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The Journal of Immunology, 2001, 167: 2112-2120.
Copyright © 2001 by The American Association of Immunologists

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

Arne Forsgren, Marta Brant, Andrea Möllenkvist, Anthony Muyombwe, Håkan Janson, Nicolas Woin and Kristian Riesbeck2

Department of Medical Microbiology, Malmö University Hospital, Lund University, Malmö, Sweden


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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 ~200 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.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
M;-2qoraxella catarrhalis is a Gram-negative diplococcus that for a long time was considered a relatively harmless commensal in the respiratory tract. At present, it is the third most frequent cause of otitis media and also a significant agent in sinusitis and lower respiratory tract infections in adults with pulmonary disease. M. catarrhalis is also one of the most common inhabitants of the pharynx of healthy children (1, 2).

Since the discovery of the first Ig-binding bacterial protein, Staphylococcus aureus protein A (SpA)3 (3), this protein has been extremely well characterized (4). The ability of SpA to bind the Fc part of IgG is well known, but SpA also binds a fraction of Ig molecules of all classes, due to the so-called "alternative" binding, which represents an interaction with the VH chains (5). All IgG-binding capacity of S. aureus has been considered to be mediated by SpA. However, the existence of a second gene in S. aureus encoding an Ig-binding protein was recently reported (6).

Protein G isolated from group C and G streptococci of human origin has a distinct affinity for the same site on the human Fc fragment of IgG as SpA and also interacts with IgG Fab (7, 8). An M-like protein, protein H, isolated from group A streptococci (Streptococcus pyogenes) is able to compete for the same region of IgG-Fc with SpA and protein G (9). S. pyogenes produce an antigenically and functionally heterogenous group of M-like proteins with different Ig-binding specificities. Proteins expressed by some strains bind IgA instead of IgG or both IgG and IgA (10). Protein Bac, or the B Ag, is an IgA-binding protein expressed by certain strains of group B streptococci (11). Finally, protein L, a surface component of Peptostreptococcus magnus, has affinity for all classes of Ig through an interaction with determinants present in the variable region of {kappa} L chains (12).

In contrast to Gram-positive bacteria, nonimmune Ig binding to Gram-negative bacteria are more rare. However, reports on bovine IgM binding to Brucella abortus, equine IgG binding to Taylorella equigenitalis, and bovine IgG and IgM binding to Haemophilus somnus have been published (13, 14, 15). An Ig FcR has also been purified from H. somnus (16). Two decades ago, H. influenzae and M. catarrhalis were shown to display a strong affinity for soluble human IgD (17). IgD binding at the cellular level explains the strong mitogenic effects on human lymphocytes by H. influenzae and M. catarrhalis (18, 19, 20). In addition, it was demonstrated that M. catarrhalis stimulates the proliferation of high density (mature) B lymphocytes expressing high levels of IgD and that soluble nonmitogenic mAbs reactive with human IgD selectively inhibit the B lymphocyte response. Inhibition by anti-IgD mAb presumably resulted from covering/capping surface IgD on B lymphocytes, thereby eliminating the bacteria-dependent stimulatory signal delivered through the B cell receptor (BCR) IgD. An IgD-binding outer membrane protein (OMP) from H. influenzae (protein D) was isolated and cloned and shown to be an important pathogenicity factor (21, 22, 23, 24). However, protein D does not bind to the majority of IgD myelomas tested, and it was suggested that encapsulated H. influenzae of serotype b expresses an additional IgD receptor (25).

The present work describes the isolation and cloning of the M. catarrhalis IgD-binding protein (MID). We also characterize the Ig-binding properties of this molecule, which were found to be different compared with previously isolated Ig-binding bacterial proteins. MID has similarities but nonidentity with previously described proteins from M. catarrhalis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents

M. catarrhalis strain Bc5 was a clinical isolate from a nasopharyngeal swab culture taken by our department (Department of Medical Microbiology, Malmö University Hospital, Lund University, Malmö, Sweden). The human purified Ig preparations IgG1-{kappa}, IgG1-{lambda}, IgG2-{kappa}, IgG2-{lambda}, IgG3-{kappa}, IgG3-{lambda}, IgG4-{kappa}, IgG4-{lambda}, IgA1-{kappa}, IgA1-{lambda}, IgA2-{kappa}, IgA2-{lambda}, IgM-{kappa}, IgM-{lambda}, IgD-{kappa}, IgD-{lambda}, IgE-{kappa}, and, finally, IgD myeloma whole sera IgD-{kappa} and IgD-{lambda} were purchased from The Binding Site (Birmingham, U.K.). The IgD standard serum OTRD 02/03 was obtained from Behringswerke (Marburg, Germany). Myeloma whole sera IgD-{lambda} A, IgD-{lambda} B, IgG A, IgG B, IgG C, IgM, IgA A, and IgA B were obtained from the Department of Clinical Chemistry at Malmö University Hospital. HRP-conjugated goat anti-human IgD was obtained from BioSource International (Camarillo, CA). FITC-conjugated mouse anti-human IgD, unlabeled rabbit anti-human IgD, HRP-conjugated rabbit anti-human IgA, IgG, and IgM, as well as HRP-labeled rabbit anti-mouse Ig were purchased from DAKO (Gentofte, Denmark). Goat anti-human IgD and HRP-conjugated rabbit anti-human polyvalent Igs were from Sigma (St. Louis, MO). PE-conjugated mouse anti-human CD3 and CD19 were obtained from BD Biosciences (San Jose, CA). Mouse mAbs 17C7 (UspA) and 10F3 (CopB) (26, 27) were kindly provided by Dr. E. J. Hansen (Department of Microbiology, University of Texas, Dallas, TX).

SDS-PAGE and detection of proteins on membranes (Western blot)

SDS-PAGE was run at 150 constant voltage using 10% (bis)Tris gels with running (MES), sample (lithium dodecyl sulfate), and transfer buffer as well as a blotting instrument obtained from NOVEX (San Diego, CA). Samples were regularly heated at 100°C for 10 min and, in some experiments, at 100°C for 1 h or at 70°C for 10 min. Gels were stained with Coomassie brilliant blue R-250 (28) (Bio-Rad, Sundbyberg, Sweden). Electrophoretical transfer of protein bands from the gel to an Immobilon-P membrane (Millipore, Bedford, MA) was conducted at 30 V for 2–3 h. After transfer, the immobilon-P membrane was blocked in PBS with 0.05% Tween 20 (PBS-Tween) containing 5% milk powder. After several washings in PBS-Tween, the membrane was incubated with purified IgD myeloma protein (0.5 µg/ml human IgD-{kappa} myeloma; The Binding Site) in PBS-Tween including 2% milk powder for 1 h at room temperature. HRP-conjugated goat anti-human IgD diluted 1/1000 was added after several washings in PBS-Tween. After incubation for 40 min at room temperature and several additional washings in PBS-Tween, development was performed with ECL Western blotting detection reagents (Amersham Pharmacia Biotech, Uppsala, Sweden).

To determine the isoelectric point, a first-dimension isoelectric focusing (IEF) was conducted using the IPGphor IEF system (Amersham Pharmacia Biotech). Sample application (before rehydration) and rehydration of immobilized pH gradient (IPG) strips (7 cm, pH 3–10, nonlinear) was performed according to the manufacturer’s instructions. The rehydration buffer consisted of 8 M urea, 0.5% (w/v) 3-[(3 cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 0.2% (v/v) IPG buffer, 15 mM DTT, and bromophenol blue (29). The IEF was run at 20°C, and the following settings were used: 30 V for 12 h, 200 V for 1 h, 500 V for 1 h, 1000 V for 30 min, and 8000 V for 2 h. IPG strips were first equilibrated with 65 mM DTT in equilibration buffer consisting of 6 M urea, 30% (w/v) glycerol, 2% (w/v) SDS, 50 mM Tris-base (pH 8.8), and bromophenol blue for 15 min at 20°C. The second equilibration was performed with 22 mM iodoacetamide in equilibration buffer. IPG strips were transferred to 10% SDS-polyacrylamide gels that were further handled as described above. To calibrate the gels, a two-dimensional SDS-PAGE standard was used (Bio-Rad, catalog number 161-0320).

ELISA

Extracts of M. catarrhalis diluted in 5-fold steps in 0.1 M Tris-HCl (pH 9.0) were added in 100-µl volumes to microtiter plates (F96 Maxisorb; Nunc, Roskilde, Denmark) and incubated at 4°C overnight. After washing 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-{kappa} myeloma protein, (0.05 µg) in 100 µl PBS-Tween containing 1.5% OVA was added to each well, and after incubation for 1 h at room temperature, the plate was washed four times with PBS-Tween. After incubation with HRP-conjugated goat anti-human IgD diluted 1/1000 and subsequent washings, plates were developed and measured at 450 nm. In another set of experiments, purified MID in volumes of 100 µl was added to microtiter plates. After incubation, washing, and blocking, dilutions of human myeloma sera in a volume of 100 µl were added, and after incubation and washing, isotype-specific detection Abs for respective myeloma sera were supplemented as described above. In a third set of experiments, microtiter wells were coated with highly purified IgD myeloma protein, and after washing and blocking, HRP-labeled anti-Ig Abs were added.

Dot blot assays

Purified MID (0.0005–0.2 µg) in a volume of 100 µl in 0.1 M Tris-HCl (pH 9.0) was manually applied to nitrocellulose membranes by using a dot blot apparatus (Schleicher & Schüll, Dessel, Germany). After saturation, the membranes were incubated for 2 h at room temperature in PBS-Tween containing 1% OVA and 5% milk powder and washed four times with PBS-Tween. Human myeloma protein (0.5 µg) in 100 µl PBS-Tween was added and, after 2 h of incubation, followed by several washings in PBS-Tween, HRP-labeled anti-human L chains ({kappa} and {lambda}) (DAKO) diluted 1/200 were used as secondary Abs. In another set of experiments, dilutions of human myeloma sera in a volume of 100 µl in 0.1 M Tris-HCl (pH 9.0) were first applied to the membranes. After saturation, incubations, blocking, and washing, steps were performed as described above. Thereafter, 125I-labeled protein MID probe (5–10 x 105 cpm/ml) in PBS-Tween was added. After overnight incubation, the membrane was washed four times with PBS-Tween, air-dried, and exposed to Kodak CEA.C x-ray films (Eastman Kodak, Rochester, NY) at -70°C using an intensifying screen.

Extraction and purification of IgD-binding protein

M. catarrhalis bacteria (1–5 x 1011 CFU/ml) were suspended in 0.05 M Tris-HCl buffer (pH 8.8) containing 3% Empigen (Calbiochem, Bedford, MA). In some experiments, Empigen was replaced by CHAPS (Sigma), N-octyl-p-D-glucosidase (Bachem, Budendorf, Switzerland), or Triton X-100 (Sigma). All these detergents at a concentration of 3% were tested with or without 0.01 M EDTA. The bacterial suspensions were mixed by magnetic stirring for 2 h at 37°C. After centrifugation at 8000 x g for 20 min at 4°C, the supernatants were filtrated with sterile filters (0.45 µm; Sterivex-HV; Millipore). M. catarrhalis extract in 3% Empigen was applied to a Q-Sepharose column (Amersham Pharmacia Biotech) equilibrated with 0.05 M Tris-HCl (pH 8.8) containing 0.1% Empigen. The column was eluted using a 0- to 1-M NaCl linear gradient in the same buffer. Fractions showing the most IgD binding were pooled, dialyzed in Spectra/Por membrane tubes (Spectrum, Houston, TX; molecular mass cut-off, 25 kDa) against 0.05 M Tris-HCl (pH 8.8), concentrated on YM100 disc membranes (Amicon, Beverly, MA; molecular mass cut-off 100 kDa), and then applied to a Sephacryl S-400 high resolution column (20 by 900 mm; Amersham Pharmacia Biotech) and equilibrated with 0.05 M Tris-HCl (pH 8.8) containing 0.1% Empigen. Fractions containing the strongest IgD-binding activity were concentrated and rechromatographed as described above.

Peptide cleavage and amino acid sequence analysis

Purified MID in 0.05 M Tris-HCl (pH 8.8) containing 0.1% Empigen was treated with trypsin or chymotrypsin in an enzyme:protein ratio of 1:10 at 37°C overnight. The cleavage mixtures were subjected to SDS-PAGE, and peptide bands transferred to Immobilon-P membranes were automatically sequenced. To get an N-terminal sequence of the protein, deblocking of intact MID from a possible pyroglutamate group was attempted (30, 31). Automated amino acid sequence analysis was performed with an Applied Biosystems (Foster City, CA) 470A gas-liquid-solid-phase sequenator (32).

Labeling of protein MID

Purified MID was radioiodinated (Amersham, Little Chalfont, U.K.) to high specific activity (0.05 mol iodine per mol protein) with lactoperoxidase (33). FITC (Sigma) was conjugated to purified MID using a standard protocol. Briefly, MID (2 mg/ml) in 0.1 M carbonate buffer (pH 9.5) was incubated with 0.15 µg/ml FITC solubilized in DMSO. After 45 min at room temperature and constant stirring, the sample was diluted and subjected to a PD10 column (Amersham Pharmacia Biotech) pre-equilibrated with PBS (pH 7.4). The resulting MID-FITC was used for binding studies.

DNA isolation and sequencing

DNA was extracted from M. catarrhalis Bc5 using a genomic DNA preparation kit (Qiagen, Hilden, Germany). Degenerate primers were synthesized according to the amino-terminal sequences from four peptide fragments (Table IGo). In some of the PCR (High Fidelity PCR System; Roche, Bromma, Sweden), specific primers were used in combination with the degenerate ones. DNA sequences flanking the central region of the gene, from where the peptide fragments originated, were isolated using inverse PCR (IPCR) (34). Genomic DNA was cleaved with the following restriction enzymes, which were used separately: EcoRV, SphI, and PstI for the isolation of the start codon; and AccI, AsuI, and HincII for the isolation of the stop codon sequences. The resulting fragments were religated upon 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 primers were designed and used in a long-template PCR (Expand Long-Template PCR System; Roche). All PCR products were cloned into pPCR-Script-Amp (Stratagene, La Jolla, CA) and sequenced. The signal peptide was deduced using the SignalP V1.1 Prediction Server Center for Biological Sequence Analysis (http://www.cbs.dtu.dk/services/SignalP/).


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  		Table I. Amino acid sequences derived from highly purified MID after protease digestions1

 
Expression of the MID gene product and cell fraction

The complete 6.4-kb open reading frame of the mid gene was ligated into 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 logarithmic-growth phase followed by addition of isopropyl-1-thio-{beta}-D-galactoside (IPTG). After 4 h of induction, 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).

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 x 105 in 100 µl) 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-{kappa} at 37°C for 30 min and thereafter directly added to the cells followed by incubation on ice. After two washes, 10 µg/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
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
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. 1Go). Most IgD-binding material was eluted in this first peak immediately after the void volume. MID was further purified by rechromatography of the first peak under the same conditions.



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  		FIGURE 1. Chromatography and rechromatography on a Sephacryl S-400 column of Empigen-soluble extract from M. catarrhalis after ion exchange chromatograpy. The solid line indicates protein content of the first chromatography, and the broken line indicates rechromatography of the first peak. V0, Void volume.

 
Fig. 2GoA shows that after purification, MID appeared as two bands, one 200-kDa band and a second band with an apparent molecular mass of >1000 kDa. Western blot experiments were performed to confirm that MID was not identical with the previously described OMPs UspA1 and 2, with an apparent molecular mass varying from 350 to 720 kDa, (37) or CopB, with a molecular mass of 80 kDa (26). The crude Empigen extract of M. catarrhalis or partly purified preparations of MID were subjected to SDS-PAGE, transferred to Immobilon-P filters, and blotted with Abs to those Moraxella proteins and also with human IgD. As can be seen in Fig. 2GoA, 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 molecular mass of >1000 kDa (Fig. 2GoB). When MID instead was heat treated at 100°C for 10 min to 1 h, the typical double-band pattern appeared.



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  		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 70°C or 100°C for 10 min. A, Two gels were run simultaneously; one was stained with Coomassie brilliant blue (Stain), and one was blotted onto Immobilon-P membranes, probed with human IgD-{kappa} myeloma protein (IgD), anti-UspA ({alpha}Usp), or anti-CopB ({alpha}B) mAbs followed by incubation with appropriate HRP-conjugated secondary Abs. B, The gel was stained with Coomassie brilliant blue. Molecular mass of marker proteins is indicated to the left in both panels.

 
Three attempts were made to determine the amino-terminal amino acid sequence of purified MID. Approximately 1000 pmol MID was applied each time in an automated amino acid sequencer. Inasmuch as no amino acid phenylthiohydantoin derivatives were obtained, the amino-terminal end of the single MID polypeptide chain was probably blocked. It was recently determined that the Moraxella UspA1 and UspA2 proteins, which are also resistant to Edman degradation, contained a pyroglutamyl residue that was removed by the treatment with pyroglutamate aminopeptidase (38). However, when MID purified from M. catarrhalis or recombinant MID was treated with this enzyme according to two different protocols (twice for each method) and then subjected to Edman degradation, no N-terminal amino acid sequence was obtained.

IgD-binding properties of MID

Crude Empigen extracts of M. catarrhalis and highly purified MID subjected to SDS-PAGE and transferred to filters were exposed to highly purified commercially available Ig preparations representing all human Ig classes and subclassses. Only the two IgD preparations interacted with the MID bands in a fashion as shown in Fig. 2Go (Table IIGo). When dot blot experiments were performed and purified MID in dilutions was first added to membranes and purified human myeloma proteins and secondary Abs were subsequently applied, only the two IgD myelomas interacted with MID. One of the two myelomas detected as little as 0.001 µg of MID on the membrane. The specificity of the interaction between MID and IgD was further verified by first adding dilutions of myeloma sera to the filters and then radiolabeled MID in other dot blot experiments. In Fig. 3Go, it is demonstrated that MID effectively bound four IgD myeloma sera. A distinct reaction could be detected in the range of 0.03–4 µg of IgD. For the IgD standard serum (Behringswerke), reactivity was seen at even lower concentrations (data not shown). In contrast, six different Ig myeloma sera representing IgG, IgA, and IgM showed no visible reaction with MID at 4 µg. MID was also bound to the surface of an ELISA plate, and the same myeloma sera were added in two-step dilutions followed by isotype-specific detection Abs. The values were at least 16-fold higher for IgD than for the other isotypes (IgG, IgA, and IgM). However IgA, IgG, and IgM myeloma sera showed background values that can be explained by a weak cross-reactivity for antisera against these isotypes with IgD. Totally, MID (in different experiments) was shown to bind seven of seven IgD sera or preparations and none of 22 non-IgDs.


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  		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

 


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  		FIGURE 3. Binding of MID to human myeloma sera representing different Ig classes. All sera were diluted in 2-fold steps (4–0.03 µg) and applied to a nitrocellulose membrane. The following myeloma whole sera were used; IgD-{kappa}, IgD-{lambda}, IgD Behringswerke (IgD B.W.), IgD-{lambda} A, IgD-{lambda} B, IgG A, IgG B, IgG C, IgM, IgA A, and IgA B. After saturation, washing, and blocking, an 125I-MID-labeled probe was added. After overnight incubation and additional washings, specific MID-IgD binding was visualized by autoradiography.

 
Purified MID specifically attracted human soluble IgD as revealed in Western and dot blots (Table IIGo and Figs. 2Go and 3Go). To test whether MID bound to the surface-expressed B cell receptor BCR IgD, human PBLs were isolated. FITC was conjugated to MID and followed by incubation with PBLs for 45 min on ice. In parallel, PBLs were labeled with RPE-conjugated mAbs directed against the T cell marker CD3 or the B cell-specific surface Ag CD19 and subsequently analyzed by flow cytometry (Fig. 4Go). Interestingly, a large fraction of CD19+ lymphocytes bound significant amounts of MID-FITC (Fig. 4GoA), whereas T cells (CD3+ lymphocytes) only displayed a nonspecific background binding (Fig. 4GoD). The MID-FITC signal corresponded well with CD19+ cells incubated with anti-IgD mAbs revealing IgD-postive B cells (Fig. 4GoB). To further elucidate the specificity of MID-FITC binding to IgD-bearing CD19+ lymphocytes, PBLs were preincubated with a rabbit anti-human IgD Ig fraction. After incubation and washings, MID-FITC binding was analyzed by flow cytometry according to the standard procedure. The antiserum almost completely inhibited specific MID-FITC binding to the IgD BCR when compared with cells incubated with the preimmune serum. Mean fluorescence intensity decreased from 79.2–14.6 arbitrary units. Similar results were obtained with goat Igs raised against IgD (data not shown). Moreover, preincubation of MID-FITC with soluble IgD-{kappa} abolished the binding to the IgD BCR. Thus, IgD-expressing B cells promoted specific MID-FITC binding to the surface-expressed BCR IgD.



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  		FIGURE 4. IgD-bearing B cells specifically bound FITC-conjugated MID. PBLs stained with RPE-conjugated mAbs against CD19+ (A) or CD3+ (D) followed by incubation with MID-FITC were compared with PBLs incubated with anti-CD19 mAb in addition to an anti-IgD mAb (B). E, Double staining with CD3+ and anti-IgD mAb. C, PBLs preincubated with a rabbit Ig fraction against human IgD followed by addition of anti-CD19 mAb and MID-FITC. F, A control sample with no Abs or MID-FITC is also included. PBLs were isolated from heparinized human blood using LymphoPrep one-step gradients, incubated with the appropriate Abs, washed, and further incubated with MID-FITC. After final washings, PBLs were analyzed by flow cytometry. In this particular experiment, 68% of the total lymphocyte population was analyzed. Less than 2% of the cells were labeled when isomatched mAbs were included as negative controls. A preimmune rabbit serum did not significantly block MID-FITC binding to the IgD BCR (data not shown). An experiment with a typical donor of three separate ones analyzed is shown.

 
Cloning of the mid gene and analysis of the deduced amino acid sequence

Degenerate primers were designed according to the obtained amino-terminal sequences of four peptide fragments originating from MID (Table IGo) and were used in PCR in all possible combinations followed by cloning and DNA sequencing. The specific primers 2982+ and 3692- (Fig. 5Go) were synthesized using the deduced sequence of a distinctive PCR product generated with the degenerate primer pair 2629+/3693-. A PCR using the specific primers in combination with the degenerate ones (718+ and 5772-) resulted 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 2982+/945- and 3668+/120-, respectively, provided the sequence for the start codon area. In addition, IPCR on HincII-digested Moraxella genomic DNA with the primer-pair 5898+/5511- generated the 3' sequence including the stop codon.



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  		FIGURE 5. Schematic map of the mid gene showing the cloning strategy. Oligonucleotide 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 IGo and specific primers are shown by broken and solid lines, respectively.

 
The complete nucleotide sequence of mid comprised 6417 bp (Fig. 6Go). Two alternative open reading frames were revealed and start at amino acid positions 1 or 17. Consequently, the length of the mid gene product might be either 2123 or 2139 aa. In addition to a putative ribosome-binding site (AAGG), -10 (TAATTA) and -35 (TTGAAT) consensus sequence boxes were identified. Furthermore, 62 bases downstream of the TAA stop codon, an inverted repeat was found with the potential of stem-loop formation that is necessary for transcriptional termination.




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  		FIGURE 6. Nucleotide sequence of the mid gene from M. catarrhalis Bc5 together with the deduced amino acid sequence. Putative -35 and -10 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 open reading frame defined a protein with a calculated molecular mass of just below 220 kDa that readily corresponded to the empirical value of ~200 kDa found by SDS-PAGE. The protein contained several predicted coiled coil structures. The N-terminal amino acid sequence showed the typical characteristics of a signal peptide with a potential cleavage site between amino acids 66 and 67. Despite that the first amino acid after the signal peptidase cleavage site was most likely a glutamine residue, any sequence could not be determined by Edman degradation. Furthermore, no amino acid sequence was obtained after pyroglutamate aminopeptidase treatment. The predicted amino acid sequence was also subjected to a hydrophobicity profile analysis by the method of Kyte and Doolittle (39) and showed mainly hydrophilic properties, except for the putative signal peptide that was strongly hydrophobic (Fig. 7Go). The isoelectric point of MID was determined to be 6.8.



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  		FIGURE 7. The hydropathy profile of MID. The hydrophobic and hydrophilic parts of the individual amino acid residues are indicated. The predicted signal peptide is outlined. Data were obtained by using a standard method as previously described (39 ).

 
Expression of rMID in E. coli

To confirm that the cloned mid gene corresponded to the purified IgD-binding protein, the gene including the predicted signal sequence and start codon was subcloned into the expression vector pET16(b) and induced with IPTG. Bacterial cells were lysed and subfractionated, and rMID was localized by Western blots using human IgD as a probe. Important verifying characteristics of MID were provided from the expression experiments (Fig. 8Go). First, following induction, cells containing pET16-MID were able to produce rMID confirming the correct reading frame of the gene. Second, rMID (as shown by SDS-PAGE) displayed a molecular mass of ~200 kDa, corresponding to the 217 kDa calculated value from the amino acid sequence. Third, the recombinant protein was indeed the mid gene product in E. coli because its IgD-binding phenotype was confirmed by Western blot analysis. Total protein from E. coli containing the induced pET16(b) vector without insert did not display any IgD-binding capacity (data not shown). Fourth, the subcellular localization of the recombinant protein showed that MID was equally located in the cytoplasmic and the membrane fractions, but not in the periplasmic space. The localization of MID in the membrane fraction correlated very well with the known outer membrane localization in M. catarrhalis.



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  		FIGURE 8. Recombinantly expressed MID retained its IgD-binding capacity. The left panel shows a Coomassie brilliant blue-stained gel, and the right panel shows a Western blot probed with human IgD. Moraxella-derived MID protein (MID) was run and compared with cytoplasmic (C), periplasmic (P), and membrane (M) fractions. Numbers on the left indicate a molecular mass standard. E. coli BL21DE3 containing pET16-MID was induced for 4 h by IPTG. Cellular fractions were collected, and proteins were separated by two SDS-PAGEs that were run in parallel and either stained with Coomassie brilliant blue or blotted onto an Immobilon-P membrane. The membrane was probed with human IgD followed by incubation with a HRP-conjugated secondary Ab.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present investigation describes the isolation, purification, characterization, cloning, and expression of a protein named MID, a novel Ig-binding protein of M. catarrhalis that has affinity for human IgD. Early studies demonstrated that the OMPs from a diverse collection of Moraxella isolates exhibit a high degree of similarity (40). Investigators have primarily focused their research efforts on a selected group of proteins (2). Recent studies have demonstrated that the high molecular mass surface Ag, termed UspA or HMW-OMP, is actually comprised of two different proteins. These proteins are named UspA1 and UspA2 (27, 38). The apparent molecular mass of these OMPs is >350–700 kDa as determined by SDS-PAGE analysis. Reduction with formic acid yields bands of ~120–140 kDa, suggesting that the UspA proteins form an oligomeric complex composed of several monomeric subunits (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 99–111 and 74–105 kDa, respectively (44).

MID is obviously not identical with previously well-characterized OMPs of M. catarrhalis. It is not recognized by mAbs derived against the UspA or CopB outer membrane Ags. MID also has a different migration pattern in SDS-PAGE and a different composition as shown by amino acid and DNA sequence analysis. In addition, MID shows no similarity with other Ig-binding proteins including protein D from H. influenzae. However, homology analyses using the GAP software from Mologen (Berlin, Germany) revealed that MID in parts has homology (48%) with USPA1 and 2. MID appeared as a 200-kDa band in accordance with the molecular mass from the deduced amino acid sequence, but also as an extra band with an estimated molecular mass of >1000 kDa. The extra band indicates that native MID is an oligomeric complex in a similar fashion as UspA (37), probably due to several stretches of coiled coil structures. This is further supported by the fact that MID was eluted immediately after the void volume from a Sephacryl S-400 column with a fractionation range of up to 8000 kDa. In a recent patent publication, an OMP of M. catarrhalis with a molecular mass of ~200 kDa was isolated (45). A sequence encoding a protein of ~200 kDa was also provided. However, that protein sequence is not identical with the sequence provided by us and shows only 53.5% identity with MID. The protein was shown to be immunogenic, but no further biological functions were presented. In addition, a 200-kDa protein is associated with hemagglutinating M. catarrhalis (46). However, hemagglutinins are not universally expressed by M. catarrhalis clinical isolates (47). In contrast, MID can be detected in all strains of M. catarrhalis (>100; our unpublished data), albeit with various molecular masses ranging from ~180 to 220 kDa. Thus, MID does not seem to be identical with either the hemagglutinin or the 200-kDa protein.

Human IgD, unlike other Igs, has been poorly studied due to the low concentration in normal serum and its great susceptibility to proteolytic degradation, which makes it further difficult to purify and quantify (48). In a study of the sera from ~50 myeloma patients, fragmented IgD was present in all cases, and usually this was the predominant form. In a study from Putnam’s laboratory (49), high pressure liquid chromatography was used to investigate the mechanism and rate of limited proteolytic cleavage of IgD and also to identify and quantify the reaction products. Within 1–5 min, tryptic digestion of native IgD almost quantitatively yields a labile Fab, a stable Fc fragment, and a highly charged peptide from the hinge region. A galactosamine-rich glycopeptide from the hinge region increases inversely as the Fab is largely degraded to a series of peptides within 1 h. In contrast, Fc fragments and the highly charged peptide resist proteolysis for >24 h.

The difference in magnitude in MID-binding capacity for different IgD preparations as shown in Fig. 3Go is most likely due to different degrees of partial degradation. The commercially available sera and IgD preparations used by us were stored according to the instructions of the manufacturers, and all sera from the hospital laboratory were stored at -20°C or -70°C. Despite this, we have seen a decreasing IgD reactivity with MID over time that indicates degradation.

The susceptibility of IgD to proteolytic degradation was not only an obstacle in our characterization of the interaction with MID. As a matter of fact, the decreasing reactivity over time supports the results that constant structures of the Fab region on the IgD molecule is responsible for the binding to M. catarrhalis (17). In reproducible experiments, when Fab and Fc portions of IgD were produced, radiolabeled, and tested for binding to H. influenzae and M. catarrhalis, Fab portions bound more strongly to the bacteria than did the Fc fragments. It was also suggested that the CH1 region in the IgD molecule was responsible for the interaction with these bacteria. The fact that the experiments by Forsgren and Grubb (17) were performed with radiolabeled Ig fragments was probably an advantage. If, instead, the interaction would have been studied with an Ab directed to IgD, the Fab interaction could have been overseen because commercially available antisera against IgD have been prepared against the Fc fragment rather than intact IgD (48). The exact localization in the IgD molecule of the MID-binding structure remains to be characterized.

Considering the problems with soluble IgD, our flow cytometric studies with cell-bound IgD are encouraging (Fig. 4Go). The cells used have always been fresh, and the reactivity with MID was reproducible. Naive resting B cells display both IgM and IgD BCRs, whereas immature newly formed B cells are only IgM positive. The membrane-bound Ag receptor is identical with the secreted counterpart, except for the BCR consists of an additional stretch of hydrophobic amino acids in its C terminus. Because sensitivity to proteolysis is a hallmark of IgD, the membrane-bound receptor for Ag on the surface of differentiating B lymphocytes, proteolysis is most likely associated with the biological function of IgDs. In this process, membrane-bound IgD is believed to undergo proteolysis to form Fab and Fc portions. The Fab part is then degraded, whereas the Fc fragment is endocytosed together with the Ag. M. catarrhalis selectively binds the IgD BCR, and proliferation of human B lymphocytes follows its interaction with cell surface IgD and MHC class I molecules (17, 18, 19). In the present study, we showed that MID bound all surface-expressed IgD BCRs on B lymphocytes isolated from human peripheral blood (Fig. 4Go). When PBLs were preincubated with a rabbit anti-serum against the IgD BCR followed by incubation with MID-FITC, a faint residual binding was observed, although the mean fluorescence intensity significantly decreased from 79.2 to 14.6 (Fig. 4Go, A and C). However, it is evident that MID is not responsible for the earlier-observed MHC class I-binding activity of M. catarrhalis (20), because no specific binding occurred to T lymphocytes (Fig. 4GoD).

An increased number of IgD immunocytes have been observed in the lymphoid tissue from nasopharyngeal tonsils, lacrimal and parotid glands, and lactating mammary glands, as compared with spleen lymph nodes and glandular tissue of the gastrointestinal tract (50, 51). Moreover, in patients with selective IgA deficiency, the majority of B lymphocytes found around the upper respiratory glands belong to the IgD class (51). A substantial local synthesis of IgD both in nasopharynx and in the middle ear cavity has also been observed (52, 53). In ~20% of middle ear effusions examined, a content of >600 mg/L of IgD could be calculated. These observations indicate that IgD may play a significant role in the humoral immunity of secretions from the upper respiratory tract. Interestingly, H. influenzae and M. catarrhalis are frequently colonizing the upper respiratory tract and are also important pathogens in, for example, acute otitis media. Thus, it is tempting to speculate that a bacteria-IgD interaction on lymphocytes and in secretion of the mucous membranes may play an important role in the pathogenesis and host defense in upper respiratory tract infections. MID will provide an excellent tool for the study of these interactions.


   Footnotes
 
1 This work was supported by grants from the Alfred Österlund Foundation, the Anna and Edwin Berger Foundation, the Crafoord Foundation, the Greta and Johan Kock Foundation, the Magnus Bergvall Foundation, the Swedish Medical Research Council, and the Cancer Foundation at Malmö University Hospital. Back

2 Address correspondence and reprint requests to Dr. Kristian Riesbeck, Department of Medical Microbiology, Malmö University Hospital, Lund University, S-205 02 Malmö, Sweden. E-mail: kristian.riesbeck{at}mikrobiol.mas.lu.se Back

3 Abbreviations used in this paper: SpA, Staphylococcus aureus protein A; BCR, B cell receptor; OMP, outer membrane protein; MID, M. catarrhalis IgD-binding protein; IEF, isoelectric focusing; IPG, immobilized pH gradient; CHAPS, 3-[(3 cholamidopropyl)dimethylammonio]-1-propanesulfonate; IPCR, inverse PCR; IPTG, isopropyl-1-thio-{beta}-D-galactoside. Back

Received for publication March 13, 2001. Accepted for publication June 4, 2001.


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