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. 2004 Aug;112(4):583–589. doi: 10.1111/j.1365-2567.2004.01914.x

Multiple cleavage sites for polymeric immunoglobulin receptor

Masatake Asano *, Nobuko Takenouchi-Ohkubo *, Naoyuki Matsumoto *, Yoshitaka Ogura *, Hirofumi Nomura *, Hisashi Suguro †, Itaru Moro *
PMCID: PMC1782529  PMID: 15270729

Abstract

Human polymeric immunoglobulin receptor (pIgR) was expressed in baby hamster kidney (BHK) cells using a recombinant vaccinia virus transfection system. Cleavage of pIgR on the cell surface was partially inhibited by the proteinase inhibitor, leupeptin. We addressed the question whether some particular regions of pIgR could affect the efficient cleavage of this molecule, with the following results: (1) a mutant lacking the entire cytoplasmic region resulted in release of secretory component (SC) into the culture supernatant much faster than wild-type; (2) a pIgR mutant lacking the entire extracellular domain 6, the region containing the susceptible cleavage sites, could be cleaved and released as a mutant SC. The transport kinetics of this mutant between endoplasmic reticulum (ER) and Golgi or Golgi and the cell surface was equivalent to wild-type pIgR. Our results indicate that although the main cleavage site is in domain 6, at least one other cleavage site may exist.

Keywords: pIgR, BHK, cleavage, vaccinia virus

Introduction

The mucosal immune system is the first line of defence against a variety of antigens.1–3 The main players in this system are the polymeric immunoglobulins (pIgs). PIgs produced by plasma cells must be transported across the epithelial cells in order to exert their protective effects against environmental antigens.4 The pIgs are captured by polymeric immunoglobulin receptor (pIgR) expressed on the basolateral surface of the glandular epithelial cell and transcytosed to the apical surface. On the apical surface of polarized epithelial cells, the extracellular domain of pIgR is cleaved by unidentified proteinase(s) and released with pIgs as secretory immunoglobulins. In addition, proteolyic cleavage of pIgR occurs irrespective of the binding of the ligand. Thus, the pIgR itself is transcytosed to the apical cell surface without the binding of pIgs and is released as a free secretory component (SC). The biological role of free SC has been recently reviewed.5

The alignment of amino acid sequences of pIgR from several species defines a conserved domain structure, the pIg-binding site, and a putative cleavage site in the extracellular portion.6 In addition to these, the cytoplasmic portion of pIgR contains important signals for endocytosis and transcytosis. Although there are several important questions concerning the pIgR, the identification of the responsible proteinase(s) cleaving the extracellular portion of the pIgR has not been extensively studied.

In this report, by transiently expressing several pIgR deletion mutants, we sought to define particular regions of the pIgR that influence the efficient cleavage and release of pIgR. We demonstrate here that deletion of the region of the pIgR susceptible to proteinase cleavage failed to completely abolish the release of free SC. We hypothesize that cleavage of pIgR might occur at least two different sites.

Materials and methods

Cell culture

Baby hamster kidney (BHK) cells were grown in Dulbecco's modified Eagle's minimal essential medium (DMEM) supplemented with 10% fetal calf serum (10% FCS-DMEM; Invitrogen Corp., San Diego, CA). Cells were plated at 5 × 105 on 35 mm-dishes on the day before transfection.

DNA construction

For the construction of the cytoplasmic domain deletion mutant (ΔCP), a 2090 bp EcoRI–Aor51HI fragment was excised from human pIgR cDNA (kindly provided by Dr P. Brandtzaeg (LIIPAT, Institute of Pathology, National Hospital, University of Oslo, Oslo, Norway)7 and ligated to the polymerase chain reaction (PCR) fragment containing the transmembrane region. The primers used were as follows; 5′-CCAGCGCTGGTCTCCACCC-3′, 5′-CCAAGCTTCTAGGCCACCCCCAC-3′).

For the construction of both transmembrane and cytoplasmic domain deletion mutants (ΔTMCP), the EcoRI–SplI fragment of the pIgR containing the extracellular region was ligated to the PCR fragment which contained domains 5 and 6 of the pIgR. The primers used were as follows; 5′-GGTCGTACGAGAAATACTGG-3′, 5′-CCGTCGACCTATCTGGAGCTTCCACC-3′.

For the construction of the cleavage region deletion mutant (ΔCL), pIgR cDNA containing domains 5 and 6 was amplified using the following primers; 5′-GGTCGTACGAGAAATACTGG-3′, 5′- CCCAGCGCTCTCCCCGCTGCCTTCCTCTC-3′. This fragment was inserted into the SplI and Aor51HI site of the pIgR cDNA. All of the constructs were inserted into pT7-blue vector (Invitrogen) and used as transfection vectors.

Transfection and metabolic labelling

Infection with recombinant vaccinia virus and transfection of BHK cells were performed as described.8–10 Briefly, before infection, cells were washed with OPTI-MEM (Invitrogen) once and then infected with vaccinia T7 RNA polymerase recombinant virus (vTF7-3)8 at a multiplicity of 10 plaque forming units/cell in 0·5 ml of OPTI-MEM for 30 min with intermittent rocking in a 37° CO2 incubator. The infection medium was removed and the cells were washed with OPTI-MEM twice. Cells were then transfected with wild-type or mutant pIgR plasmids using the Lipofectamine plus transfection method (Invitrogen) according to the manufacturer's procedure. Briefly, 1 µg of each plasmid DNA was mixed in 100 µl of OPTI-MEM with 6 µl of Plus reagent, and at the same time 6 µl of Lipofectamine was mixed with 100 µl of OPTI-MEM. Fifteen min later, the medium containing DNA was mixed with Lipofectamine medium and incubated at room temperature for 15 min. The transfection medium was then applied to BHK cells and incubated for 5·5 hr in a CO2 incubator at 37°. After transfection, the cells were washed with labelling medium (Sigma Chemical Co., St. Louis, MO) and incubated in the same medium for 15 min for starvation. The cells were then metabolically labelled with 30 µCi/ml of Tran-[35S]-label (ICN Biochemicals Inc., Costa Mesa, CA) for the indicated times at 37°.

Immunoprecipitation

For leupeptin treatment experiments, the transfectants were metabolically labelled for 15 min and further cultured in the presence or absence of 10 µg/ml of leupeptin (Sigma). For detection of intracellular pIgR and secreted SC in the culture supernatants, the transfectants were labelled for 1 hr. After washing with 10% FCS-DMEM, the cells were further cultured for 16 hr with the same medium. At this time point, cell lysates and culture supernatants were prepared and immunoprecipitated. Briefly, the cells were lysed with 500 µl of lysis buffer (50 mm Tris, pH 7·5, 150 mm NaCl, and 0·5% Triton-X-100). The cell lysates and the culture supernatants were cleared by centrifugation (14 000 g for 1 min) and transferred to new tubes. The polyclonal rabbit anti-human SC was purchased from DAKO Cytomation (Kyoto, Japan) and 5 µl of this antibody were incubated with samples for 1 hr followed by 10 µl of protein G-Sepharose (Amersham, Piscataway, NJ) for 1 hr at 4°. After washing the pellets with 500 µl of cell lysis buffer three times, the pellets were loaded onto 8% sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) gels. After electrophoresis, the gels were immersed in fluorography solution (125 mm sodium salicylate, 30% methanol) for 30 min at room temperature, dried for 2 hr with a gel dryer (Bio-Rad, Hercules, CA) and exposed to X-ray film (X-OMAT AR, Kodak, Tokyo, Japan) for 18 hr.

Western blotting

After transfection, cell lysates were prepared by using 150 µl of cell lysis buffer and 20 µl of supernatants were used for Western blotting as described previously.11 The polyclonal rabbit anti-human SC antibody (DAKO Cytomation) was used as the first antibody. Horseradish peroxidase (HRP)-conjugated goat anti-rabbit immunoglobulin G (IgG; H + I) used as the secondary antibody was purchased from Jackson ImmunoResearch Laboratories (West Grove, PA). The membranes were developed using ECL reagents (Amersham).

Detection of surface-expressed pIgR

The transfectants were metabolically labelled for 1 hr and chased with 10% FCS-DMEM for 1 hr. The cells were transferred to ice and washed with ice-cold phosphate-buffered saline (PBS). The cells were incubated with ice-cold 10% FCS-DMEM supplemented with 5 µl of a polyclonal rabbit anti-human SC (DAKO Cytomation) antibody for 30 min. The solution containing antibody was discarded and the cells were washed with ice-cold PBS once and cell lysates were prepared. The samples were centrifuged at 14 000 g for 1 min and transferred to new tubes. Ten µl of protein G-Sepharose was added to the samples and incubated for 1 hr at 4°. The samples were washed with cell lysis buffer and loaded onto 8% SDS–PAGE gels.

Results

PIgR cleavage on BHK cells is leupeptin-dependent

In a previous report, we demonstrated that the pIgR expressed on BHK cells following transfection using a recombinant vaccinia virus system could be cleaved and released into the culture medium as free SC.11 However, BHK cells are not polarized epithelial cells and it is to be expected that the properties of pIgR cleavage might be different from glandular epithelial cells. In several other experimental systems, it has been reported that the cleavage of pIgR on the apical surface of the epithelial cells could be partially prevented by the proteinase inhibitor leupeptin.12,13 To ascertain whether pIgR cleavage on the surface of BHK cells is leupeptin-dependent, we cultured pIgR transfectants in the presence or absence of leupeptin.

Wild-type pIgR transfectants were metabolically labelled for 15 min and chased for 16 hr with or without 10 µg/ml of leupeptin. After the chase, the cell lysates (lower panel) and the culture supernatants (upper panel) were collected and the released free SC was immunoprecipitated as described in Materials and Methods. The amount of pIgR released into the culture supernatant with and without leupeptin was compared. As shown in Fig. 1, the amount of free SC in culture supernatants was reduced when the cells were cultured in the presence of leupeptin. Approximately 70% of release was inhibited by leupeptin treatment. Conversely, the amount of cellular pIgR was higher in the presence of leupeptin, indicating that pIgR release from transfected BHK cells was leupeptin sensitive. These findings indicated that the proteinase responsible for the cleavage of pIgR might be shared between polarized epithelial cells and BHK cells.

Figure 1.

Figure 1

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pIgR cleavage on the surface of BHK cells is leupeptin-dependent. The pIgR transfectants were metabolically labelled with [35S]-cysteine. Cells were then further cultured with or without 10 µg/ml of leupeptin for 16 h at 37°. The cell lysates and culture supernatants were prepared and immunoprecipitataed with polyclonal rabbit antihuman SC antibody, followed by protein G-Sepharose. In the presence of leupeptin, 70% of SC release was inhibited. Reciprocally, 70% more pIgR was detected in cell lysates.

The expression of pIgR mutants in BHK cells

In order to examine whether some particular regions of the pIgR contribute to efficient cleavage, we constructed several deletion mutants of human pIgR as in Fig. 2.

Figure 2.

Figure 2

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Schematic diagram of the deletion mutants of pIgR. The bold region (69 amino acids) represents the extracellular Domain 6 and all the amino acids in this domain were deleted in ΔCL mutant. The conserved susceptible cleavage site within this area is depicted as ▪. The thick region (22 amino acids) highlighted by the vertical lines represents the transmembrane region and the following sequence (103 amino acids) corresponds to the intracellular region. In the ΔTMCP mutant, both the transmembrane and intracellular regions were deleted. For ΔCP mutant, only the intracellular region was deleted. GSRDVSLAKADAAPDEKVLDSGFREIENKAIQDPRLFAEEKAVADTRDQADGSRASVDSGSSEEQGGSSLVSTLVPLGLVLAVGAVAVGVARARHRKNVDRVSIRSYRTDISNSREFGANDNMGASSITQETSLGGKEEFVATTESTTETKEPKKAKRSSKEEAEMAYKDFLLQSSTVAAEAQDGPQEA.

Because it has been reported that the cytoplasmic region of the pIgR has several important signals for transcytosis or endocytosis14–18 we investigated whether this portion influences the secretion of free SC in our transient transfection system. For this purpose, we constructed the cytoplasmic region-deleted mutant ΔCP.

The transmembrane region of the pIgR is expected to be indispensable for the insertion and the retention of pIgR in the plasma membrane. When the transmembrane region in addition to the cytoplasmic region was deleted, could this pIgR be processed more efficiently than wild-type pIgR? In order to answer this question, we constructed a deletion mutant lacking the transmembrane and cytoplasmic regions. The mutant was designated ΔTMCP.

Several reports have indicated that the extracellular region proximal to the transmembrane region of pIgR contained the susceptible cleavage site(s) for the proteinase specific for pIgR.4,6 Although the precise cleavage site and the responsible proteinase have not been identified, if this proteinase-sensitive region was deleted, could the pIgR be cleaved by proteinase and released into the culture medium? For this, we constructed the ΔCL mutant which lacks the cleavage-susceptible region but retains the other region intact. As multiple C-termini have been found in free SC19 and the susceptible cleavage sites distributed from K559 to E593, we deleted the region from Y553 to R627, the entire domain 6 region. The deleted amino acid sequences for each mutant were depicted in the figure legends to Fig. 2.

Each plasmid was transfected into BHK cells and cell lysates were then prepared and subjected to Western blotting to establish whether these mutants could be expressed in BHK cells. As the antibody used in Western blotting was raised against human free SC, all of the mutant proteins should be recognized by this antibody. The results are given in Fig. 3, which shows that all of the mutants were successfully expressed in BHK cells. Each mutant had a reduced molecular weight corresponding to the length of the deletion (wild-type 120 000 MW, ΔCP 108 000 MW, ΔTMCP 106 000 MW, ΔCL 113 000 MW).

Figure 3.

Figure 3

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Expression of wild-type and mutants of pIgR. The wild-type, ΔCP, ΔTMCP and ΔCL mutants were transfected into BHK cells. 5·5 hr after transfection, cell lysates were prepared and loaded onto 8% SDS–PAGE gels. Western blotting was performed using polyclonal rabbit anti-human SC antibody followed by HRP-conjugated goat anti-rabbit IgG (H + I) antibody. The expression levels of the mutant proteins were equivalent to wild-type protein.

The release of the extracellular portion of pIgR

We next investigated whether the pIgR protein from these mutants could be cleaved and released into the culture supernatants. Transfectants of each mutant were metabolically labelled by Tran-[35S]-label and cultured for 16 hr. After cultivation, cell lysates and media were collected and subjected to immunoprecipitation.

In the wild-type pIgR transfectant, 120000 and 118 000 MW bands in cell lysate, and an 85 000 MW band considered to be free SC in culture medium, were observed (Fig. 4). The amount of free SC secreted was approximately 60% of the total labelled-pIgR. In contrast, we could not detect pIgR mutant proteins in cell lysates of ΔCP and ΔTMCP transfectants. In the culture supernatants, however, 85 000 MW and 86 000 MW bands were detected in ΔCP and ΔTMCP transfectants, respectively. It has been hypothesized that the ΔTMCP mutant could no longer be incorporated into the plasma membrane and it could not be cleaved by proteinase (The release of ΔTMCP mutant to the culture medium was much faster than wild-type pIgR, data not shown). The ΔCP mutant, however, lacked only the cytoplasmic region and had an intact transmembrane region. It could be incorporated into the plasma membrane and cleaved by proteinase. As a consequence, the ΔCP transfectant released the same molecular weight free SC as the wild-type transfectant and the ΔTMCP mutant released a slightly larger protein into the culture medium. Moreover, the deletion of the cytoplasmic region allowed the secretion of all labelled pIgR into the culture medium, indicating that the cytoplasmic portion may have some positive effect on the retention of the pIgR molecule inside the cell.

Figure 4.

Figure 4

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Release of free SC from wild-type- and mutant-transfectants. The wild-type- and mutant-transfectants were metabolically labelled as in Fig. 1 and further cultured with 10% FCS-DMEM for 16 hr. The cell lysates and culture supernatants were then immunoprecipitated with polyclonal rabbit anti-human SC and protein G-Sepharose. The samples were loaded onto 8% SDS–PAGE gels. In cell lysates of ΔCP and ΔTMCP mutants, no labelled protein was detected. In the culture supernatant of the ΔCL mutant, two different molecular weight bands were detected.

In the ΔCL mutant, a 113 000 MW band was detected in the cell lysates indicating the successful expression of the mutant protein. Because the ΔCL mutant lacked the susceptible cleavage region of pIgR, we expected that no cleavage of the pIgR would occur on the plasma membrane and therefore no protein would be released into the culture supernatant. Surprisingly, however, we detected bands of 84 000 MW and 113 000 MW in the culture supernatant. The intensity of the bands was, however, very low (less than 10% of the total labelled protein). As we could not detect these extra bands in wild-type pIgR transfectants, we can probably exclude the possibility that these two bands were non-specifically precipitated proteins.

ΔCL mutant can be transported from endoplasmic reticulum (ER) to Golgi with an efficiency equivalent to that of wild-type pIgR

To determine the transport efficiency between ER and Golgi of the wild-type and ΔCL mutant, each construct was transfected into BHK cells, which were then metabolically labelled for 15 min. After labelling, the cells were chased for the indicated times and immunoprecipitated as in Fig. 5(a). At the end of labelling (0 min), pIgR of both wild-type and ΔCL mutants were precipitated as a single band of 118000 and 111 000 MW, respectively. After 30 min of chase, both proteins were precipitated as two bands. PIgR of both wild-type and the ΔCL mutant began to change from the ER to the Golgi form at 30 min of chase. After 60 min of chase, all labelled protein of both wild-type and ΔCL mutant matured to the Golgi form, indicating that the ΔCL mutant has the same ER to Golgi transport efficiency as wild-type pIgR. Although the ER to Golgi transport efficiency of wild-type and ΔCL mutant was equal, we still could not exclude the possibility that the ΔCL mutant has certain defects in intracellular transport between the Golgi apparatus and the cell surface. To test this possibility, we compared surface expression levels of wild-type and ΔCL mutant transfectants.

Figure 5.

Figure 5

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(a) The ΔCL mutant has the same ER–Golgi transport rate as wild-type pIgR. The wild-type and ΔCL mutant were transfected into BHK cells and metabolically labelled with [35S]-cysteine for 15 min. The cell lysates were prepared at the indicated times and subjected to immunoprecipitation. The samples were loaded onto 8% SDS–PAGE gels. The molecular maturation kinetics were identical in both wild-type and ΔCL mutant. At 0 min of chase, only single bands were observed. At 30 min of chase, both ER and Golgi forms were detected and at 1 hr of chase all labelled protein were matured to the Golgi form.(b) Detection of cell surface-expressed wild-type and ΔCL mutant molecules. Wild-type or ΔCL mutant were transfected into BHK cells. Detection of the cell surface-expressed pIgR molecules was performed as described in Materials and Methods. The transport of the wild-type and ΔCL mutant to the cell surface was kinetically equivalent.

Cell surface expression of the ΔCL mutant

Cell surface expression of pIgR in wild-type and ΔCL mutants was examined as described in Materials and Methods. As shown in Fig. 5(b), in both transfectants, ER (118 000 MW in wild-type, 111 000 MW in the ΔCL mutant) and Golgi (120 000 MW in wild-type, 113 000 MW in ΔCL mutant) forms of each protein were detected in the cell lysates. However, on the cell surface, only the larger form of the proteins (120 000 MW in wild-type, 113 000 MW in ΔCL mutant) was detected and the level of expression was equivalent in wild-type and ΔCL mutant. In mock transfectant, we could not detect any band (data not shown), indicating that the ΔCL mutant did not have any defect in transport between the Golgi apparatus and the cell surface.

Discussion

The main function of the pIgR is to capture pIgs at the basolateral surface of glandular epithelial cells and transcytose them to the apical surface. After the pIgR–pIg complexes reach the apical surface, they must be released into the external lumen to mediate their protective function.1–4 Therefore, the proteolytic cleavage of the pIgR protein, and as a result, the release of secretory immunoglobulins is critical for proper immunological surveillance by the mucosal immune system. To date, however, the proteinase responsible for the cleavage of pIgR has not been identified. Thus, we aimed to identify the intramolecular regions contributing to the efficient release of pIgR.

In a first attempt, we tried to express human pIgR in a well-known kidney epithelial cell line, MDCK cell, however, this cell was not successfully infected by recombinant vaccinia virus. For this reason, we selected BHK cell as a host. Although BHK cells are non-polarized, they possess several features in common with polarized epithelial cells and the following facts provide a rationale for research on the proteolytic release of pIgR in non-polarized BHK cells.1) Consistent with a previous study11 we could not detect free SC in the cell lysate of pIgR transfectants, indicating that the proteolytic cleavage of pIgR occurs on the cell surface and not in the intracellular compartment.2 The cleavage of pIgR on the surface of the BHK cells was inhibited by the proteinase inhibitor leupeptin. This finding is in agreement with a report that the cleavage and release of rabbit pIgR could be inhibited by leupeptin.3,12,13 Moreover, Deitcher et al. have demonstrated that all of the transport and processing steps of the pIgR, except for transcytosis, are carried out correctly in murine fibroblasts transfected with rabbit pIgR cDNA.5,20 Furthermore, it has been demonstrated that non-polarized fibroblastic cells maintain the domain structure corresponding to the apical and basolateral cell surface domains in polarized epithelial cells.21 It has been reported that the cytoplasmic region of the pIgR contains functional signals.14–18 For instance, rabbit pIgR lacking the cytoplasmic region could not be endocytosed as efficiently as wild-type pIgR.14 In our experimental system, the ΔCP mutant was released much faster than the wild-type pIgR. After 16 hr of chase, intracellular wild-type pIgR was detected as two different molecular weight bands, which corresponded to the ER and Golgi forms, respectively. On the other hand, we could not detect any bands in the cell lysates of the ΔCP mutant. These results are consistent with the above concept. Namely, the ΔCP mutant cannot be internalized efficiently and as a result it is expressed on the cell membrane much longer. Consequently, the surface-expressed pIgR can interact more with the proteinase and more is released. In addition, in the cell lysate of the ΔCP mutant, the ER form of the pIgR molecule was completely absent, indicating that the cytoplasmic region of the pIgR might be important for the retention of the molecule in the ER. Conversely, the cytoplasmic region of the pIgR might be able to control the exocytotic rate by binding to some molecule which plays an important role for ER-Golgi transport. Although the precise mechanisms are still obscure, the cytoplasmic tail of the membrane protein has been demonstrated to be recognized by the small GTPase, Sar1, which is the driving force behind ER–Golgi protein transport.22 In fact, the pIgR cytoplasmic tail was demonstrated to interact with several proteins.23,24 In particular, binding to the small GTPase, Rab3b, is likely to be important for controlling pIgR trafficking.23 The contribution of the cytoplasmic region of pIgR to the ER-Golgi transport process is under investigation.

Although the C-terminal end of the human free SC has been reported25 the precise cleavage site of the pIgR is still unclear and several different susceptible sites have been proposed.19,26 All of them are contained within the sixth domain and the amino acid sequence of the most probable site is conserved among several mammalian species.6 If proteolytic cleavage of the pIgR takes place in this region, the release of the extracellular portion of pIgR would be completely abolished when the sixth domain is deleted (ΔCL mutant). However, this was not the case in our study. Surprisingly, even deleting the entire amino acid sequence of the sixth domain did not result in complete lack of the mutant SC secretion into the culture supernatant. A possible explanation for this may be that, in addition to the cleavage site used in wild type pIgR, the pIgR protein contains other cleavage-susceptible sites. There might also be a possibility that the release of the extracellular portion of ΔCL mutant might be due to unnatural proximity of domain 5 to the plasma membrane and the misfolding of the mutant protein exposing new sites for proteinase attack. Moreover, if the other cleavage sites exist, we could detect the different-sized SC bands in culture supernatant of wild type pIgR transfectant. However, we could only detect a single band. It might be possible that the molecular weights of the extra SC bands are close to the wild type free SC and these bands could not be distinguished from wild type free SC.

In addition to the expected size of the mutant SC a larger molecule, which is of approximately the same molecular weight as the full length ΔCL mutant, was also found in the culture supernatant. The nature of this molecule is not known.

Using mass spectrometric methods, it has been reported that human free SC collected from breast milk was composed of 585 amino acid residues of the pIgR.25 Although these investigators considered the effect of exopeptidase, their results seem to contradict our data. In our study, however, the secreted mutant SC was directly immunoprecipitated from the supernatant and was not processed for purification. Moreover, even after the susceptible region was molecularly deleted, we still found protein which was immunologically recognized as mutant free SC. Our experimental system may directly demonstrate the existence of extra cleavage sites in the pIgR molecule.

Proteolytic cleavage of the extracellular portion of the transmembrane protein has been reported in several different proteins27–29 the release of which is biologically important. The interleukin-6 (IL-6) receptor is an extensively studied membrane protein. Soluble IL-6 receptor–IL-6 complexes are known to have both agonistic and antagonistic effects. The pIgR can be transcytosed to the apical surface without pIgs and proteolytically cleaved and released as free SC. Biological activity of free SC in the external fluids has been reported5,30,31 and the identification of the proteolytic enzymes of pIgR will contribute to the further understanding of its biological function.

Acknowledgments

This work was supported by Research Grants from Nihon University Research Grant for Assistants, and from Uemura fund, Nihon University School of Dentistry, as well as a grant for promotion of multidisciplinary research projects and a grant from the Ministry of Education, Culture, Sports, Science, and Technology, Japan.

We are grateful to Dr P. Brandtzaeg for providing us with a human pIgR cDNA.

Abbreviations

pIgR

polymeric immunoglobulin receptor

BHK

baby hamster kidney

SC

secretory component

ER

endo-plasmic reticulum

pIgs

polymeric immunoglobulins

References

  • 1.Kraehenbuhl JP, Neutra MR. Molecular and cellular basis of immune protection of mucosal surfaces. Physiol Rev. 1992;72:853–9. doi: 10.1152/physrev.1992.72.4.853. [DOI] [PubMed] [Google Scholar]
  • 2.Lamm ME, Nedrud JG, Kaetzel CS, Mananec MB. IgA and mucosal defense. APMIS. 1995;103:241–6. doi: 10.1111/j.1699-0463.1995.tb01101.x. [DOI] [PubMed] [Google Scholar]
  • 3.Brandtzaeg P. Molecular and cellular aspects of the secretory immunoglobulin system. APMIS. 1995;103:1–19. doi: 10.1111/j.1699-0463.1995.tb01073.x. [DOI] [PubMed] [Google Scholar]
  • 4.Mostov KE. Transepithelial transport of immunoglobulins. Annu Rev Immunol. 1994;12:63–84. doi: 10.1146/annurev.iy.12.040194.000431. [DOI] [PubMed] [Google Scholar]
  • 5.Phalipon A, Corthesy B. Novel function of the polymeric Ig receptor: well beyond transport of immunoglobulins. Trends Immunol. 2003;24:55–8. doi: 10.1016/s1471-4906(02)00031-5. [DOI] [PubMed] [Google Scholar]
  • 6.Piskurich JF, Blanchard MH, Youngman KR, France JA, Kaetzel CS. Molecular cloning of the mouse polymeric Ig receptor. Functional regions of the molecule are conserved among five mammalian species. J Immunol. 1995;154:1735–47. [PubMed] [Google Scholar]
  • 7.Krajci P, Solberg R, Sandberg M, Øyen O, Jahnsen T, Brandtzaeg P. Molecular cloning of the human transmembrane secretory component (poly-Ig receptor) and its mRNA expression in human tissues. Biochem Biophys Res Commun. 1989;158:783–9. doi: 10.1016/0006-291x(89)92790-3. [DOI] [PubMed] [Google Scholar]
  • 8.Fuerst TR, Niles EG, Studier FW, Moss B. Eukariotic transient-expression system based on recombinant vaccinia virus that synthesizes bacteriophage T7 RNA polymerase. Proc Natl Acad Sci USA. 1986;83:8122–6. doi: 10.1073/pnas.83.21.8122. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Tisdale EJ, Bourne JR, Khosravi-Far RCJ, Balch WE. GTP-binding mutants of Rab 1 and Rab 2 are potent inhibitors of vesicular transport from the endoplasmic reticulum to the Golgi complex. J Cell Biol. 1992;119:749–61. doi: 10.1083/jcb.119.4.749. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Dascher C, Balch WE. Dominant inhibitory mutants of Arf 1 block endoplasmic reticulum to Golgi transport and trigger disassembly of the Golgi apparatus. J Biol Chem. 1994;269:1437–48. [PubMed] [Google Scholar]
  • 11.Matsumoto N, Asano M, Ogura Y, et al. Release of non-glycosylated polymeric immunoglobulin receptor protein. Scand J Immunol. 2003;58:471–6. doi: 10.1046/j.1365-3083.2003.01325.x. [DOI] [PubMed] [Google Scholar]
  • 12.Musil LS, Baenziger JU. Cleavage of membrane secretory component to soluble secretory component occurs on the cell surface of rat hepatocyte monolayers. J Cell Biol. 1987;104:1725–33. doi: 10.1083/jcb.104.6.1725. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Breitfeld PP, Harris JM, Mostov KE. Postendocytotic sorting of the ligand for the polymeric immunoglobulin receptor in Madin-Darby canine kidney cells. J Cell Biol. 1989;109:475–86. doi: 10.1083/jcb.109.2.475. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Mostov KE, de Bruyn Kops A, Deitcher DL. Deletion of the cytoplasmic domain of the polymeric immunoglobulin receptor prevents basolateral localization and endocytosis. Cell. 1986;47:359–64. doi: 10.1016/0092-8674(86)90592-1. [DOI] [PubMed] [Google Scholar]
  • 15.Casanova J, Apodaca G, Mostov KE. An autonomous signal for basolateral sorting in the cytoplasmic domain of the polymeric immunoglobulin receptor. Cell. 1991;66:65–75. doi: 10.1016/0092-8674(91)90139-p. [DOI] [PubMed] [Google Scholar]
  • 16.Aroeti BP, Kosen A, Kuntz ID, Cohen FE, Mostov KE. Mutational and secondary structural analysis of the basolateral sorting of the polymeric immunoglobulin receptor. J Cell Biol. 1993;123:1149–60. doi: 10.1083/jcb.123.5.1149. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Aroeti B, Mostov KE. Polarized sorting of the polymeric immunoglobulin receptor in the exocytic and endocytic pathways is controlled by the same amino acids. EMBO J. 1994;15:2297–304. doi: 10.1002/j.1460-2075.1994.tb06513.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Mostov KE, Breitfeld P, Harris JM. An anchor-minus form of the polymeric immunoglobulin receptor is secreted predominantly apically in Madin-Darby canine kidney cells. J Cell Biol. 1987;105:2031–6. doi: 10.1083/jcb.105.5.2031. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Eiffert HE, Quentin J, Decker RN, Hillemeir S, Hufschmidt M, Klingmuller D, Weber MH, Hilschmann N. The primary structure of the human secretory component and the arrangement of the disulfide bonds. Hoppe-Seyler's Z Physiol Chem. 1984;365:1489–95. [PubMed] [Google Scholar]
  • 20.Deitcher DL, Neutra MR, Mostov KE. Functional expression of the polymeric immunoglobulin receptor from cloned cDNA in fibroblast. J Cell Biol. 1986;102:911–9. doi: 10.1083/jcb.102.3.911. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Yoshimori T, Keller P, Roth MG, Simons K. Different biosynthetic transport routes to the plasma membrane in BHK and CHO cells. J Cell Biol. 1996;133:247–56. doi: 10.1083/jcb.133.2.247. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Nuoffer C, Balch WE. GTPases: multifunctional molecular switches regulating vesicular traffic. Annu Rev Biochem. 1994;63:949–90. doi: 10.1146/annurev.bi.63.070194.004505. [DOI] [PubMed] [Google Scholar]
  • 23.van Ijzendoorn S, Tuvim MJ, Weimbs T, Dickey BF, Mostov KE. Direct interaction between Rab3b and the polymeric immunoglobulin receptor controls ligand-stimulated transcytosis in epithelial cells. Dev Cell. 2002;2:219–28. doi: 10.1016/s1534-5807(02)00115-6. [DOI] [PubMed] [Google Scholar]
  • 24.Chapin SJ, Enrich C, Aroeti B, Havel RJ, Mostov KE. Calmodulin binds to the basolateral targeting signal of the polymeric immunoglobulin receptor. J Biol Chem. 1996;271:1336–42. doi: 10.1074/jbc.271.3.1336. [DOI] [PubMed] [Google Scholar]
  • 25.Hughes GJ, Frutiger S, Savoy LA, Reason AJ, Morris HR, Jaton JC. Human secretory component is composed of the first 585 amino acid residues of the polymeric immunoglobulin receptor. FEBS Lett. 1997;410:443–6. doi: 10.1016/s0014-5793(97)00629-7. [DOI] [PubMed] [Google Scholar]
  • 26.Kuhn LC, Kocher HP, Hanly WC, Cook L, Jaton JC, Kraehenbuhl JP. Structural and genetic heterogeneity of the receptor mediating translocation of immunoglobulin A dimer antibodies across epithelia in the rabbit. J Biol Chem. 1983;258:6653–9. [PubMed] [Google Scholar]
  • 27.Hooper NM, Karran EH, Turner AJ. Membrane protein secretases. Biochem J. 1997;321:265–79. doi: 10.1042/bj3210265. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Schlondorff J, Blobel CP. Metalloprotease-disintegrins: modular proteins capable of promoting cell–cell interactions and triggering signals by protein-ectodomain shedding. J Cell Sci. 1999;112:3603–17. doi: 10.1242/jcs.112.21.3603. [DOI] [PubMed] [Google Scholar]
  • 29.Rose-John S, Heinrich PC. Soluble receptors for cytokines and growth factors. generation and biological function. Biochem J. 1994;300:281–90. doi: 10.1042/bj3000281. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Hammerschmidt S, Talay SR, Brandtzaeg P, Chhatwal GS. SpsA, a novel pneumococcal surface protein with specific binding to secretory immunoglobulin A and secretory component. Mol Microbiol. 1997;25:1113–24. doi: 10.1046/j.1365-2958.1997.5391899.x. [DOI] [PubMed] [Google Scholar]
  • 31.Giugliano LG, Ribeiro STG, Vainstein MH, Ulhoa CJ. Free secretory component and lactoferrin of human milk inhibit the adhesion of enterotoxigenic Esherichia coli. J Med Microbiol. 1995;42:3–9. doi: 10.1099/00222615-42-1-3. [DOI] [PubMed] [Google Scholar]

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