From the Molecular Immunogenetics Program, Oklahoma Medical Research Foundation, Oklahoma
City, Oklahoma 73104
Polymeric immunoglobulins provide immunological protection at mucosal surfaces to which
they are specifically transported by the polymeric immunoglobulin receptor (pIgR). Using a
panel of human IgA1/IgG1 constant region "domain swap" mutants, the binding site for the
pIgR on dimeric IgA (dIgA) was localized to the C
3 domain. Selection of random peptides
for pIgR binding and comparison with the IgA sequence suggested amino acids 402-410 (QEPSQGTTT), in a predicted exposed loop of the C
3 domain, as a potential binding site.
Alanine substitution of two groups of amino acids in this area abrogated the binding of dIgA to
pIgR, whereas adjacent substitutions in a
-strand immediately NH2-terminal to this loop had
no effect. All pIgR binding IgA sequences contain a conserved three amino acid insertion, not
present in IgG, at this position. These data localize the pIgR binding site on dimeric human
IgA to this loop structure in the C
3 domain, which directs mucosal secretion of polymeric
antibodies. We propose that it may be possible to use a pIgR binding motif to deliver antigen-specific dIgA and small-molecule drugs to mucosal epithelia for therapy.
Key words:
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Introduction |
Secretory IgA (sIgA) mediates humoral immunological
defense at mucosal surfaces (1), the largest surface area of
the body normally exposed to pathogens (2). Mucosal secretion of antibody is mediated by the polymeric Ig receptor (pIgR) via a unique cellular transport process, termed
transcytosis (3). The precursor of sIgA is dIgA, secreted by
plasma cells with bound J chain (4). Compared with IgG,
IgA heavy chains have an additional COOH-terminal 18-amino acid tailpiece with a penultimate cysteine residue
(5). J chain disulfide bonds to two tailpiece cysteine residues, one on each monomeric IgA subunit, and the other
cysteines form a direct tailpiece-tailpiece disulfide bond (6-
9). The pIgR is a type I transmembrane protein with five
immunoglobulin superfamily homology domains (I-V)
constituting the extracellular region (10). Polymeric Igs
IgA and IgM are bound by pIgR at the basolateral surface
of mucosal epithelial cells, transported through these cells,
and secreted at the mucosae (3). sIgA retains the extracellular region of the pIgR, termed secretory component, covalently bound to dIgA (11), although the initial interaction with dIgA is the high-affinity noncovalent binding
of pIgR domain I (14, 15).
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Materials and Methods |
Baculovirus Expression.
Arsonate hapten-specific chimeric IgA1
and IgA1/IgG1 domain swap mutants were expressed as previously described (16, 17). Dimeric IgA was generated by coexpression of IgA with J chain. Affinity purification was carried out
on arsonate-sepharose. Antibodies were eluted with 200 mM arsanillic acid () in 200 mM Tris-HCl, pH 8.0, which was removed by extensive dialysis against PBS. Mono-
and dimeric IgA were detected by 4% nonreducing SDS-PAGE
analysis. The hexahistidine-tagged human pIgR extracellular domain was expressed in a similar manner and purified on a Ni-NTA Agarose (Qiagen) column (18).
Construction of Mutant IgA Antibodies for Baculovirus Expression.
The C
3 loop mutants L1, L2, and L3 were constructed by PCR
SOEing (splicing by overlap extension; reference 19) using the following complementary pairs of sense (S) and antisense (AS) primers: L1S 5'-GAGCCCAGCGCGGGCGCCGCCGCCTTCGCTGTG-3', L1AS 5'-CTCGGGTCGCGCCCGCGGCGGC-GGAAGCGACAC-3'; L2S 5'-TACCTGACTGCGGCAGCCGCGCAGGAGCCC-3', L2AS 5'-ATGGACTGACGCCGTC-GGCGCGTCCTCGGG-3'; and L3S 5'-CGGCAGGAGGCCGCCGCGGCCACCACCACC-3', L3AS 5'-GCCGTCCTCCGGCGGCGCCGGTGGTGGTGG-3'. The outer primers
B1-2 (5'-CCTATAACCATGGGATGGAGCTTCATC-3'), specific for the 5' leader of the VH region of this chimeric IgA heavy chain, and C
3-3' (5'-CCCTCTAGATTAGTAGCAGGTGCCGTCCAC-3'), specific for the 3' tailpiece-encoding sequence
of the IgA1 gene, were used with the above primer pairs L1-L3
to generate pairs of 5' and 3' fragments with complementary
overlaps. These fragments were gel purified then spliced in a further PCR reaction using the outer primers B1-2 and C
3-3'.
Modified IgA1 genes were cloned into the baculovirus transfer
vector using XbaI and NcoI digestion and the insert sequences
were verified. Recombinant baculovirus were produced using
the BacPAK system ().
FACS® Analysis.
Madin-Derby canine kidney (MDCK) cells
were placed in serum-free MEM plus Earle's salts (MediaTech,
Inc.) 16 h before the experiment. Cells were harvested in 10 mM
EDTA in PBS and washed in PBS 0.1% BSA. pIgR binding of
human IgA1 antibodies and mutants was assessed by incubation
of 100 µl antibody in PBS/BSA with ~106 cells for 1 h. Cells
were washed three times in PBS/BSA and bound antibody was
detected with 100 µl of an anti-human
FITC conjugate () diluted 1:100. Cells were washed as above and resuspended in 1 ml PBS/BSA. FACS® analysis was carried out on a
FACScan® instrument. Data collection and
analysis were performed with the LYSYSII ()
and WINMIDI (http://facs.scripps.edu) or with the Cellquest
programs ().
Phage Display Peptide Library Selection.
The random 40-mer
peptide library was constructed in the pCANTAB5e vector and
has an actual total diversity of 1.55 × 1010 (20). The random 40-mer is flanked by two peptide tag sequences, preceded by a leader
peptide and fused to the membrane-proximal domain of the M13
phage coat protein III. 1-2 × 106 MDCK cells were harvested in
5 ml PBS plus 10 mM EDTA at 37°C, washed twice in 15 ml
PBS, and resuspended in 1.8 ml PBS at 4°C. 100 µl phagemid library stock (4.5 × 1012 CFU) was added and incubated for 1 h at
37 or 4°C. The cells were then washed five times with 15 ml PBS
at 4°C. Bound phage were eluted with 2 ml of 0.1 M glycine/
HCl, pH 2.2, containing 0.1% BSA for 10 min and neutralized
immediately with 400 µl of 2 M Tris base. Phage rescue and amplification were carried out in Escherichia coli strain TG1 () according to standard procedures (21).
DNA Sequencing and Analysis.
DNA sequencing was carried
out on double-stranded plasmid or phagemid DNA using an ABI
377 Prism (Applied Biosystems, Inc.) automated sequencer.
Alignments of deduced peptide sequences and Ig-constant regions
were carried out using the MAP (22) and PIMA (23) software.
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Results and Discussion |
Chimeric human IgA1 (16) and a panel of IgA1/IgG1
constant region domain swap mutants (24) with murine-encoded arsonate specificity were expressed in baculovirus
as both monomer and dimer, affinity purified, and used to
define the pIgR binding site. dIgA was operationally defined as an IgA preparation generated by coexpression of
IgA with J chain. MDCK cells, transfected with rabbit
pIgR (25), were used to measure binding of recombinant IgA1 mutants to the receptor by FACS® analysis (Fig. 1 a).
Specific binding was observed with dIgA and not with monomeric IgA (Fig. 1 b), a medium control (Fig. 1 b) or IgG
(data not shown). Mutant VGAA, in which the C
1 domain was substituted with the C
1 domain, bound to the
pIgR in a manner similar to wild-type IgA1 (Fig. 1 c). The
dimeric molecule (Fig. 1 c, heavy line) bound to the receptor, whereas the monomer (light line) did not. Similarly,
the VGGA mutant, in which both C
1 and C
2 including
the hinge of IgA were replaced with the analogous domains
from IgG, bound as a dimer but not as a monomer (Fig. 1
d). Thus, the C
1 and C
2 domains of dIgA are not necessary for pIgR binding, suggesting that the presence of the
C
3 domain is required.

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Fig. 1.
Binding of monomeric and dimeric IgA/IgG domain swap
mutant antibodies to pIgR expressed on MDCK cells. (a) Staining of
MDCK cells with sheep anti-pIgR (heavy line) antiserum or normal
sheep serum (broken line) followed by anti-sheep IgG FITC conjugate.
(b) Binding of wild-type IgA monomer (thin line) or dimer (heavy line)
to pIgR on MDCK cells. (c) Binding of VGAA mutant expressed as
monomer (thin line) or dimer (heavy line) to pIgR on MDCK cells. (d)
Binding of VGGA mutant expressed as monomer (thin line) or dimer
(heavy line) to pIgR on MDCK cells. Bound IgA or IgA/G chimeric antibodies were detected by rabbit anti-human chain-FITC conjugate.
Negative controls are shown as broken lines.
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dIgA contains four C
3 domains and the covalently
bound J chain which, together with the IgA tailpiece, are
responsible for IgA polymerization. To reduce the complexity of this problem, a library of random 40-mer peptides, expressed as a phage display library (20), was selected
against pIgR-expressing MDCK cells. The goal was to
identify putative pIgR binding sites within IgA by reducing
them to a minimum peptide binding unit, a proven approach for several receptor-ligand interactions (26). Selection was carried out on live pIgR-expressing MDCK
cells in suspension with negative selection on nonreceptor-
expressing cells. Bound phage were eluted with acid or by
cell lysis. Recovery of both acid-eluted and cell-associated
phage increased gradually from ~6 × 104 to 5 × 107 CFU
over 4-6 successive rounds, indicating enrichment for specific binding clones. Individual clones were randomly selected from the final panning from the acid-eluted and
membrane-associated fractions and sequenced. Binding of
the enriched phage populations to recombinant human
pIgR, as measured by ELISA, increased with successive
rounds of panning and was inhibited by polymeric IgM (data not shown). Sequencing of phagemid DNA showed
that 20 out of 32 acid-eluted clones and 12 out of 32 cell-associated clones had open reading frames (Fig. 2). There is
little clonality among these two groups of sequences, although the A22 peptide was recovered three times. These
peptides were aligned for maximum homology with the
human IgA1 C
3 region amino acid sequence (Fig. 2) using the PIMA program (23). Many of the peptides, particularly A12 (9 out of 30 identical amino acids) (Fig. 3 a),
show homology with human IgA1 C
3 domain, prompting a further examination of the amino acid sequence and
structure in this area.

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Fig. 2.
Alignment of deduced peptide
sequences from selection of phage display peptide library against pIgR receptor-expressing
cells with the human C 3 domain amino
acid sequence. Peptides designated A or M
are from the acid-eluted and cell-associated
fractions, respectively. Numbering of IgA1
is according to reference 5.
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Fig. 3.
Comparison of IgG1 and IgA1 CH3 sequences and IgG1
structure in the area homologous to several phage-derived peptides. (a) The
A12 peptide alignment with human IgA1 and IgG1. IgGSTR indicates structural features of IgG1 where < denotes a -strand running in a descending
orientation (i.e., hinge to CH3 direction), > denotes a -strand running in
an ascending direction (i.e., CH3 to hinge direction), and - denotes a loop or
open structure (29). (b) Comparison of several mammalian IgA sequences
with the four human IgG subclasses showing the additional IgA-specific
amino acids present in the loop at positions 402-410 in the IgA sequence.
hu, human; gr, gorilla; mur, murine; rab, rabbit. (c) IgA1 C 3 mutants L1,
L2, and L3 aligned with the C 3 and C 3 wild-type sequences and C 3
structure (IgGSTR). = denotes sequence identity in the mutants, - denotes
a space introduced in the IgG sequence to maximize homology, and
IgGSTR is labeled according to panel a. Numbering of IgA1 and IgG1 is
according to references 5 and 29, respectively.
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The human C
3 domain is 40% identical and 62% homologous to the corresponding region of human IgG1 at
the amino acid level. In addition, all the sequence hallmarks of the immunoglobulin superfamily fold are conserved. Accordingly, the human IgG1 crystal structure (29)
was used to predict the likely positions of the major structural motifs (
-strands and loops) within the IgA1 sequence, an approach used previously to map the Fc
R
(CD89) binding site on IgA1 (24). Fig. 3 a shows the alignment of the peptide A12 with the IgA1 sequence and the
corresponding IgG1 sequence with its secondary structural
features. The A12 peptide is homologous to a region that
in the IgG structure forms an exposed 6-amino acid loop
between two
-strands. However, in IgA1, this area contains a 3-amino acid insertion to expand the loop to 9-amino
acids. The flanking
-strand sequences and part of the loop
are conserved between IgA and IgG, which suggests that
gross structural features are also conserved. Fig. 3 b shows
alignment of this region in the CH3 domain of five mammalian IgA molecules aligned with the four human IgG
subclasses. Despite sequence differences in the loop, all IgA
sequences have the three additional amino acids, whereas
the IgG sequences do not. Similar to IgA, the sequence of
IgM contains a 2-amino acid insertion at this site (data not shown). On the basis of these observations, three mutant
IgA1 molecules were constructed and expressed in baculovirus to examine the effect of amino acid changes in this
area on pIgR binding (Fig. 3 c). Mutations were made in
the loop itself (L1 and L3) and in the
-strand NH2-terminal to the loop (L2) as a negative control. Binding was then
measured to the physiologically relevant human receptor
by ELISA using the purified recombinant extracellular domain of human pIgR expressed in baculovirus as previously
described (18). Fig. 4 shows the binding of IgA1 monomer,
IgA1 dimer, and IgG compared with the monomeric and
dimeric forms of the L1, L2, and L3 mutants to purified
human pIgR. Only dimeric wild-type IgA1 and dimeric L2
mutant, in which the mutations are in the
-strand NH2-terminal to the loop, show binding. Mutations within the
loop itself, namely L1 and L3, abrogate the binding of the dimeric IgA1 mutant molecules to the pIgR. Similar binding patterns were obtained with the loop mutants and rabbit pIgR-expressing cells as measured by FACS® (data not
shown). These results indicate that this C
3 loop is the major binding motif for the pIgR on dIgA.

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Fig. 4.
Binding of IgA mutants L1, L2, and L3 to purified
human pIgR by ELISA. The extracellular domain of human
pIgR was purified after expression in baculovirus and coated
onto ELISA plates at 10 µg/ml.
Chimeric IgA1 and IgA1 C 3
mutants L1, L2, and L3 were expressed as both monomeric (m)
and dimeric (d) forms along with chimeric IgG1, purified and incubated
on the pIgR-coated plates to compare their abilities to bind to pIgR.
Bound antibodies were detected with anti-human light chain alkaline
phosphatase conjugate.
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IgA is, in functional terms, closely related to IgM, sharing its ability to polymerize and be secreted. However, the
overall IgA domain organization resembles that of IgG.
The presence of amino acid sequence insertions in all the
polymeric Igs that are ligands for this receptor and the absence of insertions from non-pIgR binding Igs (Fig. 3 b)
supports its role in Ig secretion. The variation in the insertion size and the actual IgA and IgM sequences may reflect
differences in fine structure of these polymeric antibodies or in their affinity for pIgR binding.
The fact that monomeric IgA is not secreted suggests
that either a conformational change induced by polymerization is required for dIgA binding to the receptor or that
the binding requires a polyvalent interaction of these C
3
sites with the receptor. The presence of J chain is required
for optimal IgA (or IgM) polymerization but its precise role
in Ig secretion remains to be elucidated. The increase in
binding observed with dimeric L3 when compared with
monomeric L3 (and to a lesser extent with the L1 mutants) suggests that J chain and/or polymerization may play a role
in binding (Fig. 4). Although amino acids 402-410 in the
C
3 domain of dIgA define a major pIgR binding site,
other dIgA structures may be involved. J chain-deficient
mice express lower levels of polymeric IgA and have impaired hepatic transport of IgA (which humans lack) but
normal levels of IgA at mucosal epithelial sites, compared
with wild-type mice (30, 31). J chain thus may not be necessary for secretion of IgA but still required for stable binding to the secretory component in the mucosal environment; however, alternative secretory mechanisms may also
be involved. Further studies are underway with peptides
and additional mutations to examine the nature of the interaction between IgA and the pIgR as well as the role of
J chain. The ability of a peptide sequence to confer mucosal
secretion upon a molecule may prove a powerful means of
delivery of therapeutic molecules to mucosal areas where they may prevent the entry of pathogens.
Address correspondence to J. Donald Capra, Oklahoma Medical Research Foundation, 825 N.E. 13th St.,
Oklahoma City, OK 73104. Phone: 405-271-7210; Fax: 405-271-8237; E-mail: jdonald-capra{at}omrf.ouhsc.edu
This study was supported in part by National Institutes of Health grant AI12127. J. Hexham was supported in part
by the Pediatric AIDS Foundation. K. White was a recipient of a Leukemia Research Foundation fellowship.
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