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The Journal of Immunology, 2007, 178: 1589-1597.
Copyright © 2007 by The American Association of Immunologists, Inc.

Secretory Antibody Formation: Conserved Binding Interactions between J Chain and Polymeric Ig Receptor from Humans and Amphibians1

Ranveig Braathen*, Valerie S. Hohman{dagger}, Per Brandtzaeg* and Finn-Eirik Johansen2,*

* Laboratory for Immunohistochemistry and Immunopathology, Institute of Pathology, University of Oslo, Rikshospitalet-Radiumhospitalet Medical Center, Oslo, Norway; and {dagger} Department of Biology, University of San Diego, San Diego, CA 92110


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Abs of the secretory Ig (SIg) system reinforce numerous innate defense mechanisms to protect the mucosal surfaces against microbial penetration. SIgs are generated by a unique cooperation between two distinct cell types: plasma cells that produce polymers of IgA or IgM (collectively called pIgs) and polymeric Ig receptor (pIgR)-expressing secretory epithelial cells that mediate export of the pIgs to the lumen. Apical delivery of SIgs occurs by cleavage of the pIgR to release its extracellular part as a pIg-bound secretory component, whereas free secretory components are derived from an unoccupied receptor. The joining chain (J chain) is crucial in pIg/SIg formation because it serves to polymerize Igs and endows them with a binding site for the pIgR. In this study, we show that the J chain from divergent tetrapods including mammals, birds, and amphibians efficiently induced polymerization of human IgA, whereas the J chain from nurse shark (a lower vertebrate) did not. Correctly assembled polymers showed high affinity to human pIgR. Sequence analysis of the J chain identified two regions, conserved only in tetrapods, which by mutational analysis were found essential for pIgA-pIgR complexing. Furthermore, we isolated and characterized pIgR from the amphibian Xenopus laevis and demonstrated that its pIg binding domain showed high affinity to human pIgA. These results showed that the functional site of interaction between pIgR, J chain and Ig H chains is conserved in these species and suggests that SIgs originated in an ancestor common to tetrapods.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Secretory IgA (SIgA)3 has been recognized as the major Ab class in mammals for >40 years. The conventional explanation for this huge SIgA production has been that it must protect the large mucosal surfaces against a many different bacterial and viral pathogens. Recently, however, the role of SIgA in host interactions with the commensal bacteria in the intestine has been revealed (1). Thus SIgA is important both for defense against mucosal pathogens but also to restrict the indigenous gut flora to the lumen.

Animals are adapted to live with vast number of bacteria colonizing their mucosal surfaces. Such mutualistic microorganisms provide benefit to the host, not only as competitors of pathogenic bacteria but also through direct positive tissue interactions (2, 3). However, if the mucosal barrier is breached the indigenous microbiota may cause life-threatening infections or a severe tissue-damaging response from the systemic immune system. In mammals, luminal bacteria induce production of IgA Abs (4) that cooperate with innate defense mechanisms on the mucosal surfaces to provide protection against pathogens and to restrict commensals to the lumen (5, 6, 7).

Plasma cells located in the mucosal lamina propria of the gastrointestinal and respiratory tracts are preferentially switched to IgA and coexpress the joining chain (J chain). Therefore, mucosal plasma cells secrete mostly polymeric IgA (pIgA; predominantly dimers) with the capacity to bind to the polymeric Ig receptor (pIgR) (8, 9). SIgA production is initiated when pIgA binds pIgR expressed on the basolateral side of epithelial cells lining the mucosal surfaces. Endocytosis and intracellular trafficking of the receptor-ligand complex to the apical plasma membrane is mediated by sorting signals in the cytoplasmic tail of the pIgR, and lumenal delivery of SIgA occurs by cleavage of the receptor near the plasma membrane (10). Covalently bound secretory component (SC; the extracellular cleaved piece of the pIgR) confers mucophilic properties and proteolytic resistance to the SIgA molecule (11). Similarly generated SIgM Abs are mainly important in the neonate and in individuals with selective IgA deficiency (12), whereas constitutive transcytosis of unoccupied pIgR leads to the secretion of free SC (12).

The J chain has been characterized in a range of species including mammals (human (13, 14), mouse (15), bovine (16), rabbit (17), and brushtail possum (18)), birds (chicken (19)), reptiles (turtle (20)), amphibians (Xenopus laevis (21) and Rana catesbeiana (22)), and cartilaginous fish (nurse shark (23) and clearnose skate (24)). In humans, this unique 15-kD polypeptide contains eight Cys residues that form three intrachain bridges and two bridges to the tailpieces of IgA or IgM in human pIgs (25). No crystal structure is available for SIgA or SIgM, but several lines of evidence suggest that J chain and pIgR interact directly, although this remains controversial (8, 9, 25, 26).

Monomeric IgA (composed of two H chains and two L chains) readily form polymers when coexpressed with a J chain (13). Earlier investigations into the function of the J chain showed that structural requirements for polymerization of IgA were less stringent than the requirements for pIgR/SC binding of the resulting polymers (27). In this study we show that J chain from several different tetrapod species is capable of inducing polymerization of human IgA and that the resulting pIgA binds human pIgR/SC. Furthermore, we identify two conserved regions in the J chain that are necessary for the latter function only. Finally we characterize the first amphibian pIgR and show that its pIg binding domain is capable of binding human pIgA. These findings reveal a remarkable conservation of protein-protein interaction sites in secretory Abs from different species, suggesting an evolutionary significance of this first-line defense system.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Plasmid construction

The plasmid for episomal expression of wild-type human J chain (pCEP4-wtJ chain) has been described previously (27); this expression vector contained ori P and encoded EBV nuclear Ag (EBNA) for episomal replication, HygR for antibiotic selection, and CMV promoter for cDNA expression. The J chain open reading frame from mouse (15), chicken (28), X. laevis (21), bullfrog (22), and nurse shark (23) was isolated by PCR and subcloned into pCEP4. Mutagenesis of the human J chain was performed by QuikChange PCR (Stratagene) or by splice overlap extension and subcloning into a HindIII- and BamHI-digested pCEP4. The entire open reading frame of all plasmids was verified by DNA sequencing (Medigenomics).

A plasmid for recombinant expression of human SC was constructed by PCR with a forward primer introducing a Kozac sequence at the ATG initiation codon and a reverse primer inserting a stop codon at the cleavage site for SC. The resulting PCR product was ligated into pCDNA3.1 directional TOPO (Invitrogen Life Technologies). A TBLASTN search with the amino acid sequence of the extracellular domain 1 (D1) of human or chicken pIgR as the query both identified an expressed sequence tag putatively encoding X. laevis pIgR. The clone (IMAGE 4031968) was obtained from Geneservice and sequenced in full (GenBank accession no. EF079076). An expression plasmid for xD1-hD2D5 SC was constructed by fusing the D1 of X. laevis pIgR with the domain 2 (D2) from human pIgR by splice overlap extension and subcloning as described above (domain boundaries are indicated in Fig. 3). An expression plasmid for hD2D5-SC was constructed by deleting the D1 from the human SC expression plasmid by QuikChange PCR.


Figure 3
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  		FIGURE 3. Alignment of pIgR from X. laevis with human, possum, and chicken pIgR. Residues identical in all sequences, highly conserved sequences, or conserved sequences are indicated by a star (*), a colon (:), and a period (.), respectively. Gaps in the alignment are indicated by hyphens. The start of each extracellular domain, the cytoplasmic tail, and the cleavage site for human pIgR is indicated. The CDR1, CDR2, and CDR3 in D1 are written in bold font. The predicted signal peptide and transmembrane region are underlined. GenBank accession numbers: Homo sapiens, NP_002635; possum (Trichosurus vulpecula), AAD41688; and chicken (Gallus gallus), AAW71994.

 
Production and purification of recombinant IgA and SC

The 5-iodo-4-hydroxy-2-nitrophenylacetyl (NIP)-specific IgA1-producing Chinese hamster ovary (CHO) cell line {alpha}4.2 (27) was cultured in Ham’s-F12 medium supplemented with 10% FCS, 2 mM glutamine, and 50 µg/ml gentamicin. Semistable transfections (episomal expression) were performed as previously described (27). Briefly, cells were seeded at 10–15% confluence in 6-well trays on day 1 and transfected on day 2 with 1.4 µg of DNA and 3 µl of FuGENE (Roche Diagnostics) according to the manufacturer’s protocol. On day 3, the cells received fresh medium and on day 4 they were trypsinized and seeded into 10-cm plates in medium supplemented with 300 µg/ml hygromycin B (Roche Diagnostics). Cells received fresh medium with hygromycin B every 3–4 days; after 10–12 days each 10-cm plate contained at least 500 hygromycin B-resistant colonies that were pooled and seeded into 10-cm plates or large flasks for IgA production. The vector pCEP4 without insert was used as negative control (mock).

IgA Abs secreted from pools of >500 transfectants were affinity-purified on a NIP-Sepharose column (13). Fractions containing monomeric or pIgA were separated by ion-exchange (MonoQ) chromatography (27). Briefly, IgA was eluted by a linear salt gradient from 2.5–500 mM NaCl in 20 mM Tris-HCl (pH 7.1) and collected in 300-µl fractions. Peak fractions containing pIgA were pooled and analyzed for molecular size by native gel electrophoresis and by ELISA for binding to free SC and for J chain content (see below).

The J558L transfectoma cell lines expressing human Fc-containing IgA1, IgM, and IgG1 have been described previously (29, 30). Crude cell supernatants were used in ELISA for SC binding analyses.

Production of recombinant human free SC and derivatives were performed essentially as described (31). Briefly, one 10-cm plate with 293E cells was transiently transfected with 6 µg of each expression vectors using FuGENE. Supernatants were harvested after 3–7 days, and the SC was precipitated by ammonium acetate, resuspended, and dialyzed against PBS.

Immunoprecipitations, gel electrophoresis, and Western blotting

Approximately 500 ng of IgA from CHO cell supernatants were immunoprecipitated with rabbit anti-human IgA (diluted 1/100; DakoCytomation) and sheep anti-rabbit Ig-coated magnetic beads (7 x 106 beads in 1 ml; Dynal). Samples were resolved by nonreducing SDS-PAGE on a 5% (w/v) acrylamide gel and transferred onto a polyvinylidene fluoride membrane (Millipore) that, after air drying, was blocked with PBST (10% skimmed milk in PBS plus 0.05% Tween 20). Subsequent incubations were all performed in PBST. The membrane was incubated with a mouse antiserum to human IgA (diluted 1/3000; gift from T. Lea, Institute of Immunology, Rikshospitalet-Radiumhospitalet Medical Center, University of Oslo, Norway) for 90 min, washed three times for 10 min, and then incubated with HRP-conjugated sheep anti-mouse IgG (diluted 1/3000; Amersham Biosciences) for 90 min. After three washes, the membrane was incubated for 10 min with SuperSignal (Pierce) and the substrate was detected with a light-sensitive camera (Chemidoc; Bio-Rad).

For analysis of NIP-purified IgA, 30 ng were resolved by nonreducing SDS-PAGE on a 5% (w/v) acrylamide gel with the Criterion system (Bio-Rad) and transferred and probed as described above except that the membrane was first incubated with a rabbit antiserum to human IgA (diluted 1/3000; DakoCytomation) and next incubated with HRP-conjugated donkey anti-rabbit Ig (diluted 1/3000; Amersham Biosciences). For J chain immunoblotting, the membrane was stripped in buffer (53 mM Tris (pH 6.8), 1.6% SDS, and 14.3 mM 2-ME) at 60°C (15 min) and then blocked again with 10% (w/v) skimmed milk in PBST and incubated with our rabbit antiserum to human J chain (previously absorbed with monomeric IgA) diluted 1/800 (32). Secondary Ab and the revealing reaction were as described above.

For native immunoblots, 60 ng of IgA was resolved on a 5% (w/v) acrylamide gel as described above. SDS was omitted from all buffers and the pH of the loading buffer was 8.5. The gel was transferred to a polyvinylidene difluoride membrane and the membrane was probed as for analysis of NIP-purified IgA as described above.

ELISAs

For all Ag-specific ELISAs, microtiter plates were coated with 3 µg/ml NIP-BSA and blocked with 1% (w/v) BSA in PBS. For total IgA quantification, the primary Ab was mAb against human IgA (diluted 1/30 000; a gift from T. Lea, Institute of Immunology, Rikshospitalet University Hospital, Oslo, Norway) and the secondary Ab was alkaline phosphatase-conjugated rabbit anti-mouse Ig Ab (diluted 1/1000; DakoCytomation).

For J chain-specific ELISA (Table I) and the SC-affinity ELISA (Figs. 1C and 2B), 800 ng/ml, 400 ng/ml, 200 ng/ml, and 100 ng/ml purified recombinant pIgA was incubated in triplicate wells overnight. For J chain-specific ELISA, the microtiter plates were washed and fixed with 2% glutaraldehyde for 30 min then incubated with 6 M urea (pH 3.0) for an additional 30 min to reveal bound J chain by denaturation (27). After washing, the plates were incubated with our rabbit antiserum to human J chain (diluted 1/300) and alkaline phosphatase-conjugated goat anti-rabbit Ig (diluted 1/2000; DakoCytomation). The J chain content of each pIgA sample was normalized to pIgA with a wild-type human J chain. For SC affinity measurement, the microtiter plates were washed and incubated with 8 µg/ml free SC purified from human colostrum (33), then rabbit antiserum to human SC (DakoCytomation; diluted 1/1000), and finally goat anti-rabbit Ig as above. The values were expressed as the slope of the linear regression of OD405 vs IgA concentration and normalized to the corresponding slope obtained for NIP-specific myeloma pIgA (27).


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  		Table I. Characteristics of J chain variants used in this study

 

Figure 1
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  		FIGURE 1. J chains from different vertebrates facilitate polymerization of human IgA and binding to human SC. A, NIP-specific recombinant IgA without a J chain (mock) or containing a J chain from different species was immunoprecipitated with polyclonal anti-human IgA serum, subjected to nonreducing SDS-PAGE, and immunoblotted with a mAb Ab against human IgA. Migration of monomers and dimers are indicated. The percentage of polymers for each sample is indicated above each lane. B, Binding of recombinant human SC to CHO cell-produced IgA with different J chains was analyzed by ELISA as described in Materials and Methods. Mean OD values + SD from quadruplicate wells in one of three similar experiments is shown. C, The polymeric fractions shown in A were purified by chromatography and tested for binding to native human secretory component in an ELISA as described in Materials and Methods. Mean ± SEM of three independent experiments. Binding values that differ from wild-type J chain or from mock control (p < 0.05; two-tailed Student’s t test) are indicated by a star (*) or a closed circle (•), respectively.

 

Figure 2
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  		FIGURE 2. Two conserved regions in the J chain are critical for binding of polymeric IgA to SC. A, The amino acid sequences of the J chains from human, mouse, chicken, bullfrog, X. laevis, and nurse shark were aligned with ClustalW. Dashes denote identity to residues in the human sequence, and dots denote gaps. Amino acid numbering, predicted beta-strands, and surface accessibility (bold font) of human J chain are indicated. SS marks the three intrachain disulfide bridges and "TP" marks the Cys paired with the secretory tail piece of IgA or IgM. The conserved N-linked glycosylation site is indicated by a star (*). Secondary structure prediction and surface were performed with JPRED (http://www.compbio.dundee.ac.uk/). B, NIP-specific pIgA containing mutants of human J chain was purified and analyzed as described in the legend to Fig. 1. Names of mutants include original amino acid (left), the position as indicated in A, and the new amino acid (right). Where several amino acids were altered, the first amino acid in the range is indicated. Mean + SEM of four independent experiments. Binding values that differ from wild-type J chain or from mock control (p < 0.05; two-tailed Student’s t test) are indicated by a star (*) or a closed circle (•), respectively.

 
ELISA for recombinant human and chimeric SC was performed by coating microtiter plates with 2 µg/ml SpsA surface protein from S. pneumoniae, which requires intact human D3D4 for SC/pIgR binding (31). Detection was with a mAb against human pIgR D2 (present in all SC variants) (1/5000; Sigma-Aldrich) followed by HRP-conjugated goat anti-mouse IgG (1/2000; Sigma-Aldrich) and color reaction with tetramethylbenzidine peroxidase substrate (Kirkegaard & Perry Laboratories).

The binding of recombinant human and chimeric SC to pIgA1 or pentameric IgM or IgG1 was performed by coating microtiter plates with NIP-BSA as described above and saturating with NIP-specific J558L transfectoma-produced Ig (~1.5 µg/ml). Dilution series of SC were added and detected as in ELISA for recombinant SC. For the binding of recombinant human and chimeric SC to CHO-produced IgA containing human, mouse, chicken, Xenopus, bullfrog, or nurse shark J chain, CHO cell supernatants were precipitated with 40% (w/v) ammonium acetate, resuspended in PBS, and the concentration was adjusted to 1.5 µg/ml. ELISA for SC binding analysis was performed as described above.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
J chains from divergent tetrapods support pIgA and SIgA formation with human IgA and human secretory component (SC)

We coexpressed the J chain from human, mouse, chicken, bullfrog, X. laevis or nurse shark with human IgA and analyzed the secreted IgA for molecular size and pIgR affinity (Fig. 1). Although the human J chain induced the greatest level of polymerization, the J chain from mouse, chicken, or Xenopus also induced significant pIgA formation (Fig. 1A; Table I). We then tested the binding of secreted IgA to recombinant human free SC (Fig. 1B). We found that the J chains from different species that induced polymerization of human IgA efficiently also were part of a polymer with high affinity to human SC. Surprisingly, the small amounts of pIgA formed in the presence of the bullfrog J chain showed significant affinity to human SC. Conversely, the small amounts of pIgA formed in the absence of J chain (or when IgA was coexpressed with nurse shark J chain) showed virtually no affinity to SC. To eliminate possible bias in the SC binding experiments due to different levels of polymerization, we purified each pIgA fraction and analyzed its ability to bind native human free SC purified from the colostrum (Fig. 1C). These results confirmed the binding pattern described above.

Two motifs in human J chain are important for pIgA affinity to pIgR/SC

We identified two regions of high amino acid conservation in the J chain from tetrapods (regions 2 and 3), but only one of them (region 2) was conserved in the shark (Fig. 2A; Table II). We tested a series of point mutations of these two regions in the human J chain as well as a third amino-terminal region predicted to be in a surface-exposed loop (region 1) for the effect on pIgA formation and SC binding (Fig. 2B).


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  		Table II. Sequence conservation of regions of J chaina

 
Several mutations in region 1 showed similar ability to support pIgA formation and SC binding as the wild-type J chain, suggesting no direct involvement of this region in polymerization or pIgR interaction. For region 2, we found that substitution of amino acids 50–52 (ISD) with Ala significantly reduced the affinity of pIgA to SC. Because glycosylation of the phylogenetically conserved Asn49 is directed by Ser51 the ISD mutant lacked the glycosylation of Asn49. However, the mutation of Asn49 to Ala (N49A) did not reduce pIgA affinity to SC, although a slight decrease in pIgA formation was seen (Table I) similar to that previously observed with mutagenesis of mouse J chain (34). Furthermore, the I50A/D52A mutant showed reduced SC affinity, although glycosylation would be maintained. Thus, glycosylation per se was apparently unimportant for the pIgR interaction, although an important motif for pIgR interaction was located in the highly conserved region 2 of the human J chain.

A region between the sixth and seventh Cys of the J chain was found to be highly conserved in all tetrapods examined, but not in the nurse shark (Table II; Fig. 2A). Again, we made several J chain mutations in this region and coexpressed the resulting J chain variants with human IgA. All mutants supported pIgA formation (Table I). When analyzing pIgA binding to human SC, we found that mutation of two Tyr residues (Tyr102 and Tyr104) to Ala was not tolerated, suggesting a critical role in pIgR interaction. Nevertheless, Phe residues were able to substitute in these positions without loss of SC binding. Therefore, we concluded that these Tyr residues are most likely required for a hydrophobic "core" rather than for direct pIgR interaction. Several different mutations that affected Arg106 significantly reduced SC binding to the pIgA produced. Substitution of this Arg for Ala or for a negatively charged Glu (R106E) had a similar effect (Fig. 2B; compare R106A with R106E). Altering the negatively charged Asp105 to Arg did not compensate for the R106E mutation (DR105RE). Therefore, it appeared that Arg-106 was essential for pIgR interaction. Although the triple mutation (RNK106AAA) tended to produce a slightly less functionally active pIgA than R106A, there was no statistical difference between these mutants. Furthermore, no other individual amino acid in this region resulted in a J chain with significant loss of function. Altogether, Arg106 was the only amino acid in region 3 found to be essential for pIgR interaction.

X. laevis pIgR binds human pIgA but not pentameric IgM

Having established that the J chains from birds and amphibians efficiently substituted for human J chain in the formation of pIgA with a pIgR docking site, we made the assumption that amphibians encode a pIgR. By BLAST search, we identified an expressed sequence tag encoding X. laevis pIgR and obtained the clone from Geneservice. The sequencing of cDNA from Xenopus revealed that its pIgR contained four extracellular Ig-like domains similar to the pIgR from chicken (Fig. 3) but in contrast to mammalian pIgR, which contains five Ig-like domains (18, 35, 36). The alignment of Xenopus pIgR with the pIgR from human, possum, and chicken and a sequence comparison of each Ig-like domain indicated that X. laevis pIgR lacks an Ig domain equivalent to mammalian D2 (Fig. 3; Table III). Interestingly, a Pro residue within the transmembrane helix present in mammalian and chicken pIgR was absent in Xenopus pIgR, suggesting that signaling through this receptor may be different in amphibians. However, the strong basolateral targeting signal present in all pIgRs was also conserved in Xenopus pIgR.


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  		Table III. Sequence comparison of the extracellular Ig-like domains from pIgRa

 
The initial noncovalent interaction of pIgA and pentameric IgM with pIgR is mediated by its extracellular D1 and the respective Ig Fc portions, including the J chain (37, 38). We wanted to test whether mammalian pIgA could bind amphibian pIgR and therefore constructed a chimeric SC molecule that contained D1 from Xenopus and D2 through D5 from human pIgR (xD1-hD2D5). The binding of this chimera to human pIgA or pentameric IgM or IgG was compared with that of human SC. The xD1-hD2D5 chimeric SC showed distinct binding to pIgA but no detectable affinity to pentameric IgM (Fig. 4A). Conversely, human SC bound both pIgA and pentameric IgM. A human SC lacking D1 showed no affinity to either ligand, demonstrating that human pIgA bound the Xenopus-human chimera because of Xenopus D1. No SC variant bound IgG (data not shown).


Figure 4
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  		FIGURE 4. D1 of X. laevis pIgR/SC mediates pIgR binding to J chain-containing IgA. A, Recombinant human SC, a chimeric SC with X. laevis D1 fused to human D2 (xD1-hD2D5-SC) and human SC lacking D1 (hD2D5-SC), were tested for binding to J558L transfectoma-produced pIgA or pentameric IgM (mouse J chain and human Fc region). B, Human SC or xD1-hD2D5 SC were tested for binding to CHO cell-produced IgA without J chain (mock) or with J chain from different species in an ELISA. Mean ± SD of quadruplicate (A) or triplicate (B) wells are shown. Each experiment shown is representative of three or more similar experiments.

 
Binding of human pIgA to Xenopus pIgR is improved by substituting human J chain with that of Xenopus

We hypothesized that the J chain contributes directly to the pIgR interaction site of pIgA. It was therefore reasonable to assume that pIgA composed of human {alpha} H chains and the Xenopus J chain would show better binding to Xenopus pIgR than pIgA containing the human or mouse J chain. Therefore, we tested the binding of xD1-hD2D5 SC to pIgA containing the human, mouse, or Xenopus J chain and did indeed find that pIgA with Xenopus J chain was the better ligand for Xenopus pIgR D1 (Fig. 4B). Furthermore, IgA without J chain showed no binding to Xenopus pIgR D1, demonstrating J chain dependence for pIgR-pIg interaction also in this species.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Knockout mice for the J chain and pIgR have shown that both genes are critical for SIg generation (9, 39). In this study, we present a mutational analysis of human J chain supporting a direct interaction between these two polypeptides. Furthermore, we identify the first amphibian pIgR and demonstrate that protein-protein interaction between pIgR and the J chain is remarkably conserved between tetrapods.

The J chain probably originated in the first jawed vertebrate because it is present in rays and sharks. Intriguingly, it appears to be deleted in bony fish; despite near completion of the genome project for several fish species, no gene resembling this unique gene has been identified. Interestingly, we found that a region critical for pIgA-pIgR interaction was highly divergent between the J chains from tetrapods and the nurse shark. Furthermore, this C-terminal region is absent from the J-chain sequence of clearnose skate, suggesting that it is not required for normal J-chain function in Chondrichthyes (24). We therefore speculate that the highly conserved J chain region in tetrapods is required for pIgR interaction, thereby allowing active export of pIgs to mucosal surfaces. Because the pIgR interaction site is not conserved in J chain from cartilaginous fish, its function here might solely be to facilitate Ig polymerization. Thus, Abs with higher avidity and better agglutinating capacity would be produced. Dooley and Flajnik (40) have proposed that absence of J chain expression in B cells that have undergone affinity maturation would provide monomers with enhanced tissue penetration in a memory response.

Because the Xenopus J chain showed high homology to the human J chain in two regions that were required for pIgR binding, we predicted that a pIgR homologue would be found in this species. Database searches identified such a gene and we deduced the primary sequence of the open reading frame by DNA sequencing of full-length cDNA. Although the Xenopus pIgR clearly has an overall structure similar to that of pIgR from other species, including four Ig domains (like chicken pIgR, whereas mammalian pIgR contain five Ig domains), a transmembrane region, and a cytoplasmic tail with a basolateral targeting motif, it was surprisingly divergent in the critical ligand-binding CDR1-like region in D1 (37, 41): the sequence VNRH, conserved in all pIgRs published to date (resequencing of rabbit pIgR has revealed this sequence in that species, although it was initially reported as VTRH) is substituted with ANKY in Xenopus pIgR (Fig. 3). Additionally, CDR1 is one amino acid longer in X. laevis than in other pIgRs published to date. However, structural modeling (Swiss Model; http://swissmodel.expasy.org/) predicted the CDR1 of Xenopus pIgR would form an {alpha} helix characteristic of the pIgR D1 (Ref. 42 and data not shown). Due to the relative divergence of CDR1, it was surprising that the Xenopus pIgR D1 mediates fairly strong binding to mammalian pIgA. A possible explanation for its failure to bind mammalian pentameric IgM is that this polymer adopts a different conformation than Xenopus J chain-containing IgM, which reportedly is hexameric (43). We have previously shown that differences in the CDR2 region between human and rabbit pIgR D1 are primarily responsible for the disparity of IgM binding between these two species; only human pIgR binds pentameric IgM with high affinity (38). It is therefore possible that differences between the CDR2 regions in Xenopus and human pIgR contribute to the failure of Xenopus pIgR to bind mammalian pentameric IgM.

An interesting difference between the mammalian pIgR on the one hand and the pIgR from chicken and Xenopus on the other is that the former contain five extracellular Ig-like domains while the latter contain four, lacking an equivalent to mammalian D2. In mammalian pIgR, D2 and D3 are encoded by a single exon, thereby diverging from the rule "one domain, one exon." Sequence comparisons of each individual Ig-like domain within and across mammalian and nonmammalian pIgRs showed that mammalian D2 is most similar to mammalian D3, suggesting that it arose by a duplication of the exon encoding D3 (Table III and data not shown). There is no remnant of such a duplication event in the chicken pIgR gene (36) or in the pIgR gene of Xenopus tropicalis (data not shown), suggesting that his duplication event occurred after mammals diverged from birds and amphibians.

Amphibians express five Ig classes: IgM, IgY, and IgX (5) and the recently identified IgF and IgD (44). IgY exists as a monomer and is the major serum Ig with functions similar to those of both IgG and IgE (5). IgM and IgX constitute the polymeric Igs in Xenopus, but only IgM polymers incorporate a J chain (43, 45, 46). IgX has been suggested to be an analog to IgA because of its ability to form polymers and its expression chiefly in the gut (46). It is an intriguing possibility that IgX could be a J chain-independent ligand for this receptor. We found that Xenopus pIgR only bound J chain-containing pIgA and that the affinity improved when this pIgR contained the Xenopus J chain compared with the human or mouse J chain, suggesting a J chain dependence for pIg-pIgR interaction also in this species. Importantly, our results demonstrated that the interaction site between pIgR and J chain-containing pIgs is conserved between mammals and amphibians.

In humans, SIgA is the dominant Ab on a biosynthetic basis and reportedly protects against Vibrio cholerae and enterotoxigenic Escherichia coli-induced diarrhea (47), in agreement with the notion that such Abs mediate mucosal immune exclusion (12). We have previously generated pIgR knockout mice and have recently shown that SIgs protect against horizontal spread of the mucosal pathogen Salmonella typhimurium even in naive animals, thereby providing herd immunity (39, 48). In addition, recent data have suggested that the large amounts of SIgA generated in the mouse gut may serve to curtail the commensal microbiota within the mucosal compartment (7, 49). Thus, mucosal immune exclusion protects against pathogens and promotes luminal habitation of the beneficial indigenous microbiota, thereby preventing adverse systemic immune reactions.

In conclusion, our structural data imply that the J chain has served two roles in the evolution of a secretory Ab system. Its Ig-joining capacity allowed regulation of polymerization to form Abs with better agglutinating capabilities, while its interaction with the pIgR allowed the export of such Abs to the mucosal surfaces where the bacterial load is high. The functional conservation between each polypeptide chain specifically involved in the generation of secretory Abs from amphibians, birds, and mammals suggests that this important first-line defense mechanism has been maintained in tetrapod evolution.


   Acknowledgments
 
We thank Prof. Itaru Moro for providing chicken J chain cDNA, Prof. Andrew Macpherson and Prof. Martin Flajnik for critical reading of the manuscript, and Kathrine Hagelsteen and Linda Solfjell for technical assistance with ELISA and mutagenesis, respectively.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Funding for this project has been provided by the Research Council of Norway. Back

2 Address correspondence and reprint requests to Dr. Finn-Eirik Johansen, Laboratory for Immunohistochemistry and Immunopathology, Institute of Pathology, University of Oslo, Rikshospitalet-Radiumhospitalet Medical Center, N-0027 Oslo, Norway. E-mail address: f.e.johansen{at}medisin.uio.no Back

3 Abbreviations used in this paper: SIgA, secretory IgA; CHO, Chinese hamster ovary; D1, domain 1; D2, domain 2; J chain, joining chain; NIP, -iodo-4-hydroxy-2-nitrophenylacetyl; pIgA, polymeric IgA; pIgR, polymeric Ig receptor; SC, secretory component. Back

Received for publication September 22, 2006. Accepted for publication November 16, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Macpherson, A. J., N. L. Harris. 2004. Interactions between commensal intestinal bacteria and the immune system. Nat. Rev. Immunol. 4: 478-485. [Medline]
  2. Backhed, F., R. E. Ley, J. L. Sonnenburg, D. A. Peterson, J. I. Gordon. 2005. Host-bacterial mutualism in the human intestine. Science 307: 1915-1920. [Abstract/Free Full Text]
  3. Rakoff-Nahoum, S., J. Paglino, F. Eslami-Varzaneh, S. Edberg, R. Medzhitov. 2004. Recognition of commensal microflora by Toll-like receptors is required for intestinal homeostasis. Cell 118: 229-241. [Medline]
  4. Cebra, J. J.. 1999. Influences of microbiota on intestinal immune system development. Am. J. Clin. Nutr. 69: 1046S-1051S. [Abstract/Free Full Text]
  5. Stavnezer, J., C. T. Amemiya. 2004. Evolution of isotype switching. Semin. Immunol. 16: 257-275. [Medline]
  6. Lamm, M. E.. 1997. Interaction of antigens and antibodies at mucosal surfaces. Annu. Rev. Microbiol. 51: 311-340. [Medline]
  7. Macpherson, A. J., T. Uhr. 2004. Compartmentalization of the mucosal immune responses to commensal intestinal bacteria. Ann. NY Acad. Sci. 1029: 36-43. [Medline]
  8. Brandtzaeg, P., H. Prydz. 1984. Direct evidence for an integrated function of J chain and secretory component in epithelial transport of immunoglobulins. Nature 311: 71-73. [Medline]
  9. Hendrickson, B. A., D. A. Conner, D. J. Ladd, D. Kendall, J. E. Casanova, B. Corthesy, E. E. Max, M. R. Neutra, C. E. Seidman, J. G. Seidman. 1995. Altered hepatic transport of immunoglobulin A in mice lacking the J chain. J. Exp. Med. 182: 1905-1911. [Abstract/Free Full Text]
  10. Mostov, K., C. S. Kaetzel. 1999. Immunoglobulin transport and the polymeric immunoglobulin receptor. P. L. Ogra, and J. Mestecky, and M. E. Lamm, and W. Strober, and J. Bienenstock, and J. R. McGhee, eds. Mucosal Immunology 133 Academic Press, San Diego.
  11. Phalipon, A., B. Corthesy. 2003. Novel functions of the polymeric Ig receptor: well beyond transport of immunoglobulins. Trends Immunol. 24: 55-58. [Medline]
  12. Brandtzaeg, P., F. E. Johansen. 2005. Mucosal B cells: phenotypic characteristics, transcriptional regulation, and homing properties. Immunol. Rev. 206: 32-63. [Medline]
  13. Johansen, F. E., I. Natvig Norderhaug, M. Roe, I. Sandlie, P. Brandtzaeg. 1999. Recombinant expression of polymeric IgA: incorporation of J chain and secretory component of human origin. Eur. J. Immunol. 29: 1701-1708. [Medline]
  14. Max, E. E., S. J. Korsmeyer. 1985. Human J chain gene. Structure and expression in B lymphoid cells. J. Exp. Med. 161: 832-849. [Abstract/Free Full Text]
  15. Matsuuchi, L., G. M. Cann, M. E. Koshland. 1986. Immunoglobulin J chain gene from the mouse. Proc. Natl. Acad. Sci. USA 83: 456-460. [Abstract/Free Full Text]
  16. Kulseth, M. A., S. Rogne. 1994. Cloning and characterization of the bovine immunoglobulin J chain cDNA and its promoter region. DNA Cell Biol. 13: 37-42. [Medline]
  17. Hughes, G. J., S. Frutiger, N. Paquet, J. C. Jaton. 1990. The amino acid sequence of rabbit J chain in secretory immunoglobulin A. Biochem. J. 271: 641-647. [Medline]
  18. Adamski, F. M., J. Demmer. 1999. Two stages of increased IgA transfer during lactation in the marsupial, trichosurus vulpecula (Brushtail possum). J. Immunol. 162: 6009-6015. [Abstract/Free Full Text]
  19. Takahashi, T., T. Iwase, T. Tachibana, K. Komiyama, K. Kobayashi, C. L. Chen, J. Mestecky, I. Moro. 2000. Cloning and expression of the chicken immunoglobulin joining (J)-chain cDNA. Immunogenetics 51: 85-91. [Medline]
  20. Iwata, A., T. Iwase, Y. Ogura, T. Takahashi, N. Matsumoto, T. Yoshida, N. Kamei, K. Kobayashi, J. Mestecky, I. Moro. 2002. Cloning and expression of the turtle (Trachemys scripta) immunoglobulin joining (J)-chain cDNA. Immunogenetics 54: 513-519. [Medline]
  21. Hohman, V. S., S. E. Stewart, C. E. Willett, L. A. Steiner. 1997. Sequence and expression pattern of J chain in the amphibian, Xenopus laevis. Mol. Immunol. 34: 995-1002. [Medline]
  22. Hohman, V. S., L. A. Steiner. 2000. The sequence of J chain in an amphibian, Rana catesbeiana. Immunogenetics 51: 587-590. [Medline]
  23. Hohman, V. S., S. E. Stewart, L. L. Rumfelt, A. S. Greenberg, D. W. Avila, M. F. Flajnik, L. A. Steiner. 2003. J chain in the nurse shark: implications for function in a lower vertebrate. J. Immunol. 170: 6016-6023. [Abstract/Free Full Text]
  24. Cannon, J. P., R. N. Haire, G. W. Litman. 2002. Identification of diversified genes that contain immunoglobulin-like variable regions in a protochordate. Nat. Immunol. 3: 1200-1207. [Medline]
  25. Johansen, F. E., R. Braathen, P. Brandtzaeg. 2000. Role of J chain in secretory immunoglobulin formation. Scand. J. Immunol. 52: 240-248. [Medline]
  26. Brandtzaeg, P.. 1974. Presence of J chain in human immunocytes containing various immunoglobulin classes. Nature 252: 418-420. [Medline]
  27. Johansen, F. E., R. Braathen, P. Brandtzaeg. 2001. The J chain is essential for polymeric Ig receptor-mediated epithelial transport of IgA. J. Immunol. 167: 5185-5192. [Abstract/Free Full Text]
  28. Takahashi, T., M. Kimura, N. Matsumoto, A. Iwata, Y. Ogura, T. Yoshida, N. Kamei, K. Komiyama, J. Mestecky, I. Moro. 2002. Cloning of the chicken immunoglobulin joining (J)-chain gene and characterization of its promoter region. DNA Cell Biol. 21: 81-90. [Medline]
  29. Braathen, R., V. Sorensen, P. Brandtzaeg, I. Sandlie, F. E. Johansen. 2002. The carboxyl-terminal domains of IgA and IgM direct isotype-specific polymerization and interaction with the polymeric immunoglobulin receptor. J. Biol. Chem. 277: 42755-42762. [Abstract/Free Full Text]
  30. Claypool, S. M., B. L. Dickinson, J. S. Wagner, F. E. Johansen, N. Venu, J. A. Borawski, W. I. Lencer, R. S. Blumberg. 2004. Bidirectional transepithelial IgG transport by a strongly polarized basolateral membrane Fc{gamma}-receptor. Mol. Biol. Cell 15: 1746-1759. [Abstract/Free Full Text]
  31. Elm, C., R. Braathen, S. Bergmann, R. Frank, J. P. Vaerman, C. S. Kaetzel, G. S. Chhatwal, F. E. Johansen, S. Hammerschmidt. 2004. Ectodomains 3 and 4 of human polymeric Immunoglobulin receptor (hpIgR) mediate invasion of Streptococcus pneumoniae into the epithelium. J. Biol. Chem. 279: 6296-6304. [Abstract/Free Full Text]
  32. Brandtzaeg, P.. 1975. Purification of J chain after mild reduction of human immunoglobulins. Scand. J. Immunol. 4: 309-320. [Medline]
  33. Brandtzaeg, P.. 1974. Human secretory component. I. Purification of free secretory component from colostrum. Scand. J. Immunol. 3: 579-588. [Medline]
  34. Krugmann, S., R. J. Pleass, J. D. Atkin, J. M. Woof. 1997. Structural requirements for assembly of dimeric IgA probed by site-directed mutagenesis of J chain and a cysteine residue of the {alpha}-chain CH2 domain. J. Immunol. 159: 244-249. [Abstract]
  35. Piskurich, J. F., M. H. Blanchard, K. R. Youngman, J. A. France, C. S. Kaetzel. 1995. Molecular cloning of the mouse polymeric Ig receptor. Functional regions of the molecule are conserved among five mammalian species. J. Immunol. 154: 1735-1747. [Abstract]
  36. Wieland, W. H., D. Orzaez, A. Lammers, H. K. Parmentier, M. W. Verstegen, A. Schots. 2004. A functional polymeric immunoglobulin receptor in chicken (Gallus gallus) indicates ancient role of secretory IgA in mucosal immunity. Biochem. J. 380: 669-676. [Medline]
  37. Bakos, M. A., A. Kurosky, R. M. Goldblum. 1991. Characterization of a critical binding site for human polymeric Ig on secretory component. J. Immunol. 147: 3419-3426. [Abstract]
  38. Roe, M., I. N. Norderhaug, P. Brandtzaeg, F. E. Johansen. 1999. Fine specificity of ligand-binding domain 1 in the polymeric Ig receptor: importance of the CDR2-containing region for IgM interaction. J. Immunol. 162: 6046-6052. [Abstract/Free Full Text]
  39. Johansen, F. E., M. Pekna, I. N. Norderhaug, B. Haneberg, M. A. Hietala, P. Krajci, C. Betsholtz, P. Brandtzaeg. 1999. Absence of epithelial immunoglobulin A transport, with increased mucosal leakiness, in polymeric immunoglobulin receptor/secretory component-deficient mice. J. Exp. Med. 190: 915-922. [Abstract/Free Full Text]
  40. Dooley, H., M. F. Flajnik. 2005. Shark immunity bites back: affinity maturation and memory response in the nurse shark, Ginglymostoma cirratum. Eur. J. Immunol. 35: 936-945. [Medline]
  41. Coyne, R. S., M. Siebrecht, M. C. Peitsch, J. E. Casanova. 1994. Mutational analysis of polymeric immunoglobulin receptor/ligand interactions. Evidence for the involvement of multiple complementarity determining region (CDR)-like loops in receptor domain I. J. Biol. Chem. 269: 31620-31625. [Abstract/Free Full Text]
  42. Hamburger, A. E., A. P. West, Jr, P. J. Bjorkman. 2004. Crystal structure of a polymeric immunoglobulin binding fragment of the human polymeric immunoglobulin receptor. Structure (Camb.) 12: 1925-1935. [Medline]
  43. Hadji-Azimi, I., M. Michea-Hamzehpour. 1976. Xenopus laevis 19S immunoglobulin. Ultrastructure and J chain isolation. Immunology 30: 587-591. [Medline]
  44. Zhao, Y., Q. Pan-Hammarstrom, S. Yu, N. Wertz, X. Zhang, N. Li, J. E. Butler, L. Hammarstrom. 2006. Identification of IgF, a hinge-region-containing Ig class, and IgD in Xenopus tropicalis. Proc. Natl. Acad. Sci. USA 103: 12087-12092. [Abstract/Free Full Text]
  45. Hsu, E., M. F. Flajnik, L. Du Pasquier. 1985. A third immunoglobulin class in amphibians. J. Immunol. 135: 1998-2004. [Abstract]
  46. Mussmann, R., L. Du Pasquier, E. Hsu. 1996. Is Xenopus IgX an analog of IgA?. Eur. J. Immunol. 26: 2823-2830. [Medline]
  47. Sanchez, J., J. Holmgren. 2005. Virulence factors, pathogenesis and vaccine protection in cholera and ETEC diarrhea. Curr. Opin. Immunol. 17: 388-398. [Medline]
  48. Wijburg, O. L., T. K. Uren, K. Simpfendorfer, F. E. Johansen, P. Brandtzaeg, R. A. Strugnell. 2006. Innate secretory antibodies protect against natural Salmonella typhimurium infection. J. Exp. Med. 203: 21-26. [Abstract/Free Full Text]
  49. Macpherson, A. J., T. Uhr. 2004. Induction of protective IgA by intestinal dendritic cells carrying commensal bacteria. Science 303: 1662-1665. [Abstract/Free Full Text]



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