Moraxella catarrhalis IgD-binding protein (MID), a 200-kDa outer membrane protein comprising 2,139 amino acids, has recently been isolated and shown to display a unique and specific affinity for human IgD. To identify the IgD-binding region, MID was digested with proteases. In addition, a series of truncated fragments of MID were manufactured and expressed in Escherichia coli followed by analysis for IgD binding in Western and dot blots. The smallest fragment with essentially preserved IgD binding was comprised of 238 amino acid residues (MID962–1200). Shorter recombinant proteins gradually lost IgD-binding capacity, and the shortest IgD-binding fragment comprising 157 amino acids (MID985–1142) displayed a 1,000-fold reduced IgD binding compared with the full-length molecule. The truncated MID962–1200 was efficiently attracted to a standard IgD serum and to purified myeloma IgD(κ) and IgD(λ) sera but not to IgG, IgM, or IgA myeloma sera. Furthermore, the fragment specifically bound to peripheral blood B lymphocytes, and the binding was inhibited by preincubation with anti-IgD-Fab polyclonal antibodies. Results obtained by introducing five amino acids randomly into MID962–1200 using transposons suggested that α-helix structures were important for IgD binding. Ultracentrifugation experiments and gel electrophoresis revealed that native MID962–1200 was a tetramer. Interestingly, tetrameric MID962–1200 attracted IgD more than 20-fold more efficiently than the monomeric form. Thus, a tetrameric structure of MID962–1200 is crucial for optimal IgD-binding capacity. Moraxella catarrhalis IgD-binding protein (MID), a 200-kDa outer membrane protein comprising 2,139 amino acids, has recently been isolated and shown to display a unique and specific affinity for human IgD. To identify the IgD-binding region, MID was digested with proteases. In addition, a series of truncated fragments of MID were manufactured and expressed in Escherichia coli followed by analysis for IgD binding in Western and dot blots. The smallest fragment with essentially preserved IgD binding was comprised of 238 amino acid residues (MID962–1200). Shorter recombinant proteins gradually lost IgD-binding capacity, and the shortest IgD-binding fragment comprising 157 amino acids (MID985–1142) displayed a 1,000-fold reduced IgD binding compared with the full-length molecule. The truncated MID962–1200 was efficiently attracted to a standard IgD serum and to purified myeloma IgD(κ) and IgD(λ) sera but not to IgG, IgM, or IgA myeloma sera. Furthermore, the fragment specifically bound to peripheral blood B lymphocytes, and the binding was inhibited by preincubation with anti-IgD-Fab polyclonal antibodies. Results obtained by introducing five amino acids randomly into MID962–1200 using transposons suggested that α-helix structures were important for IgD binding. Ultracentrifugation experiments and gel electrophoresis revealed that native MID962–1200 was a tetramer. Interestingly, tetrameric MID962–1200 attracted IgD more than 20-fold more efficiently than the monomeric form. Thus, a tetrameric structure of MID962–1200 is crucial for optimal IgD-binding capacity. Nonimmune Ig-binding proteins derived from Gram-negative bacteria are rare compared with Ig-binding proteins from Gram-positive bacteria. However, Brucella abortus interacts with bovine IgM (1Nielsen K. Duncan J.R. J. Immunol. 1982; 129: 366-369PubMed Google Scholar) andHaemophilus somnus with IgG-Fc (2Widders P.R. Smith J.W. Yarnall M. Mcguire T.C. Corbeil L.B. J. Med. Microbiol. 1988; 29: 307-311Crossref Scopus (25) Google Scholar), and some strains ofEscherichia coli exhibit IgG binding in a nonimmune manner (3Sandt C.H. Wang Y.-D. Wilson R.A. Hill C.W. Infect. Immun. 1997; 65: 4572-4579Crossref PubMed Google Scholar). In addition, Haemophilus influenzae and Moraxella catarrhalis have been shown to strongly bind human IgD (4Forsgren A. Grubb A.O. J. Immunol. 1979; 122: 1468-1472PubMed Google Scholar). It has also been demonstrated that M. catarrhalis and H. influenzae activate human B cells through interactions with surface IgD (5Forsgren A. Penta A. Schlossman S.F. Tedder T.F. Cell. Immunol. 1988; 112: 78-88Crossref PubMed Scopus (32) Google Scholar). We have recently purified and characterized a 200-kDa outer membrane protein from M. catarrhalisdesignated Moraxella IgD-binding protein (MID) 1The abbreviations used are: MID, Moraxella IgD-binding protein; BCR, B cell receptor; PBL, peripheral blood lymphocyte; HRP, horseradish peroxidase; pAb, polyclonal antibody; mAb, monoclonal antibody; PBS, phosphate-buffered saline; ELISA, enzyme-linked immunosorbent assay; FITC, fluorescein isothiocyanate; RPE, R-phycoerythrin. (6Forsgren A. Brant M. Möllenkvist A. Muyombwe A. Janson H. Woin N. Riesbeck K. J. Immunol. 2001; 167: 2112-2120Crossref PubMed Scopus (72) Google Scholar). The deduced amino acid sequence of MID consists of 2,139 residues. MID has specific IgD-binding properties and interacts with two purified human IgD myeloma proteins, four IgD myeloma sera, and one IgD standard serum. In contrast, no binding is detected to myelomas of other immunoglobulin classes including IgG, IgM, IgA, and IgE. MID also binds to the surface-expressed B cell receptor (BCR) IgD but not to other membrane molecules on human peripheral blood lymphocytes. Furthermore, MID is mitogenic for purified B cells and induces Ig production in the presence of Th2 cytokines (7Gjörloff-Wingren A. Hadzic R. Forsgren A. Riesbeck K. J. Immunol. 2002; 168: 5582-5588Crossref PubMed Scopus (50) Google Scholar). IgD is a unique immunoglobulin, which was first discovered in 1965 by Rowe and Fahley (8Rowe D.S. Fahley J.L. J. Exp. Med. 1965; 121: 171-178Crossref PubMed Scopus (177) Google Scholar). Because soluble IgD is easily degraded by proteolytic enzymes and therefore difficult to purify, the specific role of IgDs has not yet been completely defined (9Vladutiu A.O. Clin. Diagn. Lab. Immmunol. 2000; 7: 131-140Crossref PubMed Scopus (49) Google Scholar). Soluble IgD represents approximately 0.25% of the total serum immunoglobulins. In contrast, cell surface-bound IgD serves as an antigen BCR and is co-expressed with IgM on mature B lymphocytes. Interestingly, an increased number of IgD molecules has been observed in human bronchus-associated lymphoid tissue and tonsils (10Brandtzaeg P. Gjeruldsen S.T. Korsrud F. Baklien K. Berdal P. Ek J. J. Immunol. 1979; 122: 503-510PubMed Google Scholar), suggesting a possible interaction between the immune system and the IgD-binding respiratory pathogens M. catarrhalis and H. influenzae (4Forsgren A. Grubb A.O. J. Immunol. 1979; 122: 1468-1472PubMed Google Scholar, 6Forsgren A. Brant M. Möllenkvist A. Muyombwe A. Janson H. Woin N. Riesbeck K. J. Immunol. 2001; 167: 2112-2120Crossref PubMed Scopus (72) Google Scholar). In the present study, we show that a recombinant protein corresponding to a 238-amino acid sequence between positions 962 and 1200 of MID essentially has a preserved IgD-binding capacity on a molar basis compared with the fully intact MID1–2139molecule. The truncated protein formed a tetramer as revealed by gel electrophoresis and ultracentrifugation analyses. The tetrameric molecule bound IgD more than 20-fold efficiently compared with the monomeric counterpart. M. catarrhalis Bc5 was a clinical isolate from a nasopharyngeal swab culture obtained from our department (6Forsgren A. Brant M. Möllenkvist A. Muyombwe A. Janson H. Woin N. Riesbeck K. J. Immunol. 2001; 167: 2112-2120Crossref PubMed Scopus (72) Google Scholar). E. coli DH5α or BL21(DE3) (Novagen; Darmstadt, Germany) were used for most transformations. For vectors containing fragments A and B, the host BL21(DE3)-pLysS (Novagen) was used. Transformed bacteria were grown in Luria Bertani broth supplemented with kanamycin. In the case of BL21(DE3)-pLysS, chloramphenicol was also added. The expression vector pET-26b(+) was from Novagen, and the pMAL expression vector was purchased from New England Biolabs (Beverly, MA). The human IgD myeloma whole sera IgD(κ) and IgD(λ) were obtained from The Binding Site (Birmingham, England), and the IgD standard serum OTRD 02/03 was from Behringwerke (Marburg, Germany). Myeloma whole sera IgG, IgM, and IgA were included as described previously (6Forsgren A. Brant M. Möllenkvist A. Muyombwe A. Janson H. Woin N. Riesbeck K. J. Immunol. 2001; 167: 2112-2120Crossref PubMed Scopus (72) Google Scholar). Horseradish peroxidase (HRP)-conjugated goat anti-human IgD polyclonal antibodies were purchased from BioSource International (Camillo, CA), and HRP-conjugated anti-human κ- or λ-light chain polyclonal antibodies were from Dakopatts (Glostrup, Denmark). To produce a specific anti-MID902–1200antiserum, rabbits were immunized intramuscularly with 200 μg of purified recombinant protein emulsified in complete Freund's adjuvans (Difco, Becton Dickinson; Heidelberg, Germany) and boosted on days 18 and 36 with the same dose of protein in incomplete Freund's adjuvans. Blood was drawn 2 to 3 weeks later. HRP-conjugated swine anti-rabbit pAb (Dakopatts) were used as detection antibodies. Antibodies used for flow cytometry were R-phycoerythrin (RPE)-labeled anti-human CD19 (Dakopatts) mAb and the Ig-fraction of rabbit anti-human IgD-Fab (4Forsgren A. Grubb A.O. J. Immunol. 1979; 122: 1468-1472PubMed Google Scholar), which was further purified by passing through a polyclonal Ig-Sepharose column. MID was isolated and purified from M. catarrhalis Bc5 as described previously (6Forsgren A. Brant M. Möllenkvist A. Muyombwe A. Janson H. Woin N. Riesbeck K. J. Immunol. 2001; 167: 2112-2120Crossref PubMed Scopus (72) Google Scholar). Native MID in 0.05m Tris-HCl (pH 8.8) containing 0.1% Empigen® was digested with trypsin or chymotrypsin at an enzyme/protein ratio of 1:10 at 37 °C overnight or with Endoproteinase LysC at an enzyme/protein ratio of 1:50 (Sigma) at 30 °C overnight. The digestion mixtures were run on SDS-PAGE, and protein bands were then transferred electrophoretically to Immobilon-P membranes (Millipore, Bedford, MA) and analyzed for IgD binding. The fragments were sequenced with an Applied Biosystems (Foster City, CA) 470A gas-liquid-solid-phase sequenator (11Hewick R.M. Hunkapiller M.W. Hood L.E. Dreyer W.J. J. Biol. Chem. 1981; 256: 7990-7997Abstract Full Text PDF PubMed Google Scholar). The resulting sequences were for trypsin TAQANTESSIAVG, GNTATNFSVNSGDDNALIN, and QGINEDNAFVKGLEK, and for chymotrypsin PSTVKADN. When MID was digested with LysC, the sequence MID533–1172 was obtained by mass spectrometry (12Wilm M. Shevchenko A. Houthaeve T. Breit S. Schweigerer L. Fotsis T. Mann M. Nature. 1996; 379: 466-469Crossref PubMed Scopus (1515) Google Scholar). All truncated MID constructs were manufactured using PCR-amplified fragments.Taq DNA polymerase was from Roche Molecular Biochemicals, and PCR conditions were as recommended by the manufacturer. The open reading frame of the mid gene fromM. catarrhalis (pET26-MID) was used as template (6Forsgren A. Brant M. Möllenkvist A. Muyombwe A. Janson H. Woin N. Riesbeck K. J. Immunol. 2001; 167: 2112-2120Crossref PubMed Scopus (72) Google Scholar). All MID constructs, expect for MID367–590 (fragment C), were amplified by PCR using specific primers introducing the restriction enzyme sites BamHI and HindIII. Because fragment C has an internal HindIII site, an XhoI was used instead of HindIII at the 3′-end. The PCR products were cloned into pET26b(+), except for fragment I, which was cloned into pMAL-c2. To avoid presumptive toxicity, the resulting were first into the host E. coli the for the MID fragments were into the BL21(DE3) or All constructs were sequenced using the To produce recombinant proteins, bacteria were grown to followed by of with resulting in of the were and the recombinant proteins were purified on containing a (Novagen) to the for native I, which was cloned into the was purified on England The of proteins were using the The resulting proteins were then by SDS-PAGE, and dot blots. was run as described previously (6Forsgren A. Brant M. Möllenkvist A. Muyombwe A. Janson H. Woin N. Riesbeck K. J. Immunol. 2001; 167: 2112-2120Crossref PubMed Scopus (72) Google Scholar). of purified proteins were also with Tris-HCl (pH 8.8) at and run at native conditions in Tris-HCl in native (pH at using and from were with In the proteins were electrophoretically transferred for from a gel to an Immobilon-P membrane with full-length MID were at 30 overnight. the membrane was with in with 0.1% for 2 four in the membrane was with IgD myeloma serum human myeloma The Binding for HRP-conjugated goat anti-human IgD was four and for The Ig proteins were in containing and the were at the membrane was with four was with Western detection and with a Molecular of tetrameric and monomeric of MID962–1200 on and Western was with the from proteins in in of Tris-HCl (pH were to membranes Germany) using a dot To the truncated proteins with other and with full-length MID, the same on a molar basis was to the a at membranes were as the Western In the strongly IgD-binding recombinant protein MID962–1200 was in and to the were and with human myeloma sera μg with to for followed by four and with anti-human κ- and λ-light chain polyclonal antibodies. The membranes were then four and as described The interaction between IgD and the truncated proteins was also in were with an IgD myeloma serum μg of in in Tris-HCl (pH at °C overnight. The were four with and for 2 at with with four the were for at with 2 μg of the proteins in the were and with rabbit in at HRP-conjugated swine anti-rabbit pAb was and at for four the were and at peripheral blood were isolated from blood by were as described previously (6Forsgren A. Brant M. Möllenkvist A. Muyombwe A. Janson H. Woin N. Riesbeck K. J. Immunol. 2001; 167: 2112-2120Crossref PubMed Scopus (72) Google Scholar). recombinant MID962–1200 (fragment in with to was with in at and the was and to a with PBS, The resulting was used for binding μg of the fragment was with in of containing serum on for 30 followed by with specific anti-human CD19 In the were with 30 μg of anti-human IgD-Fab polyclonal antibodies for 30 on and were with recombinant MID962–1200 protein and anti-human CD19 the were analyzed in an flow analysis was in a with using an in containing used for native purified experiments were at two and was were with at or of were and the was at was for of the molar mass of the protein were by a for a to with the Google Scholar). were to the corresponding molar using the specific from the amino acid of the protein Rowe Ultracentrifugation in and of Scholar). of were to using the by of amino acids were randomly into the IgD-binding of MID (fragment using the to the Because the vector has an site, MID962–1200 was first into CA) using the restriction enzyme sites BamHI and HindIII. The was with by a purified The for at 30 °C followed by of by the for chloramphenicol the easily the was digested with BamHI and HindIII and by gel The containing were further cloned into followed by into The resulting were and the DNA was The was by digestion with DNA was and the into The resulting containing five amino acids at positions were for specific IgD-binding capacity using membranes Germany) were on the with E. coli bacteria on the membranes were with for 30 The membranes were and with IgD as described for Western blots. To the of the five amino acids, the fragment was amplified by PCR using and The resulting PCR products were sequenced using electrophoresis on a and a and of the resulting DNA sequences were using and The structure of the MID962–1200 fragment was using a at the for Google Scholar). In a we demonstrated that MID specifically binds human IgD (6Forsgren A. Brant M. Möllenkvist A. Muyombwe A. Janson H. Woin N. Riesbeck K. J. Immunol. 2001; 167: 2112-2120Crossref PubMed Scopus (72) Google Scholar). To which of MID interacts with native MID isolated from M. Bc5 was digested with or The protein mixtures were to and transferred to membranes followed by with IgD and HRP-conjugated polyclonal antibodies. bands of were amino acid positions in MID digestion in a IgD-binding and and bands that not with IgD. Furthermore, chymotrypsin a digestion with LysC a IgD-binding of the MID fragment the of the IgD-binding sequence by with the experiments that an IgD-binding was approximately between amino acid residues and To in more the MID IgD-binding region, sequences derived from the full-length MID were cloned and expressed in E. The recombinant proteins the MID and the and positions were as shown in The recombinant proteins comprising amino acid residues of the or of MID not bind IgD as revealed in Western and dot blots. In contrast, the protein fragment attracted strongly suggesting that the IgD-binding of MID was that To the sequence for the IgD the truncated was at the and of the recombinant proteins were compared with native full-length isolated fromM. The recombinant proteins were in to and with human IgD. a molar an essentially preserved IgD-binding capacity was detected for the truncated MID protein from amino acid 962 to The shortest truncated protein with IgD was between and (fragment The IgD binding was lost the was reduced to the (fragment or the was to (fragment fragment with a and a with fragment was also manufactured and However, truncated MID not with suggesting that the binding capacity was on a To further the specific IgD an was using human IgD as All of the recombinant truncated MID fragments were to followed by with a specific rabbit The was using HRP-conjugated goat anti-rabbit polyclonal antibodies. The same as with the dot was fragments and were not attracted to the the other fragments bound to a compared with full-length MID In a we with and that native MID is and as an (6Forsgren A. Brant M. Möllenkvist A. Muyombwe A. Janson H. Woin N. Riesbeck K. J. Immunol. 2001; 167: 2112-2120Crossref PubMed Scopus (72) Google Scholar). To a analysis on MID was MID, which was in the Empigen® was to at two in an with molar mass of was and is that native MID an In to full-length MID, we the truncated MID962–1200 to a The obtained were compared with the for a tetramer or The for MID962–1200 protein as as the for and tetramer are shown in A. The MID962–1200 at with an mass of 3 was with a protein tetramer MID962–1200 including a has a mass of as from the amino acid To the structure of MID962–1200 for IgD the recombinant protein was to analysis in gel electrophoresis native In addition, a corresponding IgD Western analysis was MID962–1200 as a protein with an mass of in the native gel the ultracentrifugation Thus, in to the the with the native gel suggested that MID962–1200 in native was a tetramer of four Furthermore, using native the tetrameric structure of MID962–1200 strongly bound IgD 3 To further the for a tetramer structure to an optimal IgD MID962–1200 was at or °C followed by analysis on and Western blots. a and a tetramer at °C The tetrameric structure at °C and in a monomeric which displayed a binding to IgD in Western A and To the of the tetramer to bind IgD in with the monomeric the MID962–1200 fragment was to analysis at °C in The protein was to SDS-PAGE, and the IgD binding was analyzed by Western blots. and were analyzed by and the protein of the was with the corresponding tetramer The obtained was to the of total protein on the Interestingly, IgD binding to the tetrameric monomeric were a more binding to IgD was with the tetrameric MID962–1200 The MID962–1200 sequence was to analysis A possible structure of the truncated MID including four and was sequence analysis suggested that a in the number of amino acids at the and of MID962–1200 the a IgD binding that five amino acid residues were randomly into MID962–1200 using transposons and a in A of the coli were for IgD and were were binding and When the sequence was with the five amino acids, IgD binding was completely sequence that was of the molecule was also to important for IgD binding. Interestingly, sequences included α-helix In contrast, MID962–1200 attracted IgD five amino acid residues were the or between and two sequences not containing of amino acids the of the not IgD a that was with the truncated MID fragments than analysis revealed that five amino acid residues were into sequences for α-helix that structure was Thus, the structure analysis compared with the obtained with transposons the with the truncated MID protein series human IgD but not from the other (6Forsgren A. Brant M. Möllenkvist A. Muyombwe A. Janson H. Woin N. Riesbeck K. J. Immunol. 2001; 167: 2112-2120Crossref PubMed Scopus (72) Google Scholar). To the of the interaction between IgD and recombinant a dot was The membrane was with the truncated MID protein followed by with IgG, IgM, or IgA myeloma sera. shown in with the IgD myeloma sera, binding was not detected to myeloma sera the IgG, IgM, or IgA The was to truncated MID also was attracted to the IgD MID962–1200 was to followed by with purified In cells were with monoclonal antibodies. were to flow cytometry Interestingly, MID962–1200 bound to the B To that specifically with the IgD BCR, were also with anti-human IgD-Fab polyclonal antibodies. in the IgD binding was completely the IgD BCR was by anti-IgD-Fab were in with the previously on full-length MID (6Forsgren A. Brant M. Möllenkvist A. Muyombwe A. Janson H. Woin N. Riesbeck K. J. Immunol. 2001; 167: 2112-2120Crossref PubMed Scopus (72) Google Scholar). The outer membrane protein MID from M. catarrhalis recently has been and shown to display a and specific affinity for human IgD (6Forsgren A. Brant M. Möllenkvist A. Muyombwe A. Janson H. Woin N. Riesbeck K. J. Immunol. 2001; 167: 2112-2120Crossref PubMed Scopus (72) Google Scholar). In the present we proteolytic fragments of MID and expressed proteins to that the IgD binding was essentially preserved in a truncated recombinant protein between amino acid residues 962 and 1200 of MID The mass of the smallest protein binding IgD comprised 157 amino The IgD-binding capacity of the recombinant proteins was to the binding with the the binding is on a specific structure of the IgD-binding was further by our ultracentrifugation and gel electrophoresis We have shown previously that native MID isolated fromM. in to the 200-kDa a that is an with a mass of in (6Forsgren A. Brant M. Möllenkvist A. Muyombwe A. Janson H. Woin N. Riesbeck K. J. Immunol. 2001; 167: 2112-2120Crossref PubMed Scopus (72) Google Scholar). When purified MID in the Empigen® was analyzed by an mass of was mass was with a tetramer of MID in the presence of The smallest truncated MID protein fragment with an essentially preserved IgD binding with full-length and which analyzed by ultracentrifugation in the of was shown to a tetramer native Interestingly, tetrameric and monomeric of MID962–1200 were by and analyzed for IgD the tetramer was shown to IgD more efficiently compared with the monomeric form. Thus, the tetrameric structure was more compared with the We fully that the monomeric protein was completely to a tetrameric structure the The MID for IgD binding is compared with other surface proteins that bind immunoglobulins. Nonimmune Ig binding was first observed with the of protein A that IgG A. J. J. Immunol. Google Scholar). A interacts with the of IgG, but with and J. Immunol. Google Scholar). In addition, protein A binds a of Ig molecules of classes of binding to the M. J. Immunol. Google Scholar). A is a protein of five as and an of the consists of amino When one of the were cloned and expressed in E. was discovered that protein the and J. Immunol. Google Scholar). In addition, that binding of not with binding or In to protein protein in C and binds with to human of IgG L. J. Immunol. Google Scholar). The smallest of protein binding IgG derived from the is residues and to amino acid residues in the M. S. T. H. L. S. A. PubMed Scopus Google Scholar). However, in with our with MID, the in binding compared with protein as a whole L. J. Biol. Chem. Full Text PDF PubMed Google Scholar). derived from to of IgG, IgM, and IgA L. J. Biol. Chem. Full Text PDF PubMed Google Scholar). The Ig binding is through L. J. Immunol. 1988; Google Scholar), of amino acid residues. Ig-binding protein derived from A binds to human IgA and IgG of L. J. J. Biol. Chem. Full Text PDF PubMed Google Scholar). The of protein recently has been characterized in and shown to of amino acids E. T. J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). However, protein was to the same residues as protein which not with IgA in When amino acid residues were at of the acid sequence and in a a and the resulting acid strongly with to optimal IgD binding and into possible the binding capacity of to bind of binding and formed as a of that binding The that IgD binding to truncated MID proteins 157 amino acid residues or more not that of amino acids are in the IgD binding. However, we randomly five amino acid residues using the amino acids in two sequence completely IgD binding. between the two amino acid were The with the ultracentrifugation experiments that the tetrameric structure of the molecule is of for the IgD our that the tetramer of in the of the binding In we have shown that the IgD-binding of the MID protein a 238-amino acid sequence that is not in Furthermore, tetramer is of for optimal IgD binding. We Brant for and for ultracentrifugation and Ek and of for mass spectrometry analyses.
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