile containing 0.1% trifluoroacetic acid and was eluted at a flow rate of 1 ml/min with a gradient from 20 to 25% acetonitrile in the presence of 0.1% trifluoroacetic acid. The identity of the fragment was confirmed by N-terminal sequencing in a Beckman LF-3000 sequencer. Separation of Oligomeric Forms of MBP-A—Conditions for separation of oligomers were similar to those used previously for the wild-type protein (4Wallis R. Drickamer K. J. Biol. Chem. 1999; 274: 3580-3589Google Scholar). Wild-type and mutant MBP-A preparations were dialyzed against 50 mm Tris-Cl, pH 8.2, containing 10 mm EDTA and separated at 25 °C on a Mono-Q HR-5/5 column (Amersham Biosciences) with a gradient of NaCl in this buffer, running from 0 to 0.075 m NaCl in 2 min and from 0.075 to 0.3 m NaCl over 45 min. Gel filtration chromatography was performed in a buffer containing 50 mm Tris-Cl, pH 8.2, on a BioSep S3000 column (300 × 7.8 mm) from Phenomenex (Cheshire, UK) at a flow rate of 0.5 ml/min. Elution positions of different oligomeric forms were deduced from the behavior of the wild-type protein on this column (4Wallis R. Drickamer K. J. Biol. Chem. 1999; 274: 3580-3589Google Scholar). Peptides—Peptides produced by Bachem UK Ltd. (Merseyside, UK) were adjudged to be at least 95% pure by reverse-phase high performance liquid chromatography, and their masses were verified by mass spectrometry. Complement Fixation and Binding Assays—Complement-fixing activities of wild-type and mutant MBPs were measured using a hemolytic assay in which mannan-coated sheep erythrocytes, preincubated with MBP, were incubated with guinea pig serum as the source of complement (4Wallis R. Drickamer K. J. Biol. Chem. 1999; 274: 3580-3589Google Scholar). Lysis was measured by following release of hemoglobin monitored by absorbance at 541 nm. Solid-phase competition assays were performed essentially as described (9Wallis R. Dodd R.B. J. Biol. Chem. 2000; 275: 30962-30969Google Scholar), in which polystyrene plates, coated with the N-terminal three-domain fragment of MASP-2 or MASP-1/-3 were incubated with increasing concentrations of competing ligands in the presence of [35S]MBP-A reporter ligand. Levels of radioactivity were determined using a PhosphorImager SI (Amersham Biosciences). Data were fitted to multiple ligand binding curves by nonlinear regression using MicroCal Origin. The amounts of [35S]MBP-A were below the concentrations required for half-maximal binding in all binding assays. All data represent the mean ± S.E. from at least two separate experiments. Circular Dichroism—Circular dichroism was measured on a Jasco J600 instrument in a 200-μl quartz cell with 1-mm path length. The temperature was allowed to equilibrate for 10 min between spectra. Spectra were averages of 10 scans obtained at 20 nm/min with a data collection rate of one point every 0.5 nm, a bandwidth of 1 nm, and a time constant of 2 s. Differential Scanning Calorimetry—Samples were dialyzed extensively against 0.15 m NaCl, 25 mm Na-HEPES, pH 7.8, 1 mm CaCl2. Aliquots (1 ml) of sample and dialysis buffer were degassed for 15 min prior to loading the sample and reference cells of a Calorimetry Systems Nano III calorimeter. All scans were preceded by 10-min equilibration at the starting temperature. In order to ensure the absence of gas in the samples, initial scans from 5 to 20 °C were repeated until a reproducible base line was achieved. Scans were then performed from 5 to 90 °C. Base lines were calculated using a fourth order polynomial equation to fit the data between 10 and 20 °C and between 80 and 90 °C as well as points at the minimum near 55 °C. Analytical Procedures—Polyacrylamide gel electrophoresis was performed by the method of Laemmli (19Laemmli U.K. Nature. 1970; 227: 680-685Google Scholar). Gels were stained with Coomassie Blue. In the case of the collagen-like tail, destaining was limited to two changes over a period of 20 min, as the dye rapidly dissociates from this fragment. Protein concentrations were determined by the alkaline ninhydrin method (20Hirs C.H.W. Methods Enzymol. 1967; 11: 325-329Google Scholar). Curve fitting was performed with SigmaPlot software. Interaction of MASPs with Truncated MBP-A—In a first approach to defining the region of MBP-A that binds to the MASPs, the interaction of MASP-2 with a series of C-terminal fragments of MBP-A was assessed. Truncated forms of MBP-A lacking the short N-terminal domain, the collagen-like domain up to the interruption, or the entire collagen-like domain were produced (Fig. 1A). For comparison, MBP-A Cys6 → Ser, which cannot form intertrimer disulfide bonds, was used as a source of full-length trimers that do not associate into higher oligomers (4Wallis R. Drickamer K. J. Biol. Chem. 1999; 274: 3580-3589Google Scholar). Proteins that contain part or all of the collagen-like domain were produced in a mammalian expression system to ensure proper modification of proline and lysine residues. The shortest fragment, comprising the neck and CRDs alone, does not contain any of these modifications and was produced in Escherichia coli. All of the expressed proteins were purified by affinity chromatography on mannose-Sepharose and characterized by SDS-polyacrylamide gel electrophoresis and equilibrium ultracentrifugation. Each fragment forms stable homotrimers (data not shown). Formation of MBP-A·MASP-2 complexes was quantified in solid-phase competition assays, in which MBP-A fragments compete with radiolabeled wild-type MBP-A for binding to the N-terminal portion of MASP-2 (Fig. 2). As expected from previous studies, the affinity of full-length, monomeric MBP-A for MASP-2 is substantially reduced compared with the native proteins that consist largely of higher oligomers. However, removal of the N-terminal domain and the N-terminal portion of the collagen-like domain did not further decrease the affinity of the proteins for MASP-2 (Table I). The smallest fragment, comprising the neck and CRDs alone, did not interact with MASP-2. Taken together, these results indicate that MASP-2 must bind to the C-terminal portion of the collagen-like domain, within the 12 Gly-X-Y triplets that lie between the hinge and the neck.Table IBinding properties of full-size and truncated MBPs Inhibition constants (KI) correspond to the concentration of each MBP fragment giving 50% inhibition of [35S]MBP binding to the N-terminal fragment of MASP. KI values of MBP are expressed as moles of trimeric subunits/l. Single MBP subunits or the smaller trimeric fragments bound to MASPs with lower affinities than the larger oligomers of wild-type MBP because there are binding sites for two MBP subunits on each MASP dimer (10Chen C.-B. Wallis R. J. Biol. Chem. 2001; 276: 25894-25902Google Scholar).ProteinKIRelative KIaExpressed as KI,MBP Cys6 → Ser/KI, fragmentMASP-2MASP-1/-3MASP-2MASP-1/-3μmMBP0.056 ± 0.0100.042 ± 0.00634.4 ± 10.822.8 ± 4.5MBP Cys6 → Ser1.82 ± 0.260.93 ± 0.051.01.0Truncation 12.87 ± 0.111.24 ± 0.240.64 ± 0.120.79 ± 0.20Truncation 23.13 ± 0.312.97 ± 0.600.79 ± 0.200.33 ± 0.08Truncation 3>8>3<0.2 700>850<0.003 700>800<0.003 8500.045 ± 0.013 700>850<0.003 8500.004 ± 0.001<0.001a Expressed as KI,MBP Cys6 → Ser/KI, fragment Open table in a new tab Comparable binding analysis with the N-terminal portion of MASP-1 and -3 revealed a similar but not identical pattern of results (Table II). Modification of triplet 9 again completely abolishes binding, but in this case a similar loss of binding is also observed when triplet 7 is changed. Surprisingly, changing triplet 6 to Gly-Pro-Hyp actually strengthens the interaction with the MASP-1/3 fragment. This effect is probably due to the increased stability of the triple helical form of this peptide due to the presence of the additional helix-stabilizing residues. This finding serves to emphasize that loss of binding activity resulting from changing the other triplets is probably partially compensated by increased stability of the triple helix, so the intrinsic effects of these changes are likely to be greater than what is measured in the assay. In any case, the results clearly indicate that Lys46 and Leu47 form part of the binding sites for all three MASPs, and Leu40 and Gln41 probably provide important additional contacts for MASP-1 and -3. Complement-fixing Activity by MBP-A Containing Mutations within the MASP-binding Site—In order to confirm the peptide binding results, it was of interest to assess the effect of disrupting the MASP-binding sites on complement-fixing activity of intact MBP-A. Recombinant MBP-A containing the changes Lys46 → Pro and Leu47 → Hyp was produced and purified in parallel with wild-type MBP-A, and binding to the MASPs was tested in the solid phase competition assay. No binding of the mutant protein to the N-terminal fragment of either MASP-2 or MASP-1/-3 was detected, demonstrating that the affinity in each case is reduced by more than 100-fold (Fig. 4). Complement-fixing activity was tested using MBP-dependent hemolysis of mannan-coated sheep erythrocytes. No activity was detected at the highest concentration of mutant MBP-A tested, which was more than 500-fold greater than levels of wild-type MBP-A needed to cause 50% lysis of the target cells (Fig. 5). Because the modified MBP-A still binds to mannose-Sepharose in a Ca2+-dependent manner, loss of activity is not due to a failure to bind to terminal-mannose moieties on the sheep erythrocytes. Furthermore, analysis by gel filtration and SDS-polyacrylamide gel electrophoresis showed that the mutant protein resembles wild-type MBP-A both in terms of it oligomeric and its covalent structure (data not shown). A small increase in the proportion of single MBP-A subunits in the mutant MBP-A is not sufficient to account for the decrease in activity observed. Thus, mutation of Lys46 and Leu47 within the collagen-like domain of MBP-A abolishes all detectable complement-fixing activity as a consequence of disruption of the interactions with MASPs.Fig. 5Complement activation by MBPs. Complement-fixing activity was measured by hemolysis of mannan-coated sheep erythrocytes in the presence of wild-type MBP-A (•) and MBP-A Lys46 → Pro, Leu47 → Hyp (▪).View Large Image Figure ViewerDownload (PPT) Effect of Naturally Occurring Mutations on the Binding of MASPs—Mutant forms of rat MBP-A have been created to mimic each of the naturally occurring human mutations (15Wallis R. Cheng J.Y.T. J. Immunol. 1999; 163: 4953-4959Google Scholar). In previous studies, the ability of these MBPs to interact with N-terminal fragments of MASP-1 and -2 has been investigated (9Wallis R. Dodd R.B. J. Biol. Chem. 2000; 275: 30962-30969Google Scholar). Although the mutations lie outside the proposed MASP-binding site, they decrease the affinity of MBP-A for MASP. Full-length MASPs that are catalytically inactive have recently been created by replacement of the active site serine residue with an alanine residue (10Chen C.-B. Wallis R. J. Biol. Chem. 2001; 276: 25894-25902Google Scholar). The availability of these proteins makes it possible to examine the interaction of wild-type and mutant forms of MBP-A with the full-length MASPs to confirm the effects of the mutations. A competition binding assay, in which binding of soluble [35S]MBP-A to immobilized MASPs is inhibited by unlabeled wild-type and mutant MBP-A, was utilized to quantify MASP in
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