Catarrhalis Moraxella Igd-Binding Protein from Isolation And

Isolation and Characterization of a Novel IgD-Binding Protein from Moraxella catarrhalis1

Arne Forsgren, Marta Brant, Andrea Mo¨llenkvist, Anthony Muyombwe, Håkan Janson, Nicolas Woin, and Kristian Riesbeck2

A novel surface protein of the bacterial species Moraxella catarrhalis that displays a high affinity for IgD (MID) was solubilized in Empigen and isolated by ion exchange chromatography and gel filtration. The apparent molecular mass of monomeric MID was estimated to ϳ200 kDa by SDS-PAGE. The mid gene was cloned and expressed in Escherichia coli. The complete mid nucleotide gene sequence was determined, and the deduced amino acid sequence consists of 2123 residues. The sequence of MID has no similarity to other Ig-binding proteins and differs from all previously described outer membrane proteins of M. catarrhalis. MID was found to exhibit unique Ig-binding properties. Thus, in ELISA, dot blots, and Western blots, MID bound two purified IgD myeloma proteins, four IgD myeloma sera, and finally one IgD standard serum. No binding of MID was detected to IgG, IgM, IgA, or IgE myeloma proteins. MID also bound to the surface-expressed B cell receptor IgD, but not to other membrane molecules on human PBLs. This novel Ig-binding reagent promises to be of theoretical and practical interest in immunological research. The Journal of Immunology, 2001, 167: 2112–2120.

oraxella catarrhalis is a Gram-negative diplococcus that IgG-Fc with SpA and protein G (9). S. pyogenes produce an an- for a long time was considered a relatively harmless tigenically and functionally heterogenous group of M-like proteins M commensal in the respiratory tract. At present, it is the with different Ig-binding specificities. Proteins expressed by some third most frequent cause of otitis media and also a significant strains bind IgA instead of IgG or both IgG and IgA (10). Protein agent in sinusitis and lower respiratory tract infections in adults Bac, or the B Ag, is an IgA-binding protein expressed by certain with pulmonary disease. M. catarrhalis is also one of the most strains of group B streptococci (11). Finally, protein L, a surface common inhabitants of the pharynx of healthy children (1, 2). component of Peptostreptococcus magnus, has affinity for all Since the discovery of the first Ig-binding bacterial protein, classes of Ig through an interaction with determinants present in 3 Staphylococcus aureus protein A (SpA) (3), this protein has been the variable region of ␬ L chains (12). extremely well characterized (4). The ability of SpA to bind the Fc In contrast to Gram-positive bacteria, nonimmune Ig binding to part of IgG is well known, but SpA also binds a fraction of Ig Gram-negative bacteria are more rare. However, reports on bovine molecules of all classes, due to the so-called “alternative” binding, IgM binding to Brucella abortus, equine IgG binding to Taylorella which represents an interaction with the V chains (5). All IgG- H equigenitalis, and bovine IgG and IgM binding to Haemophilus binding capacity of S. aureus has been considered to be mediated somnus have been published (13Ð15). An Ig FcR has also been by SpA. However, the existence of a second gene in S. aureus encoding an Ig-binding protein was recently reported (6). purified from H. somnus (16). Two decades ago, H. influenzae and Protein G isolated from group C and G streptococci of human M. catarrhalis were shown to display a strong affinity for soluble origin has a distinct affinity for the same site on the human Fc human IgD (17). IgD binding at the cellular level explains the fragment of IgG as SpA and also interacts with IgG Fab (7, 8). An strong mitogenic effects on human lymphocytes by H. influenzae M-like protein, protein H, isolated from group A streptococci and M. catarrhalis (18Ð20). In addition, it was demonstrated that (Streptococcus pyogenes) is able to compete for the same region of M. catarrhalis stimulates the proliferation of high density (mature) B lymphocytes expressing high levels of IgD and that soluble non- mitogenic mAbs reactive with human IgD selectively inhibit the B lymphocyte response. Inhibition by anti-IgD mAb presumably re- Department of Medical Microbiology, Malmo¬ University Hospital, Lund University, Malmo¬, Sweden sulted from covering/capping surface IgD on B lymphocytes, Received for publication March 13, 2001. Accepted for publication June 4, 2001. thereby eliminating the bacteria-dependent stimulatory signal de- The costs of publication of this article were defrayed in part by the payment of page livered through the B cell receptor (BCR) IgD. An IgD-binding charges. This article must therefore be hereby marked advertisement in accordance outer membrane protein (OMP) from H. influenzae (protein D) was with 18 U.S.C. Section 1734 solely to indicate this fact. isolated and cloned and shown to be an important pathogenicity 1 This work was supported by grants from the Alfred O¬ sterlund Foundation, the Anna and Edwin Berger Foundation, the Crafoord Foundation, the Greta and Johan Kock factor (21Ð24). However, protein D does not bind to the majority Foundation, the Magnus Bergvall Foundation, the Swedish Medical Research Coun- of IgD myelomas tested, and it was suggested that encapsulated H. cil, and the Cancer Foundation at Malmo¬ University Hospital. influenzae of serotype b expresses an additional IgD receptor (25). 2 Address correspondence and reprint requests to Dr. Kristian Riesbeck, Department The present work describes the isolation and cloning of the M. of Medical Microbiology, Malmo¬ University Hospital, Lund University, S-205 02 Malmo¬, Sweden. E-mail: [email protected] catarrhalis IgD-binding protein (MID). We also characterize the 3 Abbreviations used in this paper: SpA, Staphylococcus aureus protein A; BCR, B Ig-binding properties of this molecule, which were found to be cell receptor; OMP, outer membrane protein; MID, M. catarrhalis IgD-binding pro- different compared with previously isolated Ig-binding bacterial tein; IEF, isoelectric focusing; IPG, immobilized pH gradient; CHAPS, 3-[(3 cholamidopropyl)dimethylammonio]-1-propanesulfonate; IPCR, inverse PCR; IPTG, proteins. MID has similarities but nonidentity with previously de- isopropyl-1-thio-␤-D-galactoside. scribed proteins from M. catarrhalis.

Materials and Methods isotype-specific detection Abs for respective myeloma sera were supple- Reagents mented as described above. In a third set of experiments, microtiter wells were coated with highly purified IgD myeloma protein, and after washing M. catarrhalis strain Bc5 was a clinical isolate from a nasopharyngeal and blocking, HRP-labeled anti-Ig Abs were added. swab culture taken by our department (Department of Medical Microbiol- ogy, Malmo¬ University Hospital, Lund University, Malmo¬, Sweden). The Dot blot assays human purified Ig preparations IgG1-␬, IgG1-␭, IgG2-␬, IgG2-␭, IgG3-␬, ␮ ␮ ␭ ␬ ␭ ␬ ␭ ␬ ␭ ␬ ␭ Purified MID (0.0005Ð0.2 g) in a volume of 100 l in 0.1 M Tris-HCl IgG3- , IgG4- , IgG4- , IgA1- , IgA1- , IgA2- , IgA2- , IgM- , IgM- , (pH 9.0) was manually applied to nitrocellulose membranes by using a dot IgD-␬, IgD-␭, IgE-␬, and, finally, IgD myeloma whole sera IgD-␬ and ␭ blot apparatus (Schleicher & Schu¬ll, Dessel, Germany). After saturation, IgD- were purchased from The Binding Site (Birmingham, U.K.). The the membranes were incubated for2hatroom temperature in PBS-Tween IgD standard serum OTRD 02/03 was obtained from Behringswerke (Mar- ␭ ␭ containing 1% OVA and 5% milk powder and washed four times with burg, Germany). Myeloma whole sera IgD- A, IgD- B, IgG A, IgG B, PBS-Tween. Human myeloma protein (0.5 ␮g) in 100 ␮l PBS-Tween was IgG C, IgM, IgA A, and IgA B were obtained from the Department of added and, after2hofincubation, followed by several washings in PBS- Clinical Chemistry at Malmo¬ University Hospital. HRP-conjugated goat Tween, HRP-labeled anti-human L chains (␬ and ␭) (DAKO) diluted 1/200 anti-human IgD was obtained from BioSource International (Camarillo, were used as secondary Abs. In another set of experiments, dilutions of CA). FITC-conjugated mouse anti-human IgD, unlabeled rabbit anti- human myeloma sera in a volume of 100 ␮l in 0.1 M Tris-HCl (pH 9.0) human IgD, HRP-conjugated rabbit anti-human IgA, IgG, and IgM, as well were first applied to the membranes. After saturation, incubations, block- as HRP-labeled rabbit anti-mouse Ig were purchased from DAKO (Gen- ing, and washing, steps were performed as described above. Thereafter, tofte, Denmark). Goat anti-human IgD and HRP-conjugated rabbit anti- 125I-labeled protein MID probe (5Ð10 ϫ 105 cpm/ml) in PBS-Tween was human polyvalent Igs were from Sigma (St. Louis, MO). PE-conjugated added. After overnight incubation, the membrane was washed four times mouse anti-human CD3 and CD19 were obtained from BD Biosciences with PBS-Tween, air-dried, and exposed to Kodak CEA.C x-ray films (San Jose, CA). Mouse mAbs 17C7 (UspA) and 10F3 (CopB) (26, 27) (Eastman Kodak, Rochester, NY) at Ϫ70¡C using an intensifying screen. were kindly provided by Dr. E. J. Hansen (Department of Microbiology, University of Texas, Dallas, TX). Extraction and purification of IgD-binding protein SDS-PAGE and detection of proteins on membranes (Western M. catarrhalis bacteria (1Ð5 ϫ 1011 CFU/ml) were suspended in 0.05 M blot) Tris-HCl buffer (pH 8.8) containing 3% Empigen (Calbiochem, Bedford, MA). In some experiments, Empigen was replaced by CHAPS (Sigma), SDS-PAGE was run at 150 constant voltage using 10% (bis)Tris gels with N-octyl-p-D-glucosidase (Bachem, Budendorf, Switzerland), or Triton running (MES), sample (lithium dodecyl sulfate), and transfer buffer as X-100 (Sigma). All these detergents at a concentration of 3% were tested well as a blotting instrument obtained from NOVEX (San Diego, CA). with or without 0.01 M EDTA. The bacterial suspensions were mixed by Samples were regularly heated at 100¡C for 10 min and, in some experi- magnetic stirring for2hat37¡C. After centrifugation at 8000 ϫ g for 20 ments, at 100¡Cfor1horat70¡C for 10 min. Gels were stained with min at 4¡C, the supernatants were filtrated with sterile filters (0.45 ␮m; Coomassie brilliant blue R-250 (28) (Bio-Rad, Sundbyberg, Sweden). Sterivex-HV; Millipore). M. catarrhalis extract in 3% Empigen was ap- Electrophoretical transfer of protein bands from the gel to an Immobilon-P plied to a Q-Sepharose column (Amersham Pharmacia Biotech) equili- membrane (Millipore, Bedford, MA) was conducted at 30 V for 2Ð3 h. After brated with 0.05 M Tris-HCl (pH 8.8) containing 0.1% Empigen. The transfer, the immobilon-P membrane was blocked in PBS with 0.05% Tween column was eluted using a 0- to 1-M NaCl linear gradient in the same 20 (PBS-Tween) containing 5% milk powder. After several washings in PBS- buffer. Fractions showing the most IgD binding were pooled, dialyzed in Tween, the membrane was incubated with purified IgD myeloma protein (0.5 ␮ ␬ Spectra/Por membrane tubes (Spectrum, Houston, TX; molecular mass cut- g/ml human IgD- myeloma; The Binding Site) in PBS-Tween including 2% off, 25 kDa) against 0.05 M Tris-HCl (pH 8.8), concentrated on YM100 milk powder for1hatroom temperature. HRP-conjugated goat anti-human disc membranes (Amicon, Beverly, MA; molecular mass cut-off 100 kDa), IgD diluted 1/1000 was added after several washings in PBS-Tween. After and then applied to a Sephacryl S-400 high resolution column (20 by 900 incubation for 40 min at room temperature and several additional washings in mm; Amersham Pharmacia Biotech) and equilibrated with 0.05 M Tris- PBS-Tween, development was performed with ECL Western blotting detec- HCl (pH 8.8) containing 0.1% Empigen. Fractions containing the strongest tion reagents (Amersham Pharmacia Biotech, Uppsala, Sweden). IgD-binding activity were concentrated and rechromatographed as de- To determine the isoelectric point, a first-dimension isoelectric focusing scribed above. (IEF) was conducted using the IPGphor IEF system (Amersham Pharmacia Biotech). Sample application (before rehydration) and rehydration of Peptide cleavage and amino acid sequence analysis immobilized pH gradient (IPG) strips (7 cm, pH 3Ð10, nonlinear) was performed according to the manufacturer’s instructions. The rehydration Purified MID in 0.05 M Tris-HCl (pH 8.8) containing 0.1% Empigen was buffer consisted of 8 M urea, 0.5% (w/v) 3-[(3 cholamidopropyl)dimeth- treated with trypsin or chymotrypsin in an enzyme:protein ratio of 1:10 at ylammonio]-1-propanesulfonate (CHAPS), 0.2% (v/v) IPG buffer, 15 mM 37¡C overnight. The cleavage mixtures were subjected to SDS-PAGE, and DTT, and bromophenol blue (29). The IEF was run at 20¡C, and the fol- peptide bands transferred to Immobilon-P membranes were automatically lowing settings were used: 30 V for 12 h, 200 V for 1 h, 500 V for 1 h, 1000 sequenced. To get an N-terminal sequence of the protein, deblocking of V for 30 min, and 8000 V for 2 h. IPG strips were first equilibrated with intact MID from a possible pyroglutamate group was attempted (30, 31). 65 mM DTT in equilibration buffer consisting of 6 M urea, 30% (w/v) Automated amino acid sequence analysis was performed with an Applied glycerol, 2% (w/v) SDS, 50 mM Tris-base (pH 8.8), and bromophenol blue Biosystems (Foster City, CA) 470A gas-liquid-solid-phase sequenator (32). for 15 min at 20¡C. The second equilibration was performed with 22 mM iodoacetamide in equilibration buffer. IPG strips were transferred to 10% Labeling of protein MID SDS-polyacrylamide gels that were further handled as described above. To calibrate the gels, a two-dimensional SDS-PAGE standard was used (Bio- Purified MID was radioiodinated (Amersham, Little Chalfont, U.K.) to Rad, catalog number 161-0320). high specific activity (0.05 mol iodine per mol protein) with lactoperoxi- dase (33). FITC (Sigma) was conjugated to purified MID using a standard ELISA protocol. Briefly, MID (2 mg/ml) in 0.1 M carbonate buffer (pH 9.5) was Extracts of M. catarrhalis diluted in 5-fold steps in 0.1 M Tris-HCl (pH ␮ 9.0) were added in 100- l volumes to microtiter plates (F96 Maxisorb; Table I. Amino acid sequences derived from highly purified MID after Nunc, Roskilde, Denmark) and incubated at 4¡C overnight. After washing protease digestionsa the plate four times in PBS-Tween, blocking buffer (PBS-Tween containing 1.5% OVA) was added. The plate was incubated for 1 h at room temperature and further washed four times with PBS-Tween. IgD-␬ my- Peptide Sequence Protease eloma protein, (0.05 ␮g) in 100 ␮l PBS-Tween containing 1.5% OVA was added to each well, and after incubation for1hatroom temperature, the TAQANTESSIAVG Trypsin plate was washed four times with PBS-Tween. After incubation with HRP- GNTATNFSVNSGDDNALIN Trypsin conjugated goat anti-human IgD diluted 1/1000 and subsequent washings, QGINEDNAFVKGLEK Trypsin plates were developed and measured at 450 nm. In another set of experi- PSTVKADN Chymotrypsin ␮ ments, purified MID in volumes of 100 l was added to microtiter plates. a MID was incubated with trypsin or chymotrypsin followed by separation on After incubation, washing, and blocking, dilutions of human myeloma sera SDS-PAGE. Resulting peptides were transferred to nylon membranes and automati- in a volume of 100 ␮l were added, and after incubation and washing, cally sequenced by Edman degradation. 2114 Moraxella catarrhalis IgD-BINDING PROTEIN

Table II. Summary of Western and dot blot analyses showing the binding specificity of highly purified commercially available myeloma IgD preparations against a crude Empigen extract of M. catarrhalis and highly purified MIDa

200-kDa Protein in Ig Crude Extract Puriﬁed MID

IgD-␬, IgD-␭ ϩϩ IgG1-␬, IgG1-␭ ϪϪ IgG2-␬, IgG2-␭ ϪϪ IgG3-␬, IgG3-␭ ϪϪ IgG4-␬, IgG4-␭ ϪϪ IgA1-␬, IgA1-␭ ϪϪ IgA2-␬, IgA2-␭ ϪϪ IgM-␬, IgM-␭ ϪϪ IgE-␬ ϪϪ FIGURE 1. Chromatography and rechromatography on a Sephacryl a S-400 column of Empigen-soluble extract from M. catarrhalis after ion Moraxella proteins were isolated and separated on SDS-PAGE or dot-blotted directly to membranes. After gel electrophoresis, proteins were transferred to nylon exchange chromatograpy. The solid line indicates protein content of the membranes followed by blocking and washing steps. Subsequent to incubation with first chromatography, and the broken line indicates rechromatography of the different Igs indicated, HRP-conjugated-mouse anti-human polyvalent Igs were the first peak. V0, Void volume. applied. Finally, filters were developed using ECL reagents.

incubated with 0.15 ␮g/ml FITC solubilized in DMSO. After 45 min at tion enzymes, which were used separately: EcoRV, SphI, and PstI for the room temperature and constant stirring, the sample was diluted and sub- isolation of the start codon; and AccI, AsuI, and HincII for the isolation of jected to a PD10 column (Amersham Pharmacia Biotech) pre-equilibrated the stop codon sequences. The resulting fragments were religated upon with PBS (pH 7.4). The resulting MID-FITC was used for binding studies. themselves (Rapid DNA Ligation Kit; Roche), and the DNA was used in IPCR. To amplify the start and stop codon areas of the gene, specific DNA isolation and sequencing primers were designed and used in a long-template PCR (Expand Long- DNA was extracted from M. catarrhalis Bc5 using a genomic DNA prep- Template PCR System; Roche). All PCR products were cloned into pPCR- aration kit (Qiagen, Hilden, Germany). Degenerate primers were synthe- Script-Amp (Stratagene, La Jolla, CA) and sequenced. The signal peptide sized according to the amino-terminal sequences from four peptide frag- was deduced using the SignalP V1.1 Prediction Server Center for Biolog- ments (Table I). In some of the PCR (High Fidelity PCR System; Roche, ical Sequence Analysis (http://www.cbs.dtu.dk/services/SignalP/). Bromma, Sweden), specific primers were used in combination with the degenerate ones. DNA sequences flanking the central region of the gene, Expression of the MID gene product and cell fraction from where the peptide fragments originated, were isolated using inverse The complete 6.4-kb open reading frame of the mid gene was ligated into PCR (IPCR) (34). Genomic DNA was cleaved with the following restric- pET16(b) (Novagen, Darmstadt, Germany). To express the mid gene product, pET16-MID was transformed into the expression host BL21DE3, containing a chromosomal copy of the T7 RNA polymerase gene under lacUV5 control. Overexpression was achieved by growing cells to loga- rithmic-growth phase followed by addition of isopropyl-1-thio-␤-D-galactoside (IPTG). After4hofinduction, bacteria were sonicated according to a standard protocol, and the resulting proteins were analyzed by SDS- PAGE. Localization of recombinant protein from pET16-MID was conducted by osmotic shock as previously described (35).

FIGURE 2. Analysis on SDS-PAGE of fractions representing different purification steps of MID. A, The fractions are shown for crude extract in 3% Empigen after an exchange chromatography on Q-Sepharose column and after the first and second gel filtrations on a Sephacryl S-400 column. B, SDS gel electrophoresis of MID protein pretreated in sample buffer at FIGURE 3. Binding of MID to human myeloma sera representing dif- 70¡Cor100¡C for 10 min. A, Two gels were run simultaneously; one was ferent Ig classes. All sera were diluted in 2-fold steps (4Ð0.03 ␮g) and stained with Coomassie brilliant blue (Stain), and one was blotted onto applied to a nitrocellulose membrane. The following myeloma whole sera Immobilon-P membranes, probed with human IgD-␬ myeloma protein were used; IgD-␬, IgD-␭, IgD Behringswerke (IgD B.W.), IgD-␭ A, IgD-␭ (IgD), anti-UspA (␣Usp), or anti-CopB (␣B) mAbs followed by incubation B, IgG A, IgG B, IgG C, IgM, IgA A, and IgA B. After saturation, washing, with appropriate HRP-conjugated secondary Abs. B, The gel was stained and blocking, an 125I-MID-labeled probe was added. After overnight in- with Coomassie brilliant blue. Molecular mass of marker proteins is indi- cubation and additional washings, specific MID-IgD binding was visual- cated to the left in both panels. ized by autoradiography. The Journal of Immunology 2115

Flow cytometry Human PBLs were isolated from heparinized blood from healthy donors as previously described (36). For flow cytometric analyses, a standard staining protocol was used with 0.5% BSA (w/v) in PBS as buffer. PBLs (2.5 ϫ 105 in 100 ␮l) were labeled with anti-CD3 or anti-CD19 mAbs with or without FITC-conjugated anti-IgD mAb on ice for 30 min according to the manufacturers’ instructions. In neutralization experiments, MID was preincubated with IgD-␬ at 37¡C for 30 min and thereafter directly added to the cells followed by incubation on ice. After two washes, 10 ␮g/ml purified FITC-conjugated MID was supplemented to the cells, followed by incubation for 45 min on ice. After final washes, 105 cells for each sample were analyzed in an EPICS XL-MCL flow cytometer (Beckman Coulter, Hialeah, Florida). Where appropriate, rabbit and goat preimmune sera and mouse IgG1 and IgG2a were included as negative controls (DAKO).

Results Extraction and purification of MID Solubilization of MID was a major obstacle in the process of purification. Among several detergents tested, only Empigen and N- octyl-p-D-glucosidase alone, at a final concentration of 3%, efficiently solubilized MID from a suspension of M. catarrhalis,as estimated by ELISA and Western blot. The two detergents were equally efficient. Triton X-100 alone did not solubilize MID, but Triton X-100 plus 0.01 M EDTA efficiently solubilized MID. CHAPS alone or CHAPS with EDTA or EDTA alone did not solubilize MID. In the following experiments, Empigen extraction was used for solubilization and subsequent purification of MID. When the Empigen extract of M. catarrhalis was applied to a Q-Sepharose column, all IgD-binding material was eluted from the column with 0.1% Empigen in 0.05 M Tris-HCl (pH 8.8). No additional IgD-binding material could be eluted when a NaCl gradient up to 1 M was applied to the same column. After concentration of the IgD-binding material obtained after separation on the Q-Sepharose column, fractionation of the extract was achieved by gel filtration in the presence of 0.1% Empigen on a Sephacryl S-400 column (Fig. 1). Most IgD-binding material was eluted in FIGURE 4. IgD-bearing B cells specifically bound FITC-conjugated ϩ this first peak immediately after the void volume. MID was further MID. PBLs stained with RPE-conjugated mAbs against CD19 (A)or CD3ϩ (D) followed by incubation with MID-FITC were compared with purified by rechromatography of the first peak under the same PBLs incubated with anti-CD19 mAb in addition to an anti-IgD mAb (B). conditions. E, Double staining with CD3ϩ and anti-IgD mAb. C, PBLs preincubated Fig. 2A shows that after purification, MID appeared as two with a rabbit Ig fraction against human IgD followed by addition of anti- bands, one 200-kDa band and a second band with an apparent CD19 mAb and MID-FITC. F, A control sample with no Abs or MID- molecular mass of Ͼ1000 kDa. Western blot experiments were FITC is also included. PBLs were isolated from heparinized human blood performed to confirm that MID was not identical with the previ- using LymphoPrep one-step gradients, incubated with the appropriate Abs, ously described OMPs UspA1 and 2, with an apparent molecular washed, and further incubated with MID-FITC. After final washings, PBLs mass varying from 350 to 720 kDa, (37) or CopB, with a molecular were analyzed by flow cytometry. In this particular experiment, 68% of the mass of 80 kDa (26). The crude Empigen extract of M. catarrhalis total lymphocyte population was analyzed. Less than 2% of the cells were or partly purified preparations of MID were subjected to SDS- labeled when isomatched mAbs were included as negative controls. A preimmune rabbit serum did not significantly block MID-FITC binding to the PAGE, transferred to Immobilon-P filters, and blotted with Abs to IgD BCR (data not shown). An experiment with a typical donor of three those Moraxella proteins and also with human IgD. As can be seen separate ones analyzed is shown. in Fig. 2A, MID (as revealed by IgD binding) migrated differently from the OMPs UspA and CopB. After pretreatment at 70¡C for 10 min, purified MID migrated as a single band with an apparent

FIGURE 5. Schematic map of the mid gene showing the cloning strategy. Oligonu- cleotide primers used for DNA ampliﬁcation are indicated by arrows placed above (PCR) and below (IPCR) the relevant sequences. Degenerated primers based upon the amino acid sequences outlined in Table I and spe- ciﬁc primers are shown by broken and solid lines, respectively. 2116 Moraxella catarrhalis IgD-BINDING PROTEIN

FIGURE 6. Nucleotide sequence of the mid gene from M. catarrhalis Bc5 together with the deduced amino acid sequence. Putative Ϫ35 and Ϫ10 regions, a possible ribosome binding site (rbs), the predicted signal peptide (underlined), and two alternative start codons at amino acid positions 1 and 17 are indicated. The stop codon and the inverted repeat are also shown. The Journal of Immunology 2117

FIGURE 6. (continued)

molecular mass of Ͼ1000 kDa (Fig. 2B). When MID instead was values that can be explained by a weak cross-reactivity for antisera heat treated at 100¡C for 10 min to 1 h, the typical double-band against these isotypes with IgD. Totally, MID (in different exper- pattern appeared. iments) was shown to bind seven of seven IgD sera or preparations Three attempts were made to determine the amino-terminal and none of 22 non-IgDs. amino acid sequence of purified MID. Approximately 1000 pmol Purified MID specifically attracted human soluble IgD as re- MID was applied each time in an automated amino acid sequencer. vealed in Western and dot blots (Table II and Figs. 2 and 3). To Inasmuch as no amino acid phenylthiohydantoin derivatives were test whether MID bound to the surface-expressed B cell receptor obtained, the amino-terminal end of the single MID polypeptide BCR IgD, human PBLs were isolated. FITC was conjugated to chain was probably blocked. It was recently determined that the MID and followed by incubation with PBLs for 45 min on ice. In Moraxella UspA1 and UspA2 proteins, which are also resistant to parallel, PBLs were labeled with RPE-conjugated mAbs directed Edman degradation, contained a pyroglutamyl residue that was against the T cell marker CD3 or the B cell-specific surface Ag removed by the treatment with pyroglutamate aminopeptidase CD19 and subsequently analyzed by flow cytometry (Fig. 4). In- (38). However, when MID purified from M. catarrhalis or recom- terestingly, a large fraction of CD19ϩ lymphocytes bound signif- binant MID was treated with this enzyme according to two differ- icant amounts of MID-FITC (Fig. 4A), whereas T cells (CD3ϩ ent protocols (twice for each method) and then subjected to Edman lymphocytes) only displayed a nonspecific background binding degradation, no N-terminal amino acid sequence was obtained. (Fig. 4D). The MID-FITC signal corresponded well with CD19ϩ IgD-binding properties of MID cells incubated with anti-IgD mAbs revealing IgD-postive B cells (Fig. 4B). To further elucidate the specificity of MID-FITC bind- Crude Empigen extracts of M. catarrhalis and highly purified MID ing to IgD-bearing CD19ϩ lymphocytes, PBLs were preincubated subjected to SDS-PAGE and transferred to filters were exposed to with a rabbit anti-human IgD Ig fraction. After incubation and highly purified commercially available Ig preparations represent- washings, MID-FITC binding was analyzed by flow cytometry ing all human Ig classes and subclassses. Only the two IgD prep- according to the standard procedure. The antiserum almost com- arations interacted with the MID bands in a fashion as shown in pletely inhibited specific MID-FITC binding to the IgD BCR when Fig. 2 (Table II). When dot blot experiments were performed and compared with cells incubated with the preimmune serum. Mean purified MID in dilutions was first added to membranes and puri- fluorescence intensity decreased from 79.2Ð14.6 arbitrary units. fied human myeloma proteins and secondary Abs were subse- Similar results were obtained with goat Igs raised against IgD (data quently applied, only the two IgD myelomas interacted with MID. not shown). Moreover, preincubation of MID-FITC with soluble One of the two myelomas detected as little as 0.001 ␮g of MID on IgD-␬ abolished the binding to the IgD BCR. Thus, IgD-express- the membrane. The specificity of the interaction between MID and ing B cells promoted specific MID-FITC binding to the surface- IgD was further verified by first adding dilutions of myeloma sera to the filters and then radiolabeled MID in other dot blot experi- expressed BCR IgD. ments. In Fig. 3, it is demonstrated that MID effectively bound four IgD myeloma sera. A distinct reaction could be detected in the Cloning of the mid gene and analysis of the deduced amino acid range of 0.03Ð4 ␮g of IgD. For the IgD standard serum (Behrings sequence werke), reactivity was seen at even lower concentrations (data not shown). In contrast, six different Ig myeloma sera representing Degenerate primers were designed according to the obtained ami- IgG, IgA, and IgM showed no visible reaction with MID at 4 ␮g. no-terminal sequences of four peptide fragments originating from MID was also bound to the surface of an ELISA plate, and the MID (Table I) and were used in PCR in all possible combinations same myeloma sera were added in two-step dilutions followed by followed by cloning and DNA sequencing. The specific primers isotype-specific detection Abs. The values were at least 16-fold 2982ϩ and 3692Ϫ (Fig. 5) were synthesized using the deduced higher for IgD than for the other isotypes (IgG, IgA, and IgM). sequence of a distinctive PCR product generated with the degen- However IgA, IgG, and IgM myeloma sera showed background erate primer pair 2629ϩ/3693Ϫ. A PCR using the specific primers 2118 Moraxella catarrhalis IgD-BINDING PROTEIN in combination with the degenerate ones (718ϩ and 5772Ϫ) re- sulted in 5054 bp of the gene encoding for MID. Flanking sequences surrounding the core of the mid gene were obtained by IPCR. IPCR on EcoRV- and AsuI/AccI-digested M. catarrhalis genomic DNA with the primer pairs 2982ϩ/945Ϫ and 3668ϩ/ 120Ϫ, respectively, provided the sequence for the start codon area. In addition, IPCR on HincII-digested Moraxella genomic DNA FIGURE 8. Recombinantly expressed MID retained its IgD-binding ca- with the primer-pair 5898ϩ/5511Ϫ generated the 3Ј sequence in- pacity. The left panel shows a Coomassie brilliant blue-stained gel, and the cluding the stop codon. right panel shows a Western blot probed with human IgD. Moraxella- The complete nucleotide sequence of mid comprised 6417 bp derived MID protein (MID) was run and compared with cytoplasmic (C), (Fig. 6). Two alternative open reading frames were revealed and periplasmic (P), and membrane (M) fractions. Numbers on the left indicate a molecular mass standard. E. coli BL21DE3 containing pET16-MID was start at amino acid positions 1 or 17. Consequently, the length of induced for4hbyIPTG. Cellular fractions were collected, and proteins the mid gene product might be either 2123 or 2139 aa. In addition were separated by two SDS-PAGEs that were run in parallel and either Ϫ to a putative ribosome-binding site (AAGG), 10 (TAATTA) and stained with Coomassie brilliant blue or blotted onto an Immobilon-P Ϫ35 (TTGAAT) consensus sequence boxes were identified. Fur- membrane. The membrane was probed with human IgD followed by in- thermore, 62 bases downstream of the TAA stop codon, an incubation with a HRP-conjugated secondary Ab. verted repeat was found with the potential of stem-loop formation that is necessary for transcriptional termination. The open reading frame defined a protein with a calculated molecular mass of just below 220 kDa that readily corresponded to ond, rMID (as shown by SDS-PAGE) displayed a molecular mass the empirical value of ϳ200 kDa found by SDS-PAGE. The pro- of ϳ200 kDa, corresponding to the 217 kDa calculated value from tein contained several predicted coiled coil structures. The N-ter- the amino acid sequence. Third, the recombinant protein was in- minal amino acid sequence showed the typical characteristics of a deed the mid gene product in E. coli because its IgD-binding phe- signal peptide with a potential cleavage site between amino acids notype was confirmed by Western blot analysis. Total protein from 66 and 67. Despite that the first amino acid after the signal pep- E. coli containing the induced pET16(b) vector without insert did tidase cleavage site was most likely a glutamine residue, any se- not display any IgD-binding capacity (data not shown). Fourth, the quence could not be determined by Edman degradation. Further- subcellular localization of the recombinant protein showed that more, no amino acid sequence was obtained after pyroglutamate MID was equally located in the cytoplasmic and the membrane aminopeptidase treatment. The predicted amino acid sequence was fractions, but not in the periplasmic space. The localization of MID also subjected to a hydrophobicity profile analysis by the method in the membrane fraction correlated very well with the known of Kyte and Doolittle (39) and showed mainly hydrophilic prop- outer membrane localization in M. catarrhalis. erties, except for the putative signal peptide that was strongly hydrophobic (Fig. 7). The isoelectric point of MID was determined to Discussion be 6.8. The present investigation describes the isolation, purification, characterization, cloning, and expression of a protein named MID, Expression of rMID in E. coli a novel Ig-binding protein of M. catarrhalis that has affinity for To confirm that the cloned mid gene corresponded to the purified human IgD. Early studies demonstrated that the OMPs from a IgD-binding protein, the gene including the predicted signal se- diverse collection of Moraxella isolates exhibit a high degree of quence and start codon was subcloned into the expression vector similarity (40). Investigators have primarily focused their research pET16(b) and induced with IPTG. Bacterial cells were lysed and efforts on a selected group of proteins (2). Recent studies have subfractionated, and rMID was localized by Western blots using demonstrated that the high molecular mass surface Ag, termed human IgD as a probe. Important verifying characteristics of MID UspA or HMW-OMP, is actually comprised of two different pro- were provided from the expression experiments (Fig. 8). First, fol- teins. These proteins are named UspA1 and UspA2 (27, 38). The lowing induction, cells containing pET16-MID were able to pro- apparent molecular mass of these OMPs is Ͼ350Ð700 kDa as duce rMID confirming the correct reading frame of the gene. Sec- determined by SDS-PAGE analysis. Reduction with formic acid yields bands of ϳ120Ð140 kDa, suggesting that the UspA proteins form an oligomeric complex composed of several monomeric sub- units (37). The predicted mass of each protein, as deduced from the cloned genes, is 88 kDa and 62 kDa for UspA1 and UspA2, respectively (27). It is thought that the difference in the deduced mass and the mass determined using SDS-PAGE is due to a predicted coiled coil structure (38). CopB is an 80-kDa surface-exposed major OMP that shows a moderate antigenic conservation (41). In addition, OMP CD is a 46-kDa highly conserved protein with numerous surface-exposed epitopes (42), and OMP E is a 47-kDa protein detected on a variety of heterologous strains (43). The lactoferrin-binding and transferrin-binding proteins have molecular sizes of 99Ð111 and 74Ð105 kDa, respectively (44). MID is obviously not identical with previously well-characterized OMPs of M. catarrhalis. It is not recognized by mAbs derived FIGURE 7. The hydropathy profile of MID. The hydrophobic and hy- against the UspA or CopB outer membrane Ags. MID also has a drophilic parts of the individual amino acid residues are indicated. The different migration pattern in SDS-PAGE and a different compo- predicted signal peptide is outlined. Data were obtained by using a standard sition as shown by amino acid and DNA sequence analysis. In method as previously described (39). addition, MID shows no similarity with other Ig-binding proteins The Journal of Immunology 2119 including protein D from H. influenzae. However, homology anal- IgD (48). The exact localization in the IgD molecule of the MID- yses using the GAP software from Mologen (Berlin, Germany) binding structure remains to be characterized. revealed that MID in parts has homology (48%) with USPA1 and Considering the problems with soluble IgD, our flow cytometric 2. MID appeared as a 200-kDa band in accordance with the mo- studies with cell-bound IgD are encouraging (Fig. 4). The cells lecular mass from the deduced amino acid sequence, but also as an used have always been fresh, and the reactivity with MID was extra band with an estimated molecular mass of Ͼ1000 kDa. The reproducible. Naive resting B cells display both IgM and IgD extra band indicates that native MID is an oligomeric complex in BCRs, whereas immature newly formed B cells are only IgM pos- a similar fashion as UspA (37), probably due to several stretches itive. The membrane-bound Ag receptor is identical with the se- of coiled coil structures. This is further supported by the fact that creted counterpart, except for the BCR consists of an additional MID was eluted immediately after the void volume from a stretch of hydrophobic amino acids in its C terminus. Because Sephacryl S-400 column with a fractionation range of up to 8000 sensitivity to proteolysis is a hallmark of IgD, the membrane- kDa. In a recent patent publication, an OMP of M. catarrhalis with bound receptor for Ag on the surface of differentiating B lympho- a molecular mass of ϳ200 kDa was isolated (45). A sequence cytes, proteolysis is most likely associated with the biological encoding a protein of ϳ200 kDa was also provided. However, that function of IgDs. In this process, membrane-bound IgD is believed protein sequence is not identical with the sequence provided by us to undergo proteolysis to form Fab and Fc portions. The Fab part and shows only 53.5% identity with MID. The protein was shown is then degraded, whereas the Fc fragment is endocytosed together to be immunogenic, but no further biological functions were pre- with the Ag. M. catarrhalis selectively binds the IgD BCR, and sented. In addition, a 200-kDa protein is associated with hemag- proliferation of human B lymphocytes follows its interaction with glutinating M. catarrhalis (46). However, hemagglutinins are not cell surface IgD and MHC class I molecules (17Ð19). In the universally expressed by M. catarrhalis clinical isolates (47). In present study, we showed that MID bound all surface-expressed contrast, MID can be detected in all strains of M. catarrhalis IgD BCRs on B lymphocytes isolated from human peripheral (Ͼ100; our unpublished data), albeit with various molecular blood (Fig. 4). When PBLs were preincubated with a rabbit anti- masses ranging from ϳ180 to 220 kDa. Thus, MID does not seem serum against the IgD BCR followed by incubation with MID- to be identical with either the hemagglutinin or the 200-kDa FITC, a faint residual binding was observed, although the mean protein. fluorescence intensity significantly decreased from 79.2 to 14.6 Human IgD, unlike other Igs, has been poorly studied due to the (Fig. 4, A and C). However, it is evident that MID is not responsible low concentration in normal serum and its great susceptibility to for the earlier-observed MHC class I-binding activity of M. catarrha- proteolytic degradation, which makes it further difficult to purify lis (20), because no specific binding occurred to T lymphocytes (Fig. and quantify (48). In a study of the sera from ϳ50 myeloma pa- 4D). tients, fragmented IgD was present in all cases, and usually this An increased number of IgD immunocytes have been observed was the predominant form. In a study from Putnam’s laboratory in the lymphoid tissue from nasopharyngeal tonsils, lacrimal and (49), high pressure liquid chromatography was used to investigate parotid glands, and lactating mammary glands, as compared with the mechanism and rate of limited proteolytic cleavage of IgD and spleen lymph nodes and glandular tissue of the gastrointestinal also to identify and quantify the reaction products. Within 1Ð5 tract (50, 51). Moreover, in patients with selective IgA deficiency, min, tryptic digestion of native IgD almost quantitatively yields a the majority of B lymphocytes found around the upper respiratory labile Fab, a stable Fc fragment, and a highly charged peptide from glands belong to the IgD class (51). A substantial local synthesis the hinge region. A galactosamine-rich glycopeptide from the of IgD both in nasopharynx and in the middle ear cavity has also hinge region increases inversely as the Fab is largely degraded to been observed (52, 53). In ϳ20% of middle ear effusions exam- a series of peptides within 1 h. In contrast, Fc fragments and the ined, a content of Ͼ600 mg/L of IgD could be calculated. These highly charged peptide resist proteolysis for Ͼ24 h. observations indicate that IgD may play a significant role in the The difference in magnitude in MID-binding capacity for dif- humoral immunity of secretions from the upper respiratory tract. ferent IgD preparations as shown in Fig. 3 is most likely due to Interestingly, H. influenzae and M. catarrhalis are frequently col- different degrees of partial degradation. The commercially avail- onizing the upper respiratory tract and are also important patho- able sera and IgD preparations used by us were stored according to gens in, for example, acute otitis media. Thus, it is tempting to the instructions of the manufacturers, and all sera from the hospital speculate that a bacteria-IgD interaction on lymphocytes and in laboratory were stored at Ϫ20¡CorϪ70¡C. 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