The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

The Journal of Immunology, 2007, 178, 5682 -5689
Copyright © 2007 by The American Association of Immunologists, Inc.
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Hamuro, K.
Right arrow Articles by Suzuki, Y.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Hamuro, K.
Right arrow Articles by Suzuki, Y.

A Teleost Polymeric Ig Receptor Exhibiting Two Ig-Like Domains Transports Tetrameric IgM into the Skin1

Koji Hamuro*, Hiroaki Suetake2,*, Nil Ratan Saha3, Kiyoshi Kikuchi* and Yuzuru Suzuki*

* Fisheries Laboratory, Graduate School of Agricultural and Life Sciences, University of Tokyo, Hamamatsu, Shizuoka, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The skin mucus IgM is an important molecule in the mucosal immune system of teleost skin. However, the transport mechanism associated with this molecule has yet to be clarified. In this study, we isolated a gene encoding a polymeric Ig receptor (pIgR) from a species of teleost fish, Takifugu rubripes (fugu). This gene is known to be an Ig transporter in the intestine of mammals. Our studies further demonstrated that fugu pIgR was expressed in the skin and that a fragment of pIgR bound to tetrameric IgM in the skin mucus. These results indicate that the skin pIgR transports tetrameric IgM into the skin mucus. The fugu pIgR exhibits a unique structure containing only two Ig-like domains corresponding to domain 1 and domain 4/5 of mammalian pIgR. This structure was sufficient for successful binding to tetrameric IgM. Teleost skin thus adopts the same Ig transport system as mammalian intestine via a unique pIgR.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
In mammals, the mucosal surfaces lining the gastrointestinal, respiratory, and genitourinary tracts are continuously exposed to pathogenic microorganisms (1) and thus form the first defensive line against such invading organisms. These tissues have a mucosal immune system that involves local immune responses (2). One of the most important defensive molecules in this system is secretory Ig, predominantly IgA (3). In species of teleost fish, the skin also has important immunological roles against aquatic infectious agents, as does other mucosal tissues such as the gut (4). In fact, tetrameric IgM, but not dimeric IgA, exists in the skin and gut mucus of teleost fish (5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15). In addition, several reports clearly show that teleost mucosal IgM is produced independently to systemic Abs, indicating the existence of a mucosal immune system in the teleost (7, 8, 16, 17, 18, 19, 20). These observations lead us to speculate as to whether teleosts adopt an Ig transport system in the skin that is also present in mammalian mucosal tissues such as intestine.

In mammals and avian species, the mucosal Ig is transported by a polymeric Ig receptor (pIgR)4 through the monolayer of epithelia into the mucosal secretions (1, 2, 21, 22, 23, 24, 25). However, mammals and avian species do not express pIgR in the skin. The pIgR expressed on the basolateral membrane of the epithelial cells traps polymeric Ig (pIg) molecules that are synthesized by local Ig-producing cells and subsequently transports Ig through the cell to the mucosal surface. The pIg is released into the mucus by proteolytic cleavage via a fragment of the pIgR referred to as the secretory component (SC). Mammalian pIgR is a single transmembrane protein. The ectodomain of the pIgR exhibits five Ig-like domains (D1–5) (2, 26). In addition, bovine and rabbit also have a shorter type of pIgR exhibiting three Ig-like domains (D1, D4, and D5), created by alternative splicing (27, 28, 29, 30). In contrast, chicken pIgR consists of four Ig-like domains, corresponding to mammalian D1 and D3–5, but this is not an alternative splicing variant (25). In the present study, we investigated the pIgR in a teleost fish, the puffer fish (fugu). We showed that fugu pIgR has a unique structure and that the gene was expressed not only in the intestine but also in the skin. Although the transport of pIg via the pIgR does not occur in the keratinized skin of mammals and avian species, our results clearly show that teleost skin adopts a system similar to mammalian mucosal tissues.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Fish sampling

Fugu (Takifugu rubripes), weighing ~800 g each, were supplied from dealers and were reared in a 1000-liter tank with running seawater at 20°C.

The fish were fed once a day with commercial fish pellets. The fugu were anesthetized with 2-phenoxyethanol (200 parts per million) and the skin mucus was scraped off with a spatula and stored at –80°C until use. The skin, gill, intestine, liver, head kidney, kidney, spleen, thymus, gonad, heart, and muscle were also dissected and immediately fixed in RNA later (Ambion) to await RNA extraction. Small portions of the skin and intestine were also fixed in 4% paraformaldehyde in phosphate buffer for 2 days. Following fixation in methanol, the samples were stored at –30°C for in situ hybridization analysis.

Western blot analysis of fugu mucosal IgM

Collected skin mucus was homogenized with an equal volume of PBS (pH 7.4) containing 0.9 mM CaCl2 and 0.33 mM MgCl2 (PBS plus buffer) and then centrifuged at 15,000 x g for 30 min at 4°C. The supernatant (crude skin mucus extract) and fugu serum were subjected to SDS-PAGE on 3–10% gradient gels (ATTO) under nonreducing conditions and 10% polyacrylamide gels containing 9.7% acrylamide and 0.3% bis-acrylamide under reducing conditions. To estimate the molecular mass of the band, Prestained SDS-PAGE Standards Broad Range (Bio-Rad) was used as molecular mass marker. Western blotting was performed with a mAb raised against the fugu IgM H chain (anti-IgM, 16F3) (31). Primary Abs were biotinylated using EZ-Link Sulfo-NHS-LC-Biotin (Pierce) according to the manufacturer’s instructions. Signals were detected with 3,3'-diaminobenzidine tetrahydrochloride after treatment with a Vectastain ABC Kit (Vector Laboratories). A Western blotting with intestinal mucus IgM was performed in the same manner.

cDNA cloning of fugu pIgR

Total RNA was extracted from fugu skin by using RNA extraction solution (Isogen; Nippon Gene). First-strand cDNA was synthesized with a SMART RACE cDNA Amplification Kit (BD Clontech).

We first performed computational screening for a fugu homolog of the rat pIgR and obtained a scaffold from the fugu genome database (Fugu rubripes version 3.0, http://genome.jgi-psf.org/fugu6/fugu6.home.html). The amplified cDNA was obtained by PCR using adapter primer UPM-A (BD Clontech) and either SC-f1 primer (5'-CGTGAAATACTGGTGTCAAGGGCGCA-3') for 3'-RACE or SC-r1 primer (5'-CCGGAGTCCGCCTCCGTCAGGTTGTT-3') for 5'-RACE. Amplification was performed over 30 cycles of: 95°C for 10 s, 60°C for 10 s, and 72°C for 2 min. The final elongation reaction was conducted at 72°C for 3 min. Each PCR product was ligated into the pCR II-TOPO vector (Invitrogen Life Technologies). Plasmid DNA was then purified and sequenced with an Applied Biosystems PRISM 310 Genetic Analyzer (Applied Biosystems). A homology search of the deduced amino acid sequence was conducted with the BLAST 2.0 program accessed via DDBJ (http://www.ddbj.nig.ac.jp/search/blast-j.html) (32). Multiple alignment of sequences was conducted using the Clustal W program (http://www.ddbj.nig.ac.jp/search/ex_clustalw-j.html) (33) with slight modifications. Putative signal peptide, transmembrane, and cytoplasmic regions were predicted based on SignalP (http://cbs.dtu.dk/services/SignalP/) (34) and SOSUI (http://bp.nuap.nagoya-u.ac.jp/sosui/) (35) programs.

RT-PCR analysis

Total RNAs of the head kidney, kidney, spleen, thymus, intestine, skin, liver, gill, gonad, and muscle from fugu were isolated as described above. One microgram of total RNA treated with DNase RQ1 (Promega) was reverse-transcribed into cDNA using an ExScript RT reagent Kit (Takara) according to the manufacturer’s protocol. PCR was conducted with specific primers, SC-mf1 (5'-GGTCACATGACCAAGGAGACCAGCAGA-3') and SC-mr1 (5'-CTGTGCATGGTGGAAACATGACCACC-3') for 37 cycles of 95°C for 10 s, 60°C for 5 s, and 72°C for 1 min. The PCR products were analyzed by 1% agarose gel electrophoresis and stained with ethidium bromide. These primers were designed in separate exons to distinguish the band amplified from the genomic DNA.

Northern blot analysis

Poly(A)+ RNA from skin, intestine, and muscle was purified using Oligotex-dT30<Super> (Takara) according to the manufacturer’s instructions. Four micrograms of poly(A)+ RNA from each tissue was separated on a 1.5% formamide-agarose denaturing gel. Northern blot analysis was then performed according to the method of Tsutsui et al. (36). The probe used was a partial cDNA encoding fugu pIgR Ig-like domains.

In situ hybridization

Digoxigenin-labeled cRNA probes were synthesized with fugu pIgR cDNA using a digoxigenin RNA Labeling Kit (Roche Diagnostics) in accordance with manufacturer’s instructions. In situ hybridization for pIgR and secretory IgM (sIgM) was performed according to the method of Saha et al. (37).

Western blot analysis of fugu pIgR

Anti-fugu pIgR antiserum (anti-pIgR) was produced by genetic immunization. The fugu pIgR cDNA was ligated into the eukaryotic expression vector pcDNA3 (Invitrogen Life Technologies). The plasmid was injected i.m. into the hind leg (100 µg/wk) of 7-wk-old female ddY mice six times once a week. Blood samples were collected from the retro-orbital venous plexus of anesthetized (ether-induced) mice before each DNA inoculation. Serum was separated and stored at –20°C. To examine the specificity of the antiserum, recombinant fugu pIgR was produced from the Ag using the bacterial expression vector pGEX-6P-2 (Amersham Biosciences) and Escherichia coli BL21 (DE3)-competent cells (Novagen). Sera from immunized and normal mice were tested against fugu skin mucus and the lysate of recombinant fugu pIgR by Western blotting under reducing conditions. In addition, we performed Western blotting using anti-pIgR absorbed with the recombinant fugu pIgR or recombinant fugu lymphoid T cell protein tyrosine kinase (Lck) as irrelevant protein to examine the specificity of anti-pIgR. To detect the binding of fugu SC with IgM in the skin mucus, Western blotting was also performed with anti-pIgR and anti-IgM under reducing and nonreducing conditions as described above.

Immunoprecipitation

Anti-IgM was coupled to the swollen protein A-Sepharose 4 Fast Flow beads (Amersham Biosciences) according to the manufacturer’s instructions. The beads were incubated for 4 h at 4°C with crude fugu skin mucus extract. Then, the beads were washed three times with 1% BSA in PBS, followed by three additional washes with PBS. After washing, the bound proteins were eluted into SDS-PAGE sample buffer (50 mM Tris-HCl (pH 6.8), 1% SDS, 10% glycerol, 6% 2-ME, and 0.01% bromphenol blue). Eluted fractions were then separated by SDS-PAGE on a 10% polyacrylamide gel under reducing conditions. Western blotting was performed with anti-pIgR as described above. Reciprocally, anti-pIgR was also used for immunoprecipitation of pIg and the SC complex, and western blotting was performed with anti-IgM.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Tetrameric IgM in fugu mucus

To confirm whether tetrameric IgM exists in the skin mucus of fugu, as it does in other teleost species, we performed Western blottings of fugu skin mucus using a mAb raised against the fugu IgM H chain (anti-IgM). The fugu serum was also subjected to Western blotting for comparison with mucus IgM. As shown in Fig. 1A, lane M, a specific band was observed in the skin mucus using SDS-PAGE under reducing conditions. The molecular mass of the band is equal to that of the IgM H chain in fugu serum (Fig. 1A, lane S). Under nonreducing conditions, a specific band in the skin mucus was also observed at a similar position to that of serum IgM (Fig. 1B). Comparing to the size of fugu serum IgM (31), the molecular mass of skin mucus IgM was predicted as 750–800 kDa. These results indicate that tetrameric IgM exists in fugu skin mucus. In addition, tetrameric IgM was also detected in the intestinal mucus (data not shown).


Figure 1
View larger version (20K):
[in this window]
[in a new window]

  		FIGURE 1. Western blot analysis of fugu IgM. Analysis of skin mucus (M) and serum (S) was performed under reducing (A) and nonreducing conditions (B). Blots were developed with a mAb (16F3) against the fugu IgM H chain.

 
Local production of fugu sIgM in the skin and intestine

To confirm the local production of sIgM in the skin, we examined the expression of the sIgM H chain gene in the skin. We observed cells that express the sIgM H chain gene in skin tissue (Fig. 2). These cells were mainly distributed along the basal membrane. In the intestine, sIgM-producing cells were detected at lamina propria, as in the case of mammals.


Figure 2
View larger version (108K):
[in this window]
[in a new window]

  		FIGURE 2. In situ hybridization of fugu secretory IgM. sIgM-expressing cells were detected in fugu skin (A) and intestine (C) using an antisense probe. No signals were detected in either fugu skin (B) or intestine (D) with a sense probe. Positive cells are shown by arrowheads. d, Dermis; mp, melanophore; ed, epidermis; lp, lamina propria; el, epithelial layer.

 
Isolation and characterization of fugu pIgR

To find the pIgR gene in the teleost genome, we performed a BLAST search of the fugu genome database using the rat pIgR cDNA sequence. We identified a candidate gene that was homologous to mammalian pIgR. We isolated a cDNA (AB186753) that encodes a fugu putative pIgR from the skin. The putative fugu pIgR cDNA consists of 1392 bp, including an open reading frame of 984 bp that encodes for 328 aa and 73- and 335-bp 5'- and 3'-untranslated regions, respectively. BLASTX searches revealed that the fugu cDNA exhibited the highest e-values with chicken pIgR following mammalian pIgRs. The fugu pIgR contains two Ig-like domains, a transmembrane region, and a cytoplasmic tail (Fig. 3). The first Ig-like domain of the fugu putative pIgR had higher sequence identities with D1 (21–23%) than any other domains of vertebrate pIgRs (14–22%). The second domain of fugu pIgR shows the highest identity with the chicken D5 (19%) among chicken domains (17–18%). The number and the location of cysteine residues in both Ig-like domain were conserved with D1, D3, and D4 of other vertebrate pIgRs, but differing from those in D5 with two extra cysteine residues (Fig. 3). Two distinctive amino acids sequences, i.e., KXWC and DXGXYXC, were found in both domains of fugu pIgR. These sequences are conserved in D1, D4, and D5 of higher vertebrate pIgRs. These data suggest that the first Ig-like domain of fugu pIgR corresponds to D1 and that the second Ig-like domain would be an equivalent of chicken and mammalian D4 and/or D5. Tentatively, we designated the second domain as D4/5. The transmembrane region predicted by the SOSUI program showed low amino acid identity with that of mammalian and chicken pIgR, but this highly hydrophobic region would work as a transmembrane region. A cytoplasmic tail of fugu pIgR is also dissimilar to that of chicken and mammalian pIgRs, however, this region contains two conserved serine residues. One of them corresponds to mammalian Ser664 that is important for internalization of pIgR (38). This suggests that the cytoplasmic tail of fugu pIgR is also involved in the internalization of the pIgR-polymeric IgM complex. From these data, we concluded that fugu possesses a pIgR that contains two Ig-like domains and thus differed from mammalian pIgR, which has either five or three domains (2) and chicken pIgR which has four domains (25).


Figure 3
View larger version (38K):
[in this window]
[in a new window]

  		FIGURE 3. Alignment of the amino acid sequences of fugu pIgR, mammalian pIgRs, and chicken pIgR. The sequence of human (NM_002644), bovine (S48841), rabbit (X00412), chicken (AY233381), and fugu pIgRs were aligned using CLUSTAL W software. Transmembrane domains are shown in boxes. Conserved cysteine residues are boxed in black. Asterisks indicate the positions of fully conserved residues.

 
Genomic organization of the fugu pIgR

To confirm the gene structure of fugu pIgR, we compared the cDNA and genomic sequences of fugu pIgR and found that the fugu pIgR gene is composed of eight exons and seven introns (Fig. 4). The first Ig-like domain is encoded by exon 2, and the second is encoded by exons 3 and 4. Exon 6 encodes the transmembrane region. Gene prediction analysis failed to detect any coding sequence for Ig-like domains in all introns of the fugu pIgR gene. We concluded that this gene encodes a single pIgR with two Ig-like domains.


Figure 4
View larger version (18K):
[in this window]
[in a new window]

  		FIGURE 4. Schematic structure of the fugu and human pIgR genes along with their respective mRNAs. A, Domain structure of fugu pIgR mRNA. B, Intron-exon distribution in the fugu pIgR gene. C, Intron-exon distribution in the human pIgR gene. D, Domain structure of the human pIgR mRNA. SP, Signal peptide; TM, transmembrane region; Cy, cytoplasmic domain.

 
Expression of fugu pIgR gene

RT-PCR analysis showed that the fugu pIgR gene is strongly expressed in the spleen, thymus, skin, gill, and intestine and that the weak expression is also observed in some tissues (Fig. 5A). The fugu pIgR gene was expressed in almost similar tissues to those of mammals and chickens. Skin, gill, and intestine expressed the pIgR gene in fugu. These tissues are mucosal tissues like mammalian intestine, and polymeric IgM has been detected in the teleost skin and intestinal mucus (Fig. 1A and Refs. 5, 6, 7, 8, 9, 10, 11, 12, 13, 14). This suggests that pIgR in these tissues participates in the transport of polymeric IgM into mucus. In addition, fugu lymphoid organs expressed the pIgR gene like those of the chicken and mammalian species (2, 25), but its function remains unknown like that in the higher vertebrates.


Figure 5
View larger version (51K):
[in this window]
[in a new window]

  		FIGURE 5. RT-PCR and Northern blot hybridization of the fugu pIgR. A, RT-PCR of fugu pIgR and EF-1{alpha} was performed in various organs. B, Northern blot hybridization of the fugu pIgR was analyzed in skin, intestine, and muscle.

 
To investigate the splicing variant of fugu pIgR, Northern blot analysis was performed. Northern blot analysis confirmed the existence of a single pIgR mRNA in the skin and intestine (Fig. 5B).

To identify fugu pIgR-producing cells in the skin and intestine, we conducted in situ hybridization analysis. In the fugu skin, most of the epithelial cells were pIgR-expressing cells. No expression was detected in either the mucosal cells, dermal cells, or melanophore cells (Fig. 6). Similarly, expression of the fugu pIgR gene was detected in epidermal cells of the intestine (Fig. 6).


Figure 6
View larger version (135K):
[in this window]
[in a new window]

  		FIGURE 6. In situ hybridization of the fugu pIgR gene. pIgR-expressing cells were detected in fugu skin (A) and intestine (C) using an antisense probe. Using the sense probe, no signals were detected in fugu skin (B) and intestine (D). d, Dermis; mp, melanophore; ed, epidermis; lp, lamina propria; el, epithelial layer.

 
Fugu SC-IgM complex in the skin mucus

If the skin mucus IgM of fugu is transported by pIgR in a similar manner to that of mammals, a fragment of the pIgR (SC) should bind to the tetrameric IgM in the skin mucus. To detect fugu SC in the skin mucus, we raised antiserum against fugu pIgR (anti-pIgR) by genetic immunization. This antiserum exhibited specific reactivity to recombinant fugu pIgR, but normal mouse serum did not react with the recombinant protein (Fig. 7A). In addition, Western blot reaction was inhibited by addition of recombinant fugu pIgR to the antiserum but not by recombinant fugu Lck (Fig. 7A). Using this antiserum, we performed Western blot analysis of the skin mucus under reducing conditions and observed a specific band with the predicted molecular mass of ~60 kDa (Fig. 7B). This result demonstrated that the skin mucus contains SC. To confirm whether the fugu SC binds to skin mucus IgM, we first analyzed the molecular mass of proteins that were reactive to the antiserum specific to pIgR by Western blotting under nonreducing conditions and found a specific band around 800 kDa. We performed Western blotting under nonreducing conditions using anti-IgM and detected a single band at a similar molecular mass (Fig. 7B). The Western blot patterns are consistent with the predicted size of the SC-tetrameric IgM complex in the skin mucus under nonreducing conditions. Normal mouse serum did not react with fugu skin mucus (Fig. 7B). For further confirmation, we performed immunoprecipitation experiments. The skin mucus IgM complex was precipitated by anti-IgM, and the presence of SC in the precipitate was confirmed by Western blotting using anti-pIgR (Fig. 7C). Reciprocally, skin mucus IgM was also precipitated by anti-pIgR (Fig. 7D). After precipitated by anti-pIgR or anti-IgM, supernatant did not contain IgM (Fig. 7, C and D). These results clearly showed that fugu SC binds to the tetrameric IgM and transports it into the skin mucus. This is the first report concerning the functional characterization of the Ig receptor in a teleost species.


Figure 7
View larger version (28K):
[in this window]
[in a new window]

  		FIGURE 7. Western blot analysis of SC. A, Western blotting was performed using anti-pIgR, normal mouse serum (NMS), anti-pIgR with recombinant fugu pIgR (r-pIgR), and anti-pIgR ({alpha}-pIgR) with recombinant fugu Lck (r-Lck) under reducing conditions. B, Western blot analysis of skin mucus performed using anti-pIgR and anti-IgM under reducing and nonreducing conditions. Normal mouse serum did not react against skin mucus under reducing conditions. C, Western blot analysis of immunoprecipitate by anti-IgM from skin mucus, and the supernatant was performed with anti-pIgR under reducing conditions. D, Reciprocally, Western blot analysis of immunoprecipitate by anti-pIgR from skin mucus and the supernatant was performed with anti-IgM under reducing conditions.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The existence of tetrameric IgM in the skin mucus of teleost has been well documented (5, 7, 8, 10, 11, 13, 14, 15, 16, 17). However, the transport system associated with tetrameric IgM in this tissue has yet to be defined. In this study, we have demonstrated that the teleost pIgR transports tetrameric IgM into the skin mucus in a manner similar to the way mammalian pIgR transports IgM into the intestinal mucus. Immunoprecipitation analysis indicates that pIgR is the only way to secrete the IgM into the skin mucus in fugu. The fugu pIgR was expressed along the epithelial cells of the skin (Fig. 6). We have also shown that lymphocytes synthesizing sIgM are located in the epithelium near the basal membrane of the skin (Fig. 2). The source of mucosal pIg in the mammalian gut is the lymphocytes in the lamina propria (3). These data suggest that lymphocytes in the skin are likely to produce skin mucus IgM in the teleost. In fact, Ab titers in the teleost skin mucus change independently of those in the serum (7, 8, 16, 17, 18, 19, 20). It follows therefore that the local sIgM-producing cells and pIgR would contribute to mucosal immunity in teleost.

The fugu pIgR consisted of only two Ig-like domains differing from mammalian pIgR with five or three domains (2) and chicken pIgR with four (25) (Fig. 8A). In mammals, D1 and D5 are known to participate in the binding to pIg (2). The first and the second Ig-like domains of fugu pIgR were aligned to D1 and D5 of higher vertebrate pIgR, respectively (Fig. 3). The first domain of fugu pIgR would be a counterpart of D1 of higher vertebrate pIgR, but it is controversial which domain in chicken and mammalian pIgRs is an equivalent of the second domain. Amino acids identities of the second domain showed similar levels with any domains of mammalian and chicken. This analysis did not show clear evidence for domain designation of the domain. However, two distinctive amino acids sequences, i.e., KXWC and DXGXYXC, were found only in D1, D4, and D5 of chicken and mammalian pIgRs. The latter sequences in D4 and D5 are specific, i.e., DXGFYWC in D4 and DXGWYWC in D5 (Fig. 3). The second domain of fugu pIgR has KKWC and DTGWYWC like D5. The second domain of fugu conserved four cysteine residues like the D1, D3, and D4. Chicken and mammalian D5 have six cysteine residues with an additional two. In mammals, four conserved cysteine residues are known to form two disulfide bonds linking two beta-sheets (2, 39). One of two extra cysteine residues in D5 is responsible for covalent binding with IgA in mammals (2). Our result suggests that in teleost species, the extra cysteine residues are not necessary to bind with tetrameric IgM. The second domain binds with tetrameric IgM in a different manner from D5 with dimeric IgA, and this last domain is not always a functional equivalent of mammalian D5. These studies also lead us to speculate that these extra cysteine residues in D5 might have coevolved with the dimeric IgA. Taken together, the second domain of fugu pIgR has both features of D4 and D5 of higher vertebrate pIgRs, suggesting that this is an ancestral domain of D4 and D5. Thus, we designated it as the fugu D4/5. We demonstrated that this two-Ig-like domain structure of fugu pIgR is sufficient for binding with tetrameric IgM. Further study is required to clarify how fugu pIgR binds with polymeric IgM.


Figure 8
View larger version (26K):
[in this window]
[in a new window]

  		FIGURE 8. A, Structure model of the pIgR. The Ig-like domains of pIgR are indicated by numbers. Ig-like domains that contain pIg-binding regions are represented by a bold line. Alternative splicing of D2 and D3 is represented by the dotted line. B, A putative binding model of pIg/pIgR. i, Dimeric IgA with the standard-type pIgR in mammals; ii, pentameric IgM with the mammalian standard-type pIgR or shorter-type pIgR in rabbits; iii, trimeric IgA with the chicken pIgR; and iv, tetrameric IgM with the fish pIgR. Variable and constant regions of Ig are shown in boxes and constant regions containing pIgR binding sites are gray. Ig-like domains of the pIgR are indicated by numbers. The D1 and D5 of pIgR exhibiting pIg-binding regions are represented by bold lines. The distances between the D1 binding sites and D5 binding site are represented by arrows. Binding with joining chains is not shown here.

 
Considering the structure of fugu pIgR, a short pIgR with two or three Ig-like domains would be sufficient for binding with tetrameric or pentameric IgM. In contrast, dimeric or trimeric IgA would require a longer pIgR with four or five Ig-like domains. This would be reasonable because the distance between the two pIgR binding sites in dimeric or trimeric IgA is longer than that in tetrameric or pentameric IgM (Fig. 8B). This was supported by the report of Nordehaug et al. (40) that reported that when D2 and D3 were deleted from the human pIgR, then binding occurred only to IgM but not to IgA (40). In accordance with the conversion of mucosal Ig from polymeric IgM to polymeric IgA during evolution, pIgR would increase the number of Ig-like domains. Comparison of the genomic organization of fugu and human pIgR showed that the fugu pIgR gene does not possess additional exons corresponding to three domains (Fig. 4). This result suggests that these exons of higher vertebrates had been inserted between the D1 and D4/5 during evolution. In the Ig superfamily proteins, each exon corresponds to a separate functional domain (41), indicating that the increase of domains would occur through the introduction of exons (39, 41). D1, D4, and D5 of pIgRs share the KXWC and DXGXYXC sequences, suggesting that these domains arose from ancestral domain by duplication. Interdomain comparison of chicken and mammalian pIgRs showed that chicken D4 is more similar to mammalian D5 than D1–D3 (25) and that fugu D4/5 has both features of D4 and D5. Thus, it is reasonable to assume that D4 and D5 arose from the tandem duplication of D4/5. The study of chicken pIgR gene suggests the occurrence of D2–D3 duplication (25). These duplication events would make a primitive pIgR longer during evolution. To clarify the process of molecular evolution in the domain structure of pIgR, it is necessary to characterize and compare pIgR structure in a variety of different species such as amphibians, reptiles, and cartilaginous fish.

In mammals, the joining (J) chain is a part of pIg and binds to D1 of pIgR (42, 43, 44, 45, 46, 47). Moreover, it is indispensable for Ig transport by pIgR (2). However, teleost J chain has not been reported until now. In the present study, we demonstrated that pIg transport by pIgR in the teleost species for the first time. In addition, the J chain was found recently in the cartilaginous fish (48). These studies strongly suggest that the J chain also exists in teleosts and participates in the Ig transport by pIgR.

In the mammalian species, the secretion of pIg to the external surface of the body is observed only in exocrine glands such as the mammary gland and lachrymal gland (2). In the evolutionary process leading to the land animal from ancestors living in an aquatic environment, the skin defense mechanisms are likely to have undergone significant change, from a mucosal system to keratinization of the skin epidermis. The fish body covered with a mucosal immune system may represent a primitive state of ancestral vertebrates, and the skin system may be lost during amniote lineage in accordance with adaptation to the ground environment. To test this hypothesis, an investigation of outgroups to animals, including amniotes and teleosts, is necessary. A highly useful outgroup in this respect is the cartilaginous fish, whose lineage separated from that of their common ancestors (amniotes and teleosts) ~530 million years ago.


   Acknowledgment
 
We acknowledge Dr. T. Miyadai (Fukui Prefectural University, Obama, Japan) for the provision of a mAb against fugu IgM.


   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 The sequence presented in this article has been submitted to GenBank under the accession no. AB176853. Back

2 Address correspondence and reprint requests to Dr. Hiroaki Suetake, Fisheries Laboratory, Graduate School of Agricultural and Life Sciences, University of Tokyo, 2971-4 Bentenjima, Maisaka, Hamamatsu, Shizuoka, Japan. E-mail address: asuetake{at}mail.ecc.u-tokyo.ac.jp Back

3 Current address: Molecular Genetics Department, Benaroya Research Institute at Virginia Mason, 1201 9th Avenue, Seattle, WA 98101. Back

4 Abbreviations used in this paper: pIgR, polymeric Ig receptor; D, Ig-like domain; pIg, polymeric Ig; SC, secretory component; sIgM, secretory IgM. Back

Received for publication December 29, 2005. Accepted for publication February 16, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Kaetzel, C. S.. 2005. The polymeric immunoglobulin receptor: bridging innate and adaptive immune responses at mucosal surfaces. Immunol. Rev. 206: 83-99. [Medline]
  2. Ogra, P. L., J. Metstecky, M. E. Lamm, W. Strober, J. Bienenstock, J. R. MacGhee. 1999. Mucosal Immunology 2nd Ed. Academic, San Diego, CA. eds.
  3. Woof, J. M., J. Mestecky. 2005. Mucosal immunoglobulins. Immunol. Rev. 206: 64-81. [Medline]
  4. Ingram, G. A.. 1980. Substances involved in the natural resistance of fish to infection. J. Fish Biol. 16: 23-60.
  5. Di-Conza, J. J., W. J. Halliday. 1971. Relationship of catfish serum antibodies to immunoglobulin in mucus secretions. Aust. J. Exp. Biol. Med. Sci. 49: 517-519. [Medline]
  6. Fletcher, T. C., A. White. 1973. Antibody production in the plaice (Pleuronectes platessa L.) after oral and parenteral immunization with Vibrio anguillarum antigens. Aquaculture 1: 417-428.
  7. Lobb, C. J., L. W. Clem. 1981. Phylogeny of immunoglobulin structure and function: XI. Secretory immunoglobulins in the cutaneous mucus of the sheepshead Archosargus probatocephalus. Dev. Comp. Immunol. 5: 587-596. [Medline]
  8. Rombout, J. W., L. J. Blok, C. H. Lamers, E. Egberts. 1986. Immunization of carp (Cyprinus carpio) with a Vibrio anguillarum bacterin: indications for a common mucosal immune system. Dev. Comp. Immunol. 10: 341-351. [Medline]
  9. Hart, S., A. B. Wrathmell, T. A. Dogget, J. E. Harris. 1987. An investigation of the biliary and intestinal immunoglobulin and the plasma cell distribution in the gall bladder and liver of the common dogfish Scyliorhinus canicula L. Aquaculture 67: 147-155.
  10. Itami, T., Y. Takahashi, T. Okamoto, K. Kubono. 1988. Purification and characterization of immunoglobulin in skin mucus and serum of Ayu. Nippon Suisan Gakkaishi 54: 1611-1617.
  11. Al-Harbi, A. H., B. Austin. 1993. Purification of macroglobulins from the serum, and skin and gut mucus of turbot (Scophthalmus maximus L.) immunized with lipopolysaccaride (LPS) from a fish-pathogenic Cytophaga-like bacterium (CLB). Bull. Eur. Assoc. Fish Pathol. 13: 40-44.
  12. Rombout, J. H., N. Taverne, M. van de Kamp, A. J. Taverne-Thiele. 1993. Differences in mucus and serum immunoglobulin of carp (Cyprinus carpio L.). Dev. Comp. Immunol. 17: 309-317. [Medline]
  13. Zilberg, D., P. H. Klesius. 1997. Quantification of immunoglobulin in the serum and mucus of channel catfish at different ages and following infection with Edwardsiella ictaluri. Vet. Immunol. Immunopathol. 58: 171-180. [Medline]
  14. Hou, Y. Y., Y. Suzuki, K. Aida. 1999. Effects of steroid hormones on immunoglobulin M (IgM) in rainbow trout, Oncorhynchus mykiss. Fish Physiol. Biochem. 20: 155-162.
  15. Hatten, F., A. Fredriksen, I. Hordvik, C. Endresen. 2001. Presence of IgM in cutaneous mucus, but not in gut mucus of Atlantic salmon, Salmo salar, serum IgM is rapidly degraded when added to gut mucus. Fish Shellfish Immunol. 11: 257-268. [Medline]
  16. Lobb, C. J.. 1987. Secretory immunity induced in catfish. Ictalarus punctatus, following bath immunization. Dev. Comp. Immunol. 11: 727-738. [Medline]
  17. Joosten, P. H. M., E. Tiemersma, A. Threels, C. Caumartin-Dhieux, J. H. Rombout. 1997. Oral vaccination of fish against Vibrio anguillarum usingalginate microparticles. Fish Shellfish Immunol. 7: 471-485.
  18. Cain, K. D., D. R. Jones, R. L. Raison. 2000. Characterisation of mucosal and systemic immune responses in rainbow trout (Oncorhynchus mykiss) using surface plasmon resonance. Fish Shellfish Immunol. 10: 651-666. [Medline]
  19. Maki, J. L., H. W. Dickerson. 2003. Systemic and cutaneous mucus antibody responses of channel catfish immunized against the protozoan parasite Ichthyophthirius multifiliis. Clin. Diagn. Lab. Immunol. 10: 876-881. [Medline]
  20. Esteve-Gassent, M. D., B. Fouz, C. Amaro. 2004. Efficacy of a bivalent vaccine against eel diseases caused by Vibrio vulnificus after its administration by four different routes. Fish Shellfish Immunol. 16: 93-105. [Medline]
  21. Nagura, H., P. K. Nakane, W. R. Brown. 1979. Translocation of dimeric IgA through neoplastic colon cells in vitro. J. Immunol. 123: 2359-2368. [Abstract/Free Full Text]
  22. Verbeet, M. P., H. Vermeer, G. C. Warmerdam, H. A. de Boer, S. H. Lee. 1995. Cloning and characterization of the bovine polymeric immunoglobulin receptor-encoding cDNA. Gene 164: 329-333. [Medline]
  23. 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]
  24. Taylor, C. L., G. A. Harrison, C. M. Watson, E. M. Deane. 2002. cDNA cloning of the polymeric immunoglobulin receptor of the marsupial Macropus eugenii (tammar wallaby). Eur. J. Immunogenet. 29: 87-93. [Medline]
  25. 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]
  26. Mostov, K. E., M. Friedlander, G. Blobel. 1984. The receptor for transepithelial transport of IgA and IgM contains multiple immunoglobulin-like domains. Nature 308: 37-43. [Medline]
  27. Mostov, K. E., J. P. Kraehenbuhl, G. Blobel. 1980. Receptor-mediated transcellular transport of immunoglobulin: synthesis of secretory component as multiple and larger transmembrane forms. Proc. Natl. Acad. Sci. USA 77: 7257-7261. [Abstract/Free Full Text]
  28. Kuhn, L. C., J. P. Kraehenbuhl. 1981. The membrane receptor for polymeric immunoglobulin is structurally related to secretory component: isolation and characterization of membrane secretory component from rabbit liver and mammary gland. J. Biol. Chem. 256: 12490-12495. [Abstract/Free Full Text]
  29. Kuhn, L. C., H. P. Kocher, W. C. Hanly, L. Cook, J. C. Jaton, J. P. Kraehenbuhl. 1983. Structural and genetic heterogeneity of the receptor mediating translocation of immunoglobulin A dimer antibodies across epithelia in the rabbit. J. Biol. Chem. 258: 6653-6659. [Abstract/Free Full Text]
  30. Kulseth, M. A., P. Krajci, O. Myklebost, S. Rogne. 1995. Cloning and characterization of two forms of bovine polymeric immunoglobulin receptor cDNA. DNA Cell Biol. 14: 251-256. [Medline]
  31. Miyadai, T., M. Ootani, D. Tahara, K. Aoki, T. Saitoh. 2004. Monoclonal antibodies recognising serum immunoglobulins and surface immunoglobulin-positive cells of puffer fish, torafugu (Takifugu rubripes). Fish Shellfish Immunol. 17: 211-222. [Medline]
  32. Altschul, S. F., T. L. Madden, A. A. Schaffer, J. Zhang, Z. Zhang, W. Miller, D. J. Lipman. 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25: 3389-3402. [Abstract/Free Full Text]
  33. Thompson, J. D., D. G. Higgins, T. J. Gibson. 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22: 4673-4680. [Abstract/Free Full Text]
  34. Bendtsen, J. D., H. Nielsen, G. von Heijne, S. Brunak. 2004. Improved prediction of signal peptides: SignalP 3.0. J. Mol. Biol. 340: 783-795. [Medline]
  35. Hirokawa, T., S. Boon-Chieng, S. Mitaku. 1998. SOSUI: classification and secondary structure prediction system for membrane proteins. Bioinformatics 14: 378-379. [Abstract/Free Full Text]
  36. Tsutsui, S., S. Tasumi, H. Suetake, Y. Suzuki. 2003. Lectins homologous to those of monocotyledonous plants in the skin mucus and intestine of puffer fish, Fugu rubripes. J. Biol. Chem. 278: 20882-20889. [Abstract/Free Full Text]
  37. Saha, N. R., H. Suetake, Y. Suzuki. 2005. Analysis and characterization of the expression of the secretory and membrane forms of IgM heavy chains in the puffer fish, Takifugu rubripes. Mol. Immunol. 42: 113-124. [Medline]
  38. Casanova, J. E., P. P. Breitfeld, S. A. Ross, K. E. Mostov. 1990. Phosphorylation of the polymeric immunoglobulin receptor required for its efficient transcytosis. Science 248: 742-745. [Abstract/Free Full Text]
  39. Barclay, A. N.. 2003. Membrane proteins with immunoglobulin-like domains: a master superfamily of interaction molecules. Semin. Immunol. 15: 215-223. [Medline]
  40. Norderhaug, I. N., F. E. Johansen, P. R. Krajci, P. Brandtzaeg. 1999. Domain deletions in the human polymeric Ig receptor disclose differences between its dimeric IgA and pentameric IgM interaction. Eur. J. Immunol. 29: 3401-3409. [Medline]
  41. William, A. F., A. N. Barclay. 1988. The immunoglobulin superfamily: domains for cell surface recognition. Annu. Rev. Immunol. 6: 381-405. [Medline]
  42. Kobayashi, K., J.-P. Vaerman, H. Bazin, A.-M. Lebacq-Verheyden, J. F. Heremans. 1973. Identification of J-chain in polymeric immunoglobulins from a variety of species by cross-reaction with rabbit antisera to human J-chain. J. Immunol. 111: 1590-1594. [Abstract/Free Full Text]
  43. Koshland, M. E.. 1985. The coming of age of the immunoglobulin J chain. Annu. Rev. Immunol. 3: 425-453. [Medline]
  44. Mikoryak, C. A., M. N. Margolies, L. A. Steiner. 1988. J chain in Rana catesbeiana high molecular weight Ig. J. Immunol. 140: 4279-4285. [Abstract]
  45. 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]
  46. 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]
  47. 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]
  48. 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]



This article has been cited by other articles:


Home page
 J. Immunol.Home page
Z. Wei, Q. Wu, L. Ren, X. Hu, Y. Guo, G. W. Warr, L. Hammarstrom, N. Li, and Y. Zhao
Expression of IgM, IgD, and IgY in a Reptile, Anolis carolinensis
J. Immunol., September 15, 2009; 183(6): 3858 - 3864.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Hamuro, K.
Right arrow Articles by Suzuki, Y.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Hamuro, K.
Right arrow Articles by Suzuki, Y.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS