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Department of Microbiology, Montana State University, Bozeman, MT 59717
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We applied phage-display technology to search for structural equivalents of microbial polysaccharide epitopes. We had previously identified carbohydrate epitopes from Candida albicans and group B streptococci (GBS) that elicit protective Ab responses, as well as other carbohydrate epitopes that elicit nonprotective Abs. The Abs used to select the phage were of the IgM class, which are typical of T-independent, anti-carbohydrate responses. While using these IgM Abs to select for carbohydrate-mimetic phage (8), we identified a population of phage that bind to all IgM Abs regardless of antigenic specificity, presumably recognizing determinants in the constant regions. We characterized these phage and demonstrated, surprisingly, that the phage and peptides derived from them can inhibit the interaction between Ag and IgM Ab.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The J404 PDPL we used in our studies was constructed by one of the authors (J.B.B.) and is described elsewhere (1, 9, 10). The library displays random nonamer peptides from the N-terminal portion of the pIII capsid protein of kanamycin-resistant, filamentous bacteriophage M13KBst. The J404 library contains an estimated 5 x 108 different nonapeptides at high titer (1 x 1013 pfu/ml).
Abs
Anti-Candida IgM mAbs B6.1 and B6 were isolated as previously described (11) and produced in serum-free medium (BG 101 Liquid Kit; Irvine Scientific, Santa Ana, CA). The Abs were concentrated by ammonium sulfate precipitation and exhaustively dialyzed against Dulbecco’s PBS (DPBS) (Sigma, St. Louis, MO). Anti-GBS IgM mAbs S7, S9, and S10 (12) were prepared as mouse ascites, and the IgM fraction was isolated by distilled water dialysis. S7 and S9 are directed against carbohydrate Ags, while S10 is specific for the GBS Ibc protein. Monoclonal anti-GBS mAbs 1B1 and 2A6 are specific for the GBS type III capsular polysaccharide (8), as is the IgM S9 (12). Additional mAbs and polyclonal Abs were used to assess peptide binding and Ig class specificity. Murine IgM mAbs used included: H9 and C6 (13); mt9A, mt5A, mt6F, P3 x 63 (14); 2B3.1, A4.1, C3.4, JD3 (M. Riesselman and J.E.C., unpublished observations), and T17 directed against the synthetic peptide (Y,E)-A-K– (15). Murine IgG mAbs included 6C5-H4 (a gift from Kevin C. Hazen, University of Virginia, Charlottesville, VA), 44.1 (9), and 924 (16). Polyclonal preparations included murine ascites and rabbit and human sera.
Preparation of selection matrices
Ab affinity matrices for interaction with the J404 PDPL were mAb B6.1 adsorbed to polystyrene dishes, mAb B6.1 conjugated to Sepharose 4B (CL-4B-200; Sigma), or mAb S10 conjugated to Sepharose beads. Polystyrene dishes (Falcon 35 mm; Becton Dickinson, San Diego, CA) were coated for 2 h at room temperature with 1 mg mAb B6.1/ml DPBS and washed five times with cold DPBS. For mAb-conjugated Sepharose matrices, the Sepharose 4B was activated with cyanogen bromide as previously described (17) and coated with mAb B6.1 or S10 (3 mg per ml packed beads, 16 h at 4°C), washed, and blocked in 1% BSA (ICN Biomedicals, Aurora, OH) in DPBS before incubation with phage.
Interaction of the J404 library with IgM mAb matrices
All Ab-coated dishes and beads were preblocked for 1 h in
DPBS plus 1% BSA before incubation with the library. Three independent
selections (mAb B6.1-dish, MAb B6.1-Sepharose, and mAb S10-Sepharose)
were performed with aliquots from the J404 PDPL. Manipulation of the
nonapeptide PDPL and appropriate Escherichia coli K91 host
cells were essentially as described (9). Briefly, aliquots
of the nonapeptide PDPL (
7.5 x 1011
phage) were diluted in phage buffer (50 mM Tris-HCl, pH 7.5, 150 mM
NaCl, 0.5% v/v Tween 20 and 0.1–1.0% BSA) and incubated with the
blocked matrices (16 h with rotation, 4°C). After thorough washing
with phage buffer, bound phage were eluted with 0.1 M glycine buffer,
pH 2.2, and immediately neutralized by addition of Tris base.
A few microliters of the eluted phage were removed for titering, and the remaining phage were amplified in "starved" E. coli K91 to a titer of 1012 to 1013 pfu/ml. Subsequent selections were conducted against the same IgM mAb as the initial selection. Half of the amplified phage were diluted in phage buffer and incubated with a fresh mAb-coated selection matrix for a second round of affinity selection. The eluted phage were titered and amplified as above, and half were subjected to a third round of selection.
Analysis of the third-round phage pools
The third-round selection pools of phage were analyzed by
sequencing of phage clones as follows. An appropriate dilution of phage
pool was plated, single plaques were excised, and phage minipreps
(9) were prepared in kanamycin-containing Luria broth and
harvested to provide single-stranded template DNA for sequencing with
Sequenase v.2.0 (USB/Amersham, Arlington Heights, IL). The phage
templates were primed with a gene III-specific primer, which anneals
50 nt from the 27-mer insert as described (9). Clones
were tested for binding to IgM mAbs by plaque lifts, immunoblots, and
ELISA.
Preparation and analysis of immunoblots
SDS-PAGE, transfer of proteins, and immunoblot preparations were essentially as described (18). Briefly, phage samples were electrophoresed in 12.5% polyacrylamide gels, and proteins were transferred to nitrocellulose membranes (NCM) (BA83; Schleicher and Schuell, Keene, NH) with a semidry blotting system (MilliBlot-SDE; Millipore, Bedford, MA). NCM were briefly rinsed in transfer buffer and dried.
For immunoblotting, membranes were rehydrated in water, washed for 10 min in DPBS, and blocked (1–2 h, 23°C) in a fresh solution of DPBS containing 5% (w/v) nonfat milk and 0.1–0.2% Tween 20 (BLOTTO). BLOTTO was used for all Ab dilutions and wash steps. Blocked membranes were incubated overnight (4°C) with monoclonal (5 µg/ml or a 1:200 dilution of ascites preparations) or polyclonal Abs (1:200 dilution of serum samples). Blots were washed three times (10 min each) and incubated with alkaline phosphatase (AP) or HRP-conjugated secondary Abs. AP-conjugate incubations were followed by washes (3 x 15 m) in 0.1 M Tris base, pH 9.5, 0.1 M NaCl, 1 mM MgCl2, and development with nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate-p-toluidine. Blots incubated with HRP conjugates were immersed in 3,3'-diaminobenzidine plus H2O2 in 50 mM Tris-HCl, pH 7.4, for detection.
Plaque lifts of phage clones were prepared by placing NCM circles on overlay plates for 1–2 h and washing three times with TBS. Lifts were blocked and processed as described above for Western blots.
ELISA
ELISA was used to measure phage binding to IgM and inhibition of IgM Ab binding to Ag. ELISA to determine phage binding to Ab were performed as follows. Immulon II microtiter plates (Dynatech, Chantilly, VA) were coated with 0.2–1.0 µg mAb per well and blocked with PBS/1% BSA for 2 h at room temperature. Varying concentrations of phage or keyhole limpet hemocyanin (KLH)-conjugated peptides derived from the phage were added to wells in a 100-µl volume and incubated. Plates were washed with PBS 0.1% Tween 20, and rabbit anti-M13 antiserum (a gift from Al Jesaitis, Montana State University, Bozeman, MT) or rabbit anti-KLH antiserum was added and the plates were incubated. Plates were washed, and then AP-conjugated anti-rabbit Ig (Zymed, South San Francisco, CA) was added. We previously determined that this antiserum does not react with mouse Ig. Following an incubation, the plates were washed and the substrate p-nitrophenyl phosphate (Sigma) was added. The absorbance at 405 nm (A405) was measured using an EL-320 Microplate reader (Bio-Tek, Winooski VT).
ELISA assays for phage inhibition of mAb interaction with C. albicans Ags were conducted by coating microtiter wells with 0.5 µg (based on dry weight) C. albicans cell wall extract (19), blocking 1 h with 1% Ficoll 400 in TBS, and then blocking 2 h in BLOTTO. The inhibition assay was performed by either preincubating the Ab with phage or by adding the phage to microtiter wells after the Ab solution was present. For the preincubation inhibition assays, mAbs were diluted (0.25 µg/ml) in BLOTTO alone or mixed with 1.8 x 1012 phage/ml phage and rotated for 1 h before placing 100 µl of the mixture into the cell wall extract-coated wells (2 h). For the later addition of phage, mAbs were incubated in wells for 1 h, and then phage was added to the microtiter wells and additionally incubated for 1 h. Wells were washed, and an HRP-conjugated goat anti-mouse polyvalent Ig was added and incubated 2–4 h. Substrate containing O-phenylenediamine, H2O2, in 0.1 M sodium citrate, pH 5.0, was added, and the color was developed for 10–30 min. Reactions were stopped by the addition of 10% H2SO4, and A490 was measured.
GBS were coated onto microtiter wells using polyl-lysine and glutaraldehyde as described elsewhere (8). Plates were blocked with 1% BSA. IgM anti-GBS Abs were mixed with phage or with KLH-conjugated peptides derived from the phage and incubated in microtiter wells at 40 for 16 h. The plates were washed and incubated with AP-conjugated anti-Ig for 6 h, followed by washing and the addition of p-nitrophenyl phosphate. A405 was then determined.
Latex agglutination for measurement of rheumatoid factor (RF)
Latex beads coated with aggregated IgG were obtained from REFSCAN Kit (Becton Dickinson, Cockeysville, MD). Serum containing high titered RF activity were either contained within the kit or were obtained from the Division of Rheumatology, University of Utah School of Medicine (Salt Lake City, UT). Pretitered dilutions of serum were premixed with phage and then added to the latex beads. The mixture was stirred and allowed to incubate with slow shaking for 20 min before photography.
Flow cytometry for cell surface IgM
Murine B lymphoma cells expressing cell surface IgM (designated 5F5) or differentiated to express IgA (4F10) (20, 21) were incubated with phage in PBS/BSA/0.1% sodium azide, washed, incubated with rabbit anti-M13 antiserum, washed, and then stained with FITC-conjugated anti-rabbit Ig (Cappell Laboratories, Durham, NC). Alternatively, cells were directly stained with FITC-conjugated anti-mouse IgM (Cappell).
Preparation of synthetic peptide–carrier protein conjugate
The nonapeptide displayed by phage clone ed1 was chosen for conjugation to KLH with a heterobifunctional cross-linker, m-maleimidobenzoyl-N-hydroxy-succinimide ester (MBS) (Pierce, Rockford, IL). A synthetic peptide containing a 4-aa extension including cysteine for cross-linking (YDWIPSSAWGPPC) was made (Bio-Synthesis, Lewisville, TX). Briefly, 10 mg KLH (Imject KLH; Pierce) in degassed, nitrogen-sparged 0.05 M citrate-phosphate buffer, pH 5.0, was stirred gently with 2 mg cross-linker for 1 h at room temperature under a N2 cap, then passed over a Sephadex G-25 column to separate the carrier protein-linker product from unreacted cross-linker. The MBS-KLH was placed into a fresh glass tube and stirred 6 h (N2 cap, room temperature) with 5 mg of the synthetic ed1 peptide, which was dissolved in 50 µl dimethyl formamide. The sample was dialyzed against 3 L of the citrate-phosphate buffer, pH 5.0, to remove free peptide and then dialyzed against three changes of DPBS. Aliquots of the ed1-KLH-conjugate preparation were stored at -20°C before use.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Phage were selected in four distinct selections on three different
mAbs, immobilized either on Sepharose beads or on polystyrene dishes.
In each case, the final selection pools contained phage clones that
bound to multiple IgM Abs in ELISA or immunoblot analysis (see below).
Nonapeptide sequences from individual IgM-binding phage clones are
shown in Table I
. Phage displaying some
sequences, especially those designated ed1, ed4, and ps1, were isolated
considerably more frequently than the others. All sequences contain a
tryptophan residue in the C-terminal or penultimate position, and the
sequences shown in Table I are aligned to that residue. While there is
variability in the sequences capable of binding to IgM, certain motifs
are strongly represented, such as aspartate on the amino-terminal side
of the conserved tryptophan and W I S/P S/Q X D W in many clones. The
absolute or minimal sequence requirements necessary for IgM binding
activity have not been established.
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We have used immunoblotting and ELISA to demonstrate binding to IgM by phage and peptides derived from the phage. Phage clones were initially identified as binding to IgM by plaque lift immunoblots (data not shown).
Immunoblots of SDS-PAGE-separated phage proteins were used to
demonstrate that IgM binding by phage involves the chimeric
pIII-displayed peptide and also to show the specificity of binding.
IgM-binding or nonbinding irrelevant phage were subjected to SDS-PAGE,
blotted onto nitrocellulose, and were incubated with either one of two
different IgM mAbs or a murine IgG mAb. The results of the immunoblot
with the IgM mAb B6 is shown in Fig. 1.
Signal occurred at the appropriate mobility for the pIII-nonapeptide
chimeric protein in the lanes containing the IgM-binding phage
(lane 4, es81; lane 6, ed9; lane
7, ed23; lane 8, ed36; lane 9, ed42; and
lane 17, ed1), but not with any of the irrelevant phage
clones (lanes 2, 3, 5, and 10–16) or with
the parental phage M13KBst (lane 18). Duplicate blots
were prepared and tested against a second IgM mAb, B6.1, or an IgG mAb,
6C5-H4. The identical pattern was seen with the IgM, and no reactivity
was seen with the IgG (data not shown).
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). All IgM mAbs
bound the ed1 pIII-displayed nonapeptide (Fig. 2A,
lanes 1–13). Because the mAbs in Fig. 2A were
primarily directed against carbohydrate Ags, two mAbs against protein
Ags, T17 and S10, were tested for binding to ed1. Fig. 2, B
and C, lanes 1 and 2, demonstrate that
these IgMs also bind to ed1, but not to an irrelevant phage. In
lanes 3 and 4, we showed that two IgG mAbs are
not bound by ed1 (Fig. 2B), but that one of the IgGs will
bind to phage displaying the epitope recognized by that IgG (Fig. 2C). IgM Abs in a murine polyclonal ascites (Fig. 2A, lane 14) are bound by ed1. IgM Abs from human
and rabbit serum samples also bound the pIII band (Fig. 2A,
lane 15, and Fig. 2B, lane 3). The ed1
pIII did not bind IgG Abs, neither mAbs as described above, nor IgGs
found in the affinity-purified secondary Abs used for the control
strips (Fig. 2A, lanes 16 and 17, Fig. 2B, lanes 5 and 6, and Fig. 2D, lanes 1, 2, and 4).
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. Two different IgM-binding phage
(S10-4 and S10-8) were compared with three different Ab-specific phage
(8) in an ELISA in which microtiter wells were coated with
different Abs. S10-4 and S10-8 bound to wells coated with each of the
IgM, but not IgG, Abs, while the Ab-specific phage only bound to the
selecting Ab. Fig. 3 shows that phage ed1
and S10-4 bind to IgMs displaying specificity for both carbohydrate
(S9) and protein Ags (S10 and T17), but do not bind to IgG Abs. The
parental phage M13KBst and an irrelevant phage, J508,22 do not bind to
the IgMs. Because J508,22 represents the epitope bound by mAb 44.1
(9), it binds well to the IgG mAb 44.1.
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A. Seven different
IgM-binding phage and the parental phage M13KBst were serially diluted,
and the IgM binding activity was measured. The results demonstrate at
least a 10-fold difference in relative avidity between those phage with
the highest avidity (ed1, ed42) and those with the lowest (ed9).
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B and demonstrate
that binding occurred with the KLH-ed1 conjugate, but with neither KLH
nor KLH-pep2. Phage bind to cell-surface IgM
Indirect immunofluorescence and flow cytometry were used to
demonstrate that IgM-specific phage bind to the monomeric cell-surface
form of IgM (Fig. 5). Two different B
cell lymphoma lines were used, one expressing cell-surface IgM (5F5),
the other IgA (4F10). FITC-conjugated anti-mouse IgM Abs were used
to demonstrate that 5F5 does express IgM and 4F10 does not
(right panels). Binding of phage to the cells was
detected by first incubating the cells with phage, washing, and then
incubating with rabbit anti-M13 antiserum, followed by
FITC-conjugated anti-rabbit Ig. As shown in the left
panels of Fig. 5, S10-4 phage bind to the IgM-expressing
cells, but not to those with cell-surface IgA. The parental phage,
M13KBst, bind to neither (center panels).
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We next tested the ability of the IgM-binding phage to inhibit Ag binding by IgM Abs. We have performed these experiments in multiple different antigenic systems.
The ability of phage to block the binding to GBS of two anti-GBS
mAbs directed against two different carbohydrate epitopes
(12) is shown in Table III.
For each mAb, the inhibition caused by the IgM-binding phage S10-4 was
compared with the inhibition seen with mAb-specific phage described in
a previous publication (8). Phage S10-4 inhibited both
mAbs, the parental M13KBst inhibited neither, and each mAb-specific
phage inhibited its respective mAb, but not the other. The inhibition
obtained with S10-4 was equal or greater than that obtained with the
mAb-specific phage, which presumably mimics the antigenic structure and
binds within the Ab-combining site. Fig. 6 shows that both ed1 and S10-4 inhibit
the binding of two IgM Abs, one specific for carbohydrate Ag (S9) and
the other for a protein structure (S10), to GBS. However, they inhibit
neither of two IgG Abs (2A6 and 1B1) with the same antigenic
specificity as S9. The parental phage, M13KBst, does not inhibit Ag
binding at all.
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The comparative ability of different IgM-binding phage to inhibit
binding to GBS by mAb S9 is shown in Fig. 7A. The results demonstrate
that there are marked differences in the degree of inhibition by phage
with different displayed sequences, and, not surprisingly, the relative
inhibition correlates with the avidity of phage binding to the IgM Ab
(Fig. 4A). We next tested whether Ab binding to Ag could be
inhibited with a peptide corresponding to the sequence displayed by the
ed1 phage (Fig. 7B). The results showed that the KLH-ed1
conjugate inhibited binding of Ab S9 to GBS, but neither KLH alone nor
KLH conjugated to an irrelevant peptide had that effect.
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. As with the GBS, two different Abs
with different epitope specificity were tested. Inhibition of both Abs,
mAb B6.1 (Fig. 8A) and mAb B6 (Fig. 8B) was
observed with phage ed1, but not with M13KBst. Much greater inhibition
was seen when the phage and Ab were premixed and then added to Ag than
when the phage were added after the initial Ag-Ab interaction had
occurred.
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shows that ed1 phage, but not
M13KBst, inhibited the RF-induced agglutination of IgG-coated latex
beads.
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By use of ELISA, we tested the ability of phage to inhibit complement activation (22). IgM mAb and Ag were allowed to interact, then phage and human complement-containing serum was added. The binding of C1q and C3b to the immune complexes was assessed with specific antisera. There was no difference in complement binding in the presence of IgM-binding phage, control phage, or no phage (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The site of peptide binding on the IgM Abs has not been defined. Because the peptides bind to all IgMs tested, but to no Abs of other classes, it is likely that the site is located within the H chain C regions. Because the peptides bind to IgM Abs of multiple species, presumably this site is well-conserved. Because the peptides bind to monomeric IgM, the J chain is an unlikely site of binding. Because of its effect on Ag binding, it is possible that the peptide binds the Fab. To determine whether these peptides represent a portion of a known IgM ligand, we performed a BLAST search using the National Center for Biotechnology Information website; no homologous protein sequences were identified. We have experimentally demonstrated that peptide binding does not affect complement activation, suggesting that the peptide binding site is not physically close to the C1q-binding site.
Although there is variability in the sequence of the different
IgM-binding peptides, there are several well-conserved elements as
well. A tryptophan is always found in the C-terminal or penultimate
position, with an aspartate residue located on the N-terminal side of
the tryptophan in the large majority of sequences. Interestingly, when
there is not an aspartate adjacent to the C-terminal tryptophan,
elsewhere in the sequence there is an aspartate-tryptophan set,
suggesting that this may be a minimum requirement for IgM binding. Many
of the residues are hydrophobic. Clearly the avidity of binding is
influenced by the sequence of the displayed peptide (Fig. 4), with the
sequence YDWIPSSAW (ed1) having the highest binding activity for murine
IgM. Although we have shown that some of these peptides bind to IgM Abs
of other species, we have not determined whether the sequence
requirements for binding are the same for species other than mouse.
The mechanism whereby the peptides inhibit the binding of Ab to Ag is
intriguing, because the peptides do not appear to bind within the Ab V
region. Two potential mechanisms are steric hinderance or the induction
of a conformational change in the IgM molecule that hinders the binding
to Ag. Although steric hinderance has not been absolutely ruled out,
for several reasons we do not believe it is the mechanism. The peptides
bind to C-region determinants, and it is well established that
anti-isotypic Abs do not block Ag binding. While the phage
displaying the peptides are quite large and could conceivably cause
steric inhibition over a large area, the peptides conjugated to KLH are
smaller and less likely to sterically hinder. Finally, the
stoichiometry of the interaction suggests that the mechanism is more
than simple blocking of the Ag combining site on the IgM, because
virtually complete inhibition of Ag binding was seen at 1:1 molar
ratio of displayed peptide to pentamer IgM (Fig. 7A).
However, to definitively prove that the mechanism of inhibition is not
steric, we will need to construct a small soluble peptide capable of
binding to IgM and then test that molecule for inhibition of Ag
binding. If steric hinderance does not account for the inhibition of Ag
binding, then it is possible that the peptide either induces or
prevents a conformational change in the IgM molecule. There is clear
evidence that IgM molecules undergo a conformational shift upon Ag
binding (23, 24), and it is presumed that this shift, a
lifting of the Fab arms from the planar structure of the molecule,
allows for greater accessibility of the Ag for the combining sites
present on the large IgM molecule. Perhaps the IgM-binding peptides
interfere with this process. Alternatively, the peptides might insert
between unique CH1-CL1 interactions of the IgM and disrupt the
Ag-binding site.
The IgM-binding peptides may represent potential artifacts when PDPL are used to epitope map IgM Abs, because such peptides appear to bind to an IgM Ab in a manner analogous to Ag. It is only when the specificity of binding and Ag inhibition are tested on irrelevant IgM Abs that the true nature of the peptide binding is revealed.
The peptides described in this publication may have both experimental and therapeutic utility. If the affinity of interaction between peptide and IgM molecule is sufficiently high, the peptides may be used to purify IgM Abs on affinity matrices. Because the peptides bind to cell-surface IgM, they may function as agents that activate B cells. The ability of the peptides to block the interaction between IgM Abs and Ag may be used to prevent deleterious Ag-Ab interactions or dissociate IgM-containing immune complexes. Such pathologic IgM Ab interactions can occur in hemolytic transfusion reactions, autoimmune diseases such as rheumatoid arthritis, and in paraneoplastic syndromes associated with Waldenstrom’s macroglobulinemia.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Pati M. Glee, Department of Microbiology, Montana State University, P.O. Box 173520, Bozeman, MT 59717-3520. E-mail address:
3 Abbreviations used in this paper: PDPL, peptide-display phage library; A, absorbance; AP, alkaline phosphatase; DPBS, Dulbecco’s PBS; GBS, group B streptococci; KLH, keyhole limpet hemocyanin; NCM, nitrocellulose membrane; RF, rheumatoid factor.
Received for publication October 13, 1998. Accepted for publication May 10, 1999.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-chain-specific Ab.
D, Western blot strips of clone ed1. Lanes
1 and 3 were incubated with 1:500 human serum,
while control lanes 2 and 4 were
incubated in block alone. Secondary Abs were 1:1000 dilutions of
affinity-purified AP conjugates: lanes 1 and
2, goat-human 
