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Department of Medical Microbiology, Malmö University Hospital, Lund University, Malmö, Sweden
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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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 |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 
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.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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-, IgG1-
, IgG2-, IgG2-, IgG3-, IgG3-,
IgG4-, IgG4-, IgA1-, IgA1-, IgA2-, IgA2-, IgM-,
IgM-, IgD-, IgD-, IgE-, and, finally, IgD myeloma whole
sera IgD- and IgD- 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- A, IgD-
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- 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- 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 ( and ) (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 I
). 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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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-
-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- 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).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 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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A 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. 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 molecular mass of >1000 kDa (Fig. 2B). 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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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. 2
(Table II). 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. 3, 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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and Figs. 2 and 3). 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. 4). Interestingly, a
large fraction of CD19+ lymphocytes bound
significant amounts of MID-FITC (Fig. 4A), whereas T cells
(CD3+ lymphocytes) only displayed a nonspecific
background binding (Fig. 4D). The MID-FITC signal
corresponded well with CD19+ cells incubated with
anti-IgD mAbs revealing IgD-postive B cells (Fig. 4B).
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- 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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Degenerate primers were designed according to the obtained
amino-terminal sequences of four peptide fragments originating from MID
(Table I) and were used in PCR in all
possible combinations followed by cloning and DNA sequencing. The
specific primers 2982+ and 3692- (Fig. 5) 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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). 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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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. 7). The
isoelectric point of MID was determined to be 6.8.
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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. 8). 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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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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. 3 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. 4). 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. 4). 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. 4, 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. 4D).
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.
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Footnotes |
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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
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--D-galactoside.
Received for publication March 13, 2001. Accepted for publication June 4, 2001.
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Usp), or anti-CopB (
