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Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

The polymeric immunoglobulin receptor (pIgR) is a type I transmembrane protein that is expressed on the surfaces of glandular and intestinal epithelial cells. The extracellular portion of the pIgR is composed of six different domains. Domain 6 is involved in the enzymatic cleavage and release of the pIgR into the intestinal lumen as a free secretory component (fSC). A highly conserved 9-amino acid sequence is present in this region in various species. Although mutations in domain 6 are associated with particular diseases, such as IgA nephropathy and Epstein–Barr virus-related nasopharyngeal cancer, and the glutamic acid residues in the conserved 9-amino acid sequence are expected to be indispensable for the secretion of fSC, the importance of these residues has not been examined. In the present study, we attempted to examine the role of these residues in the enzymatic cleavage of the pIgR. The enzymatic cleavage of the pIgR was not affected by the presence of an alanine to valine substitution at position 580 or glutamine to alanine substitutions at positions 606 and/or 607, or the deletion of the whole 9-amino acid conserved sequence. Intriguingly, the 10 amino acid sequences flanking the N- and C-terminal ends of the conserved 9-amino acid sequence had opposite effects on pIgR cleavage. Namely, the N-terminal and C-terminal sequences enhanced and reduced pIgR cleavage efficiency, respectively. These results indicated that the pIgR can be divided into several functionally distinct regions.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

The mucosal immune system is the first line of defence against a variety of antigens [1], and polymeric immunoglobulins (pIg) are the main players in this system. The pIg produced by plasma cells in the lamina propria have to be transported across the epithelial barrier to allow them to exert their protective effects against environmental antigens [1]. These pIg are captured by polymeric immunoglobulin receptors (pIgR) expressed on the basolateral surface of the glandular and intestinal epithelium and transcytosed to the apical surface. On the apical surfaces of polarized epithelial cells, the extracellular domain of the pIgR is cleaved by unidentified proteinase(s), which results in the captured pIg being released as secretory immunoglobulins. In the absence of a pIg binding, the pIgR is transcytosed to the apical cell surface and released as a free secretory component (fSC). Alignment studies of the amino acid sequences of pIgR from several species have detected conserved domain structures. Among them, a putative cleavage site in the extracellular portion of the pIgR that is closest to the cell membrane [1] is highly conserved. In spite of the high degree of conservation in this region, the precise cleavage site remains obscure because the C-terminus of human fSC, which is derived from the extracellular portion of the pIgR, varies from Ala550 to Lys559, with Ser556 being the dominant residue [2]. In a previous study using a recombinant vaccinia virus-mediated transient expression system, we demonstrated the importance of domain 6 of the pIgR for cleavage of the receptor [3].

The identification of single-nucleotide polymorphisms in the human genome has led to the discovery of disease-susceptibility genes, and single-nucleotide polymorphisms in the pIgR gene were reported to be associated with IgA nephropathy (IgAN) [4]. Among six mutations, one mutation in domain 6 of the pIgR protein caused an alanine to valine substitution at position 580. This mutation was also found in patients with Epstein–Barr virus (EBV)-associated nasopharyngeal cancer (NPC), and it was suggested that the mutant protein might act as a receptor for EBV [5]. In both diseases, this mutation is considered to reduce the transcytosis of the pIgR molecule.

These findings prompted us to further investigate the portions of domain 6 required for the efficient cleavage and release of the pIgR. The aim of this study was to explore which regions of pIgR domain 6 influence the efficient cleavage and release of pIgR.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Cells. Chinese hamster ovary (CHO) cells were cultured with Dulbecco's minimum essential medium supplemented with 10% fetal calf serum, 50 U/ml penicillin and 50 mg/ml streptomycin (10% FCS-DMEM).

Antibody. Non-labelled or horseradish peroxidase (HRP)-conjugated rabbit anti-human SC antibodies (Ab) were purchased from DAKO (Tokyo, Japan). HRP-conjugated goat anti-rabbit IgG Ab were purchased from Jackson ImmunoResearch (Tokyo, Japan).

Plasmid construction. The EcoRI fragment of the full-length human pIgR cDNA sequence was inserted into the pcDNA3.1 vector (Invitrogen, Tokyo, Japan). The resultant plasmid was designated pcDNA-pIgR-WT and used as a template for the construction of the mutant plasmids. The QuikChange II site-directed mutagenesis kit (Stratagene, La Jolla, CA, USA) was used to delete or substitute the desired amino acids or nucleotide regions. The empty pcDNA3.1 vector was used as a mock transfection vector.

Transfection and metabolic labelling. The recombinant vaccinia virus infection and transfection method is very useful because the protein expression can be detected after 5 h of transfection. Although there are several cell lines that are incompatible to this method, we used this method for transient expression experiments. Transfection was performed as described previously [3]. After 5 h of transfection, the cells were washed with labelling medium (Sigma, St Louis, MO, USA) and incubated with 1 ml of the same medium for 15 min to induce starvation. The cells were then metabolically labelled with 30 μCi/ml of trans-[35S]-label (MP Bio Japan, Tokyo, Japan) for 30 min at 37 °C. The cells were washed with 10% FCS-DMEM once and then further incubated with the same medium for 16 h. At the end of each culture, the culture supernatant and cell lysate were harvested and subjected to immunoprecipitation. Briefly, the cells were lysed with 500 μl of lysis buffer (50 mm Tris-HCl (pH 7.5), 150 mm NaCl and 0.5% Triton X-100). The cell lysates were cleared by centrifugation (14,000 g for 1 min) and transferred to new tubes. Each sample was incubated with 1 μl of anti-SC Ab (DAKO, Tokyo, Japan) for 18 h, before being incubated with 10 μl of protein G-Sepharose (Amersham, NJ, USA) for 1 h at 4 °C. After washing the pellets with 500 μl of cell lysis buffer 5 times, the samples were subjected to 8% SDS-PAGE and autoradiographed. For ΔCL, Δ604–612, Δ594–612 and Δ604–622 mutants, the stable transfectants were established. To establish stable transfectants, each plasmid was linearized with NheI and transfected into the CHO cells with Lipofectamine plus reagent (Invitrogen). The transfectant candidates were selected with medium containing G418 (250 μg/ml) and further subjected to limiting dilution to obtain the single clones. After selection, each clone was expanded and used for experiments.

Enzyme-linked immunosorbent assay (ELISA). Ninety-six-well round-bottomed plates were coated with 50 μl of rabbit anti-human SC Ab (×1000 diluted with PBS) for 18 h at 4 °C. After the plates had been washed three times with phosphate-buffered saline (PBS), they were incubated with 200 μl of 1% bovine serum albumin (BSA)-PBS for 1 h at 37 °C. Then, the 1% BSA-PBS was discarded, and 50 μl of the culture supernatant or cell lysate was applied to the plates and incubated for 1 h at room temperature. The plates were washed three times with 0.05% Tween 20/PBS. Fifty μl of HRP-conjugated rabbit anti-human SC Ab (×1000 diluted with 1% BSA-PBS, DAKO) was added to the plates, which were then incubated for 30 min at room temperature. Next, the plates were extensively washed with 0.05% Tween 20/PBS, before the colour reaction was performed by incubating the plates with 50 μl of 1 mg/ml o-phenylenediamine (Kanto Chemical, Tokyo, Japan) in 0.1 m citrate phosphate buffer (pH 5.0) supplemented with 0.03% H2O2 for 20 min at room temperature. The reaction was stopped by adding 25 μl of 2 m H2SO4. Absorbance was measured with a microplate reader (model 3550; Bio Rad, Tokyo, Japan). Secretion efficiency was calculated as follows: secreted SC/cellular pIgR + secreted SC, and the secretion efficiency of each mutant was compared with that of the WT transfectant (100%).

Statistical analysis. The one-way ANOVA with post hoc Bonferroni multiple comparison test was used for all statistical analyses. Results are presented as mean ± SD values. P-values of <0.05 were considered statistically significant.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Construction and expression of pIgR mutants

An alanine (Ala) to valine (Val) substitution at position 580 of the human pIgR molecule was reported to be associated with IgAN and EBV-associated NPC [4, 5]. Based on these observations, we first attempted to examine the functional relevance of this Ala residue to pIgR expression and cleavage. In addition, an alignment study of the amino acid sequences of pIgR from several species revealed a potential cleavage site [1]. In humans, this site is located between glutamic acid (Glu) residues 606 and 607. The Glu were substituted for Ala individually (E606A and E607A) or in combination (E606, 607A). Figure 1 shows the positions of each amino acid residue relative to the whole pIgR molecule. The sequences of the DNA constructs and their mutations were confirmed by DNA sequencing. CHO cells were transiently transfected with the wild-type construct (WT) or one of the mutant constructs, and the expression of each mutant was examined by immunoprecipitation following metabolic labelling (Fig. 2A,B). Clear 120 and 110 kDa bands, corresponding to mature and immature pIgR molecule, respectively, were detected in the cell lysates of all transfectants, except for the mock transfectant, indicating the successful expression of each mutant in the CHO cells. The SC secretion efficiency of each transfectant was also examined. One hundred kDa bands were detected in all samples, and the intensity of the bands did not differ significantly between the transfectants. To confirm these results, we measured the amount of secreted SC by ELISA (Fig. 2C). The secretion efficiency was calculated as ((secreted fSC/cellular pIgR + secreted fSC) ×100%). This value for WT transfectant was set as 100%, and the secretion efficiencies of the mutant transfectants were calculated based on this value. As a result, we found that the secretion efficiencies of the WT and mutants were not significantly different. Taken together, the above results indicated that the examined mutations did not influence the secretion efficiency of the pIgR molecule.

image

Figure 1. The schematic structure of the pIgR. The entire amino acid sequence of domain 6 of the pIgR is shown. The shaded region corresponds to the 9-amino acid sequence. The Ala at position 580 and the Glu at positions 606 and 607 are indicated by large letters. TM: transmembrane region.

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image

Figure 2. The fSC secretion efficiency of the pIgR was not affected by mutations involving the A580, E606, or E607 residues. Chinese hamster ovary (CHO) cells were transfected with the WT or A580V mutant plasmid (A) or the E606A, E607A or E606,607A mutant plasmid (B). After being transfected, the cells were metabolically labelled with trans-[35S]-label and cultured for a further 16 h with 10% FCS-DMEM. At the end of the culture period, the culture supernatants and cell lysates were harvested and subjected to immunoprecipitation with anti-SC Ab. The precipitates were washed and subjected to 8% SDS-PAGE. The resultant gels were dried and autoradiographed. (C) After being transfected, each transfectant was cultured for 16 h with 10% FCS-DMEM. The culture supernatants and cell lysates were harvested and subjected to ELISA. The total amounts of pIgR and fSC were determined, and the fSC secretion efficiency (secreted fSC/(cellular pIgR + secreted fSC)) was calculated. The fSC secretion ratio of the WT was defined as 100%. Data are mean ± SD of three separate experiments.

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Expression of pIgR deletion mutants

Although the above mutations did not affect fSC secretion efficiency, the deletion of domain 6 of the pIgR significantly reduced SC cleavage and secretion [3]. To further explore the region required for the efficient cleavage of the pIgR, we constructed several deletion mutants (Fig. 3A). The part of the pIgR sequence that has been shaded in Fig. 1 is a 9-amino acid sequence that is highly conserved among many species [1]. This sequence is predicted to encode an α-helical region that plays an important role in pIgR cleavage. We first examined the secretion of a pIgR mutant lacking this sequence (Δ604–612). The expression level of the mutant was confirmed by Western blotting, and a single slightly faster migrating band was detected (Fig. 3B; 4th lane). Then, the transfectant was freshly plated, and the amount of secreted SC was measured by ELISA (Fig. 3C). The WT transfectant secreted 86.1 pg/ml of fSC into the culture supernatant, which corresponded to 0.3% of total pIgR (pIgR in the cell lysate of WT transfectant was 27 ng/ml). The mutant that lacked domain 6 was designated ΔCL, and its characteristics were compared with those of the WT and the other mutants. The fSC secretion efficiency of the ΔCL transfectant was extremely low (<5 pg/ml), and no fSC was detected in its supernatant. The Δ604–612 transfectant exhibited slightly reduced fSC secretion compared with the WT (76 pg/ml); however, the difference was not significant. Based on these results, we further examined the regions flanking the 604–612 region. The ten amino acid sequences on the N-terminal (594–603) and C-terminal (613–622) sides of the 604–612 region were deleted in combination with the 604–612 sequence, and the resultant mutants were designated Δ594–612 and Δ604–622, respectively. Both mutants were detected as single bands that migrated slightly faster than the WT band during Western blotting (Fig. 3B, 5th and 6th lanes). Compared with the Δ604–612 mutant, the Δ594–612 transfectant exhibited markedly reduced fSC secretion (30 pg/ml). In contrast, the fSC secretion of the Δ604–622 transfectant was significantly enhanced; that is, its total fSC secretion value was 2.2-fold higher than that of the WT (199.5 pg/ml). To confirm these results, fSC secretion efficiency was examined by metabolic labelling followed by immunoprecipitation. As shown in Fig. 3D (upper panel), the Δ604–612 transfectant displayed equivalent fSC secretion to the WT (1st and 3rd lanes). The supernatant of the ΔCL mutant did not produce any bands (2nd lane). As for the Δ594–612 transfectant, although a clear intracellular pIgR band was detected, its supernatant only produced a very faint band (4th lane). On the other hand, the supernatant of the Δ604–622 transfectant produced a clear band (5th lane), indicating that significant amount of the labelled pIgR was secreted as fSC. These results indicated that amino acids 594–603 and 613–622 have opposite effects on pIgR cleavage.

image

Figure 3. Distinct functional regions in the pIgR molecule. (A) The schematic structure of each deletion mutant. The conserved region in domain 6 of the WT pIgR is indicated by the shaded box. The ΔCL mutant lacked domain 6. The Δ604–612 mutant lacked the conserved region (a 9-amino acid sequence). The ten amino acid sequences on the N-terminal (594–612) and C-terminal sides (604–622) of the 9-amino acid sequence are shown. (B) The cell lysates of all of the transfectants were harvested and subjected to Western blotting. Western blotting was performed using anti-SC Ab as the primary Ab. (C) 5 × 105 cells of each stable transfectant was plated in 6-well plates and cultured for 16 h. At the end of the culture period, the culture supernatants were harvested, and the fSC concentrations were measured with ELISA. Data are mean ± SD of three separate experiments. (D) Each stable transfectant was metabolically labelled and subjected to immunoprecipitation. The upper panel shows the secreted fSC in culture supernatants (supernatant), and the lower panel shows the pIgR in the cell lysates (Cell lysate), respectively. P-values of <0.05 were considered statistically significant.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Epstein–Barr virus-associated NPC is an important squamous cell cancer that is endemic in South-East Asia and the Far East and is considered to be a multifactorial genetic disease [6]. Although it has not been definitively confirmed, at least two molecules, complement receptor type 2 and pIgR, have been proposed to be important for the entry of EBV into the nasopharyngeal epithelium [7]. The pIgR mediates the endocytosis and transcytosis of IgA-EBV complexes when EBV is encountered at the luminal surface. However, when the pIgR gene contains mutations, the viral translocation process can fail, resulting in EBV infection [8]. A 1739C to T mutation was found to alter the efficiency with which pIgR released IgA-EBV complex [5]. This mutation resulted in the substitution of Ala for Val at position 580, and the same mutation demonstrated a significant association with IgAN [4]. Moreover, elevated serum IgA levels are observed in IgAN. Based on these observations, we speculated that this mutation might influence the enzymatic cleavage of pIgR. A comparison of the cleavage and secretion efficiencies of the WT and A580V mutant revealed that fSC secretion was not affected by this mutation. Recently, using the Madin-Darby canine kidney cell (polarized epithelial cell) system, A580V mutation is shown to reduce pIgR transcytosis and fSC secretion efficiency [9]. These data apparently contradict to our results. We used the combination of human pIgR and hamster-derived fibroblastic cell system. Not only the cells are non-polarized, but also the enzymes contributing to the cleavage of pIgR might be different between epithelial cell and fibroblast. Although the reasons are obscure, these differences might lead to the different output.

In addition to this mutation, we attempted to examine the importance of the Glu at positions 606 and 607, which constitute the border of a potential cleavage site [1]. However, substitutions at these locations did not influence the cleavage of pIgR. Our results suggested that these Glu do not contribute to the enzymatic recognition of the pIgR molecule by the relevant enzyme(s).

The main function of the pIgR is to capture pIg at the basolateral surface of the glandular and intestinal epithelium and transcytose them to the apical surface [1]. After the pIgR-pIg complexes reach the apical surface, the pIg must be released into the external lumen to allow them to perform their protective functions [1]. Therefore, the proteolytic cleavage of the pIgR and the subsequent release of secretory immunoglobulins are critical for appropriate immunological surveillance by the mucosal immune system. Although the function of the cytoplasmic portion of the pIgR has been extensively studied [10-15], the role of its extracellular portion has never been examined in detail. In a previous study, domain 6 of the pIgR was demonstrated to be important for its effective cleavage and release [3]. In the present study, we attempted to determine the region within domain 6 that is responsible for its role in the cleavage and release of the pIgR. The 9-amino acid sequence from position 604 to position 612 in pIgR is predicted to encode an α-helical region that is critical for pIgR cleavage. Although the deletion of all 9 of these amino acids resulted in a reduction in fSC secretion efficiency, the reduction was only marginal. On the other hand, the Δ594–612 mutant exhibited significantly reduced fSC secretion efficiency. The Δ584–612 mutation did not have a marked additive effect on pIgR cleavage (data not shown) indicating that the amino acid sequence from 594 to 603 is indispensable for the enzymatic recognition of pIgR in addition to the above-mentioned 9-amino acid sequence. On the contrary, the Δ604–622 mutant displayed significantly enhanced fSC secretion, indicating that the amino acids between 613 and 622 have an inhibitory effect on fSC secretion. Taken together, our results indicated that the conserved 9-amino acid sequence of the pIgR and its flanking regions have important effects on pIgR cleavage.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

This study was supported by a Nihon University Joint Research Grant for 2010–2012 (to Dr. Okayama and Dr. Imamura); Strategic Research Base Development Program for Private Universities grants from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan, for 2010–2014 (S1001024) and 2011–2013 (23592778); funding from the Health and Labor Sciences Research Grants program and Research for International Cooperation in Medical Science; funding from the Promotion and Mutual Aid Corporation for Private Schools of Japan (2011); and a Grant-in-aid for Scientific Research (C) (2011–2013). The authors do not have any conflict of interests to declare.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References