Characterization of the Oligomer Structure of Recombinant Human Mannan-binding Lectin

Abstract

Mannan-binding lectin (MBL) belongs to a family of proteins called the collectins, which show large differences in their ultrastructures. These differences are believed to be determined by different N-terminal disulfide-bonding patterns. So far only the bonding pattern of two of the simple forms (recombinant rat MBL-C and bovine CL-43) have been determined. Recombinant MBL expressed in human cells was purified, and the structure of the N-terminal region was determined. Preliminary results on human plasma-derived MBL revealed high similarity to the recombinant protein. Here we report the structure of the N-terminal part of recombinant human MBL and present a model to explain the oligomerization pattern. Using a strategy of consecutive enzymatic digestions and matrix-assisted laser desorption ionization mass spectrometry, we succeeded in identifying a number of disulfide-linked peptides from the N-terminal cysteine-rich region. Based on these building blocks, we propose a model that can explain the various oligomeric forms found in purified MBL preparations. Furthermore, the model was challenged by the production of cysteine to serine mutants of the three N-terminally situated cysteines. The oligomerization patterns of these mutants support the proposed model. The model indicates that the polypeptide dimer is the basic unit in the oligomerization. Mannan-binding lectin (MBL) belongs to a family of proteins called the collectins, which show large differences in their ultrastructures. These differences are believed to be determined by different N-terminal disulfide-bonding patterns. So far only the bonding pattern of two of the simple forms (recombinant rat MBL-C and bovine CL-43) have been determined. Recombinant MBL expressed in human cells was purified, and the structure of the N-terminal region was determined. Preliminary results on human plasma-derived MBL revealed high similarity to the recombinant protein. Here we report the structure of the N-terminal part of recombinant human MBL and present a model to explain the oligomerization pattern. Using a strategy of consecutive enzymatic digestions and matrix-assisted laser desorption ionization mass spectrometry, we succeeded in identifying a number of disulfide-linked peptides from the N-terminal cysteine-rich region. Based on these building blocks, we propose a model that can explain the various oligomeric forms found in purified MBL preparations. Furthermore, the model was challenged by the production of cysteine to serine mutants of the three N-terminally situated cysteines. The oligomerization patterns of these mutants support the proposed model. The model indicates that the polypeptide dimer is the basic unit in the oligomerization. Mannan-binding lectin (MBL) 1The abbreviations used are: MBL, mannan-binding lectin; rMBL, recombinant MBL; rhMBL, recombinant human MBL; pMBL, plasma-derived MBL; aa, amino acid; MALDI, matrix-assisted laser desorption/ionization; MS, mass spectrometry; CL-43 and -46, collectin 43 and 46, respectively; IAA, iodoacetamide; Q-TOF, quadropole time-of-flight; DTT, dithiothreitol; HPLC, high pressure liquid chromatography.1The abbreviations used are: MBL, mannan-binding lectin; rMBL, recombinant MBL; rhMBL, recombinant human MBL; pMBL, plasma-derived MBL; aa, amino acid; MALDI, matrix-assisted laser desorption/ionization; MS, mass spectrometry; CL-43 and -46, collectin 43 and 46, respectively; IAA, iodoacetamide; Q-TOF, quadropole time-of-flight; DTT, dithiothreitol; HPLC, high pressure liquid chromatography. is a serum protein that acts in innate immunity. It is a C-type lectin that recognizes and binds to specific sugars such as d-mannose and N-acetyl-d-glucosamine present on pathogen surfaces (1Weis W.I. Drickamer K. Hendrickson W.A. Nature. 1992; 360: 127-134Crossref PubMed Scopus (842) Google Scholar). The main functions in innate immunity are 1) the opsonin effect, where it binds to the surface of the pathogen and thereby enhances its clearance from the bloodstream (2Turner M.W. Immunol. Today. 1996; 17: 532-540Abstract Full Text PDF PubMed Scopus (671) Google Scholar) and 2) the ability to activate the complement cascade via the lectin pathway (3Matsushita M. Fujita T. J. Exp. Med. 1992; 176: 1497-1502Crossref PubMed Scopus (546) Google Scholar). Activation of the complement cascade requires binding of the MBL-associated serine proteases to the oligomeric forms of MBL (3Matsushita M. Fujita T. J. Exp. Med. 1992; 176: 1497-1502Crossref PubMed Scopus (546) Google Scholar, 4Dahl M.R. Thiel S. Matsushita M. Fujita T. Willis A.C. Christensen T. Vorup-Jensen T. Jensenius J.C. Immunity. 2001; 15: 127-135Abstract Full Text Full Text PDF PubMed Scopus (319) Google Scholar, 5Thiel 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. Jensenius J.C. Nature. 1997; 386: 506-510Crossref PubMed Scopus (736) Google Scholar) and leads to the formation of the membrane attack complex that perforates the cell membrane of the pathogen. The function of MBL is thus dependent on its oligomeric structure, since the small oligomer forms act as opsonins and the large oligomer forms activate complement (6Holmskov U. Laursen S.B. Malhotra R. Wiedemann H. Timpl R. Stuart G.R. Tornoe I. Madsen P.S. Reid K.B. Jensenius J.C. Biochem. J. 1995; 305: 889-896Crossref PubMed Scopus (58) Google Scholar, 7Kurata H. Cheng H.M. Kozutsumi Y. Yokota Y. Kawasaki T. Biochem. Biophys. Res. Commun. 1993; 191: 1204-1210Crossref PubMed Scopus (57) Google Scholar, 8Super M. Gillies S.D. Foley S. Sastry K. Schweinle J.E. Silverman V.J. Ezekowitz R.A. Nat. Genet. 1992; 2: 50-55Crossref PubMed Scopus (123) Google Scholar). The overall polypeptide structure of MBL is similar to that of the other collectins (surfactant protein A, surfactant protein D, conglutinin, CL-43, liver collectin 1, and CL-46). It includes a short, cysteine-rich N-terminal stretch (aa 1–21), a collagen-like region (aa 22–81) with one interruption (aa 43–44) that causes the collagen-like structure to bend, a neck region (aa 82–115), and a carbohydrate recognition domain (aa 116–228) (9Drickamer K. Taylor M.E. Annu. Rev. Cell Biol. 1993; 9: 237-264Crossref PubMed Scopus (703) Google Scholar). This domain confers the carbohydrate specificity of MBL and is stabilized by two disulfide bonds (1Weis W.I. Drickamer K. Hendrickson W.A. Nature. 1992; 360: 127-134Crossref PubMed Scopus (842) Google Scholar). Due to the collagen-like domain, MBL forms homotrimers, designated the MBL subunit. The collagen-like structure is stabilized by the presence of hydroxyprolines and glycosylated hydroxylysines (10Ma Y. Shida H. Kawasaki T. J. Biochem. (Tokyo). 1997; 122: 810-818Crossref PubMed Scopus (37) Google Scholar). The subunit structures assemble from the C to the N terminus. The neck region initiates the folding (11Childs R.A. Feizi T. Yuen C.T. Drickamer K. Quesenberry S. J. Biol. Chem. 1990; 265: 20770-20777Abstract Full Text PDF PubMed Google Scholar), and the collagen-like region zips toward the N terminus, creating trimeric subunits. The structure is finally stabilized by intrasubunit disulfide bonds in the N-terminal region (12Hansen S. Holmskov U. Immunobiology. 1998; 199: 165-189Crossref PubMed Scopus (124) Google Scholar). The oligomer structure of MBL is similar to the structure of C1q, the primary component of the classical pathway of complement (13Ikeda K. Sannoh T. Kawasaki N. Kawasaki T. Yamashina I. J. Biol. Chem. 1987; 262: 7451-7454Abstract Full Text PDF PubMed Google Scholar), where the bouquet-like forms arise from the formation of intersubunit disulfide bonds in the N-terminal region (14Hoppe H.J. Reid K.B. Protein Sci. 1994; 3: 1143-1158Crossref PubMed Scopus (187) Google Scholar). However, whereas C1q is made up of three different polypeptide chains, MBL consists of homotrimers. In addition, C1q only exists as hexamers, whereas MBL exists as anything from dimers to octamers. The disulfide-bonding pattern of C1q thus cannot be expected to be identical to that of MBL. The elucidation of the structure of MBL is complicated by the fact that the polypeptide chain of MBL is very heterogeneous. In addition to several post-translational modifications, there are three well documented mutations in the collagen-like region (15Garred P. Thiel S. Madsen H.O. Ryder L.P. Jensenius J.C. Svejgaard A. Clin. Exp. Immunol. 1992; 90: 517-521Crossref PubMed Scopus (110) Google Scholar, 16Lipscombe R.J. Sumiya M. Hill A.V. Lau Y.L. Levinsky R.J. Summerfield J.A. Turner M.W. Hum. Mol. Genet. 1992; 1: 709-715Crossref PubMed Scopus (373) Google Scholar, 17Madsen H.O. Garred P. Kurtzhals J.A. Lamm L.U. Ryder L.P. Thiel S. Svejgaard A. Immunogenetics. 1994; 40: 37-44Crossref PubMed Scopus (456) Google Scholar). These mutations all lead to amino acid substitutions, which distort the collagen-like region and inhibit the correct formation of the oligomer forms of MBL. In addition to the heterogeneity of the polypeptide chain, promoter polymorphisms (18Madsen H.O. Garred P. Thiel S. Kurtzhals J.A. Lamm L.U. Ryder L.P. Svejgaard A. J. Immunol. 1995; 155: 3013-3020PubMed Google Scholar) result in highly variable amounts of MBL in the blood. The objective of the present work was to map the disulfide-bonding pattern of the N-terminal part of MBL. The work was performed using recombinant protein due to the availability of larger amounts and the fact that the recombinant protein would be more homogenous than MBL purified from a pool of plasma. Materials—α-Cyano-4-hydroxycinnamic acid was from Aldrich (recrystallized in boiling acetonitrile). Porcine trypsin was a gift from Novo Nordisk A/S. 3–8% NuPAGE gradient gels and 45-nm polyvinylidene difluoride membranes were from Novex. Primary anti-human MBL antibody HYB131-01 was purchased from The Antibody Shop (The State Serum Institute), and polyclonal horseradish peroxidase anti-mouse antibody was from DAKO. The SuperSignal West was from The Cell was purchased from Novex. was from was from a and all were of was by using a human cell as T. Schwaeble W. Kawasaki T. Y. K. N. Y. K. Ezekowitz Thiel S. Jensenius J.C. 2001; 1: PubMed Scopus Google Scholar). was from the cell cell using a from and with The was on a and on a from The to by and of protein were using 3–8% from Novex. of protein was and was The was a 45-nm polyvinylidene difluoride membrane using the and a of The membrane was in a with and the primary antibody was and with The membrane was and with the antibody with The protein were finally using the SuperSignal West and MBL was and to were with two of and with two of were with a were a of using of were with of trypsin as T. P. Biochem. PubMed Scopus Google Scholar). was in and to in The was and the was by by N-terminal of was in and to in of a in the was of and the was in the to the addition of The digestions were with and by of the and by of N-terminal peptides were in of and of of The digestions were and by and digestions were using the by were performed on a using a gradient from by a gradient from The used was acid in and the was acid in The of the N-terminal part of were using a were by of were in DTT, and by N-terminal of peptides was performed on a protein to the of was to the of of peptides was performed on a by the from The was with of and were of was in acid in using the of peptides was performed on a by from of was on a in a as J. R. P. J. PubMed Scopus Google Scholar). The was the with a of acid in on the was using the of all was performed using peptides of of and were using the and were using the and the of human MBL number the and MBL been T. Schwaeble W. Kawasaki T. Y. K. N. Y. K. Ezekowitz Thiel S. Jensenius J.C. 2001; 1: PubMed Scopus Google Scholar). The MBL was the the MBL is the of the the of MBL, we and to using a the to all of The were as H. and in the of the were to that the correct mutations were of of the MBL human cells was performed using the of of the to the was to cells in with with The cells were with a of the were and by of of and 1) show that the structures present in the plasma-derived of MBL are present in the of the presence of the different oligomer forms all the forms of are to the polypeptide chain of MBL with the mass of of and A, of a was using a 3–8% of a and was on a gels were and was using The Antibody and SuperSignal West of the N-terminal of to the N-terminal part of from the several different and were up However, the N-terminal peptides be from of these preparations. The N-terminal peptides were finally from a by of the were by in to the N-terminal region. The in of The mass in the N-terminal with of The and to the of from the glycosylated and due to The heterogeneity of from the in of and The of the peptides was using of and the disulfide-linked peptides were with and the was by in were and by in to disulfide-linked of the N-terminal part of MBL two and different disulfide-linked were in the as in two peptides by a disulfide the which to This the presence of a in as by the in The presence of a in the is due to of the disulfide two peptides to one The the heterogeneity in the of and by the two and to the peptides and This the presence of a and a in as by the in present in the in is a which This to one and one by a disulfide This from the of a disulfide in the of one present in the the mass two peptides is the result of the heterogeneity in the of and by the a to This lead to the of disulfide since two as by the in In to the disulfide the was with since and Drickamer R. Drickamer K. Biochem. J. 1997; PubMed Scopus Google Scholar) have that can the in rat the the and the The to two and which These two to two peptides and two the This the presence of and bonds in rhMBL, as by the two building in the of the more whereas the of is the This is to of the due to in the mass and of a one two peptides to two peptides However, was in the where was to trypsin Here one to one with one was of two and to and The was by MS, and from the mass of the be This that and are is and is by IAA, only to be the presence of a in This is by the in such peptides the are a disulfide This can to the in the in is the presence of three peptides Based on the of the mass is present in than of the The is in the that it have by of a larger oligomer by the cysteine The of is to that of collectins CL-43, due to the was of the to of binding model on the results was challenged by the production of to mutants of the N-terminal of to the production of all of the to mutants the three the three and the of their oligomerization patterns is in The and mutants polypeptide dimers and subunit forms a small of different The and all of the mutants polypeptide polypeptide The only exists as polypeptide as in the MBL is to of forms a model have to the as well as the of disulfide The results from the and the to mutants to the oligomerization model in This the formation of two of a subunit as well as a subunit. The present was performed using recombinant human mannan-binding lectin as to plasma-derived mannan-binding lectin This several from one a of MBL is more homogenous than purified from a pool of where several are to (15Garred P. Thiel S. Madsen H.O. Ryder L.P. Jensenius J.C. Svejgaard A. Clin. Exp. Immunol. 1992; 90: 517-521Crossref PubMed Scopus (110) Google Scholar, 16Lipscombe R.J. Sumiya M. Hill A.V. Lau Y.L. Levinsky R.J. Summerfield J.A. Turner M.W. Hum. Mol. Genet. 1992; 1: 709-715Crossref PubMed Scopus (373) Google Scholar, 17Madsen H.O. Garred P. Kurtzhals J.A. Lamm L.U. Ryder L.P. Thiel S. Svejgaard A. Immunogenetics. 1994; 40: 37-44Crossref PubMed Scopus (456) Google Scholar). the of are a similar on of such a large of would large amounts of and of the results be complicated by the presence of as a result of the in the human In addition, all indicates that differences with to post-translational and the and the recombinant protein. The is in human from a the human MBL of the similarity and found that is and similar to T. Schwaeble W. Kawasaki T. Y. K. N. Y. K. Ezekowitz Thiel S. Jensenius J.C. 2001; 1: PubMed Scopus Google Scholar). This the of oligomerization and the complement of the structures of and support the high similarity Furthermore, all post-translational in have been in P. H. Primary of and of Scholar), and the complement of the two are similar J.C. K. Thiel S. Biochem. PubMed Scopus Google Scholar). The MBL subunit consists of three polypeptide subunit that assemble oligomeric The collagen-like in with the neck is the of the polypeptide the subunit (10Ma Y. Shida H. Kawasaki T. J. Biochem. (Tokyo). 1997; 122: 810-818Crossref PubMed Scopus (37) Google Scholar, R.A. Feizi T. Yuen C.T. Drickamer K. Quesenberry S. J. Biol. Chem. 1990; 265: 20770-20777Abstract Full Text PDF PubMed Google Scholar). The subunit is stabilized by intrasubunit disulfide bonds in the N-terminal region (12Hansen S. Holmskov U. Immunobiology. 1998; 199: 165-189Crossref PubMed Scopus (124) Google Scholar). disulfide bonds in the N-terminal region have been to be the of the oligomeric forms (14Hoppe H.J. Reid K.B. Protein Sci. 1994; 3: 1143-1158Crossref PubMed Scopus (187) Google Scholar). The function of MBL in the is to the of oligomerization. The small oligomer structures are believed to be whereas the larger forms activate complement (6Holmskov U. Laursen S.B. Malhotra R. Wiedemann H. Timpl R. Stuart G.R. Tornoe I. Madsen P.S. Reid K.B. Jensenius J.C. Biochem. J. 1995; 305: 889-896Crossref PubMed Scopus (58) Google Scholar, 7Kurata H. Cheng H.M. Kozutsumi Y. Yokota Y. Kawasaki T. Biochem. Biophys. Res. Commun. 1993; 191: 1204-1210Crossref PubMed Scopus (57) Google Scholar, 8Super M. Gillies S.D. Foley S. Sastry K. Schweinle J.E. Silverman V.J. Ezekowitz R.A. Nat. Genet. 1992; 2: 50-55Crossref PubMed Scopus (123) Google Scholar). and two of MBL, one two N-terminal which and one three N-terminal which forms the human MBL R. Drickamer K. Biochem. J. 1997; PubMed Scopus Google Scholar, R. Ezekowitz R.A. Sastry 1995; PubMed Scopus (57) Google Scholar, R. Drickamer K. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The N-terminal disulfide-bonding pattern of the rat MBL two was by and Drickamer R. Drickamer K. Biochem. J. 1997; PubMed Scopus Google Scholar). This model a large of the polypeptide in the subunit. This pattern is identical to that determined CL-43 Holmskov U. P. J. Biochem. 1997; PubMed Scopus Google Scholar). The N-terminal disulfide binding pattern been of the three from the that the of the three N-terminal to be the whereas the two other intrasubunit bonds R. Ezekowitz R.A. Sastry 1995; PubMed Scopus (57) Google Scholar, R. Drickamer K. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). Due to the large heterogeneity of the MBL polypeptide chain, a of the of the disulfide-bonding pattern was the of the N-terminal region. been used to and of MBL K. J. Biol. Chem. Full Text PDF PubMed Google we have been to the N-terminal region using other specific and the two and a dimer of the peptides from the N-terminal region were with in of the various forms of the N-terminal region the number of This is due to the high heterogeneity of the which is in the of the peptides with trypsin and in the of several specific disulfide The on intrasubunit disulfide the peptides to a of building that the oligomer structures of The to mutants and their disulfide bonding patterns support to the results by and show that the building and intersubunit disulfide The of three the of the very complex bonding pattern in The mutants the of of the three in the complex oligomerization of The is the only that whereas forms subunit and only forms polypeptide The mutants support the of the and bonds by The the of the N-terminal in the folding of rhMBL, since only exists as polypeptide The results can be used to explain the of the different MBL oligomer the building and the bonding patterns of the to mutants a the of the of is proposed The leads to the formation of one of three subunit which the of The model indicates that the binding pattern is and the of the oligomerization and is thus to all of the different oligomer forms of MBL. The of the proposed oligomer are similar to the by and (1Weis W.I. Drickamer K. Hendrickson W.A. Nature. 1992; 360: 127-134Crossref PubMed Scopus (842) Google Scholar). that as the collagen-like region zips from the C (11Childs R.A. Feizi T. Yuen C.T. Drickamer K. Quesenberry S. J. Biol. Chem. 1990; 265: 20770-20777Abstract Full Text PDF PubMed Google Scholar) and the N-terminal the the the which a is This three to with subunits. are two of only in the in the of the two the building in and However, since are the these building of the number of This from the to to subunit and the to which to polypeptide whereas it to subunit It the to since is the the binding pattern in CL-43 and rat MBL-C is to be the formation of polypeptide whereas indicates that it be to subunit The of structure in be due to in the of the correct subunit the forms the two by the This leads to the polypeptide the of the structure the thereby the oligomerization The of subunit as the folding of one chain binding to the of a This subunit the building in and the polypeptide dimer formation in the in Based on the of the different building in and the amounts in gels we that pattern very of oligomerization can by of a number of subunits. all of the different oligomer is proposed to be in be dependent on the polypeptide dimers in a the disulfide bonding and These forms are as as the subunit forms it is and are present in the MBL the of the the presence of a cysteine in oligomer is a to R. Drickamer K. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, U. and of the Mannan-binding of Scholar). is the small of cysteine with the of The would than of the since in the proposed of in a is expected to in MBL In a with is expected to be be that the cysteine in is to be by cysteine The two in the model in have three cysteines. two are to be in the oligomerization with subunits. In with number of all three in disulfide However, in the of one cysteine be to and to be in with the building in only to up as a cysteine the of oligomeric The of MBL is with C1q, the component of the classical pathway of The two are similar in function and structure, have their C1q is of two and two by disulfide bonds in the N-terminal region. of C1q only two to the polypeptide dimer and the polypeptide dimer K.B. Biochem. J. 155: PubMed Scopus Google Scholar). of MBL a large number of different oligomer that the MBL and are R. Drickamer K. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, H. Hansen S. K. Ezekowitz Hansen Jensenius J.C. Thiel S. J. Immunol. 2001; PubMed Scopus Google Scholar, Thiel S. Wiedemann H. Timpl R. Reid K.B. J. Immunol. 1990; Google Scholar), in to the collagen-like structure and the C1q This is by the fact that the to only exists as polypeptide chain and the of the N-terminal bonding pattern of C1q MBL. MBL is to the other the polypeptide all of the collectins have a similar domain of the collectins homotrimers, their oligomer structures several CL-43 U. Willis A.C. Reid K.B. Jensenius J.C. J. Biol. Chem. 1993; Full Text PDF PubMed Google Scholar) and MBL-C R. Drickamer K. Biochem. J. 1997; PubMed Scopus Google Scholar) only as whereas surfactant protein A. K. M. W. PubMed Scopus Google Scholar) and PubMed Scopus Google Scholar) protein J. K. J. S. Nature. Scopus Google Scholar) forms a bouquet-like structure similar to C1q and MBL. is the of the two liver collectin K. Y. S. T. T. H. T. H. Y. A. T. N. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) and S. Reid K.B. K. Holmskov U. J. Immunol. PubMed Scopus Google Scholar). It is believed that the N-terminal and their bonding patterns are the in the oligomer structures of the CL-43 and the of rat MBL show identical bonding patterns R. Drickamer K. Biochem. J. 1997; PubMed Scopus Google Scholar, Holmskov U. P. J. Biochem. 1997; PubMed Scopus Google Scholar). The bonding patterns the other collectins have been The pattern cannot be on human MBL, since protein and the rat oligomer are the only collectins three N-terminal cysteines. The and mutants show oligomer that a binding pattern similar to that CL-43 and rat The fact that the in the as the is in to that the cysteine is intersubunit whereas the two intrasubunit bonds R. Ezekowitz R.A. Sastry 1995; PubMed Scopus (57) Google Scholar, R. Drickamer K. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The fact that the the subunit that there is the formation of the expected that of the subunit is more This that the number as well as the of the oligomerization pattern of The three well mutations in the collagen-like region of human MBL and are to the of the protein H. Cheng H.M. Kozutsumi Y. Yokota Y. Kawasaki T. Biochem. Biophys. Res. Commun. 1993; 191: 1204-1210Crossref PubMed Scopus (57) Google Scholar, 8Super M. Gillies S.D. Foley S. Sastry K. Schweinle J.E. Silverman V.J. Ezekowitz R.A. Nat. Genet. 1992; 2: 50-55Crossref PubMed Scopus (123) Google Scholar, R. Cheng J. Immunol. Google Scholar, Madsen H.O. R. Garred P. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The is believed to be and of the N-terminal disulfide-bonding pattern of these is by the of cysteine in the collagen-like This cysteine results in number and thereby the oligomerization in to a T. and with