Identification of a polymeric Ig receptor binding phage-displayed peptide that exploits epithelial transcytosis without dimeric IgA competition

The polymeric Ig receptor (pIgR), also called membrane secretory component (SC), mediates epithelial transcytosis of polymeric immunoglobulins (pIgs). J Chain-containing polymeric IgA (pIgA) and pentameric IgM bind pIgR at the basolateral epithelial surface. After transcytosis, the extracellular portion of the pIgR is cleaved at the apical side, either complexed with pIgs as bound SC or unoccupied as free SC. This transport pathway may be exploited to target bioactive molecules to the mucosal surface. To identify small peptide motifs with specific affinity to human pIgR, we used purified free SC and selection from randomized, cysteine-flanked 6- and 9-mer phage-display libraries. One of the selected phages, called C9A, displaying the peptide CVVWMGFQQVC, showed binding both to human free SC and SC complexed with pIgs. However, the pneumococcal surface protein SpsA (Streptococcus pneumoniae secretory IgA-binding protein), which binds human SC at a site distinct from the pIg binding site, competed with the C9A phage for binding to SC. The C9A phage showed greatly increased transport through polarized Madin-Darby canine kidney cells transfected with human pIgR. This transport was not affected by pIgA nor did it inhibit pIgR-mediated pIgA transcytosis. A free peptide of identical amino acid sequence as that displayed by the C9A phage inhibited phage interaction with SC. This implied that the C9A peptide sequence may be exploited for pIgR-mediated epithelial transport without interfering with secretory immunity.     

Secretory antibody formation: conserved binding interactions between J chain and polymeric Ig receptor from humans and amphibians

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

Synergistic effect of IL-4 and IFN-gamma on the expression of polymeric Ig receptor (secretory component) and IgA binding by human epithelial cells

The expression of secretory component (SC), the epithelial receptor for polymeric Ig, was enhanced by the addition of human rIFN-gamma or rIL-4, as revealed by the binding of radiolabeled polymeric, J chain-containing IgA or anti-SC antisera to the human colonic adenocarcinoma epithelial cell line HT-29. In combination, these cytokines exhibited a synergistic effect, and the potentiating effect of IL-4 was inhibitable by polyclonal anti-IL-4 antisera. Because the binding of radiolabeled polymeric IgA (pIgA) to HT-29 cells was inhibited by unlabeled pIgA or a polyclonal anti-SC reagent, but not by IgG, monomeric IgA, or Fab alpha fragments, we conclude that the receptor involved in the increased binding of pIgA is indeed SC. These data suggest that the expression of SC on human epithelial cells and the subsequent binding of pIgA (produced in mucosal tissues and glands by subepithelial plasma cells) is regulated by lymphokines such as IL-4 and IFN-gamma that are presumably derived from T cells found in abundant numbers in these tissues. These findings demonstrate a novel pathway of interaction between T cell products and epithelial cells that may result in enhanced translocation of large amounts of locally produced pIgA through epithelial cells into external secretions.     

Cellular location of the cleavage event of the polymeric immunoglobulin receptor and fate of its anchoring domain in the rat hepatocyte.

Transcytosis of polymeric immunoglobulin (pIg) across glandular and mucosal epithelia is mediated by a member of the immunoglobulin supergene family, the pIg receptor. During transcellular routing, the receptor is cleaved and its ectoplasmic domain, known as secretory component (SC), is released into secretions bound to pIg. Using receptor-domain-specific antibodies, we have combined cell fractionation and immunoblotting of rat liver to examine the cellular routing of the receptor, the cellular location of the cleavage event and the fate of the anchor domain. Cleavage is a late event in receptor processing. It appears to occur at the canalicular plasma membrane, since intact receptor is present in this membrane domain and no SC is detected in whole liver homogenate or in cell fractions. The membrane anchor remaining after cleavage can be recovered in bile, as well as in a low-density fraction obtained after equilibrium centrifugation of liver (microsomal fractions) on sucrose density gradients. These data suggest that the membrane-anchor domain may be internalized as well as secreted together with SC into bile.     

Solution structure of human secretory component and implications for biological function

Secretory component (SC) in association with polymeric IgA (pIgA) forms secretory IgA, the major antibody active at mucosal surfaces. SC also exists in the free form, with innate-like neutralizing properties against pathogens. Free SC consists of five glycosylated variable (V)-type Ig domains (D1-D5), whose structure was determined by x-ray and neutron scattering, ultracentrifugation, and modeling. With a radius of gyration of 3.53-3.63 nm, a length of 12.5 nm, and a sedimentation coefficient of 4.0 S, SC possesses an unexpected compact structure. Constrained scattering modeling based on up to 13,000 trial models shows that SC adopts a J-shaped structure in which D4 and D5 are folded back against D2 and D3. The seven glycosylation sites are located on one side of SC, leaving known IgA-binding motifs free to interact with pIgA. This work represents the first analysis of the three-dimensional structure of full-length free SC and paves the way to a better understanding of the association between SC and its potential ligands, i.e. pIgA and pathogenic-associated motifs.     

Characterization of a critical binding site for human polymeric Ig on secretory component

Secretory component (SC) is an integral membrane glycoprotein of secretory epithelial cells which is responsible for the specific transport of polymeric Ig (PIg) to external mucosal surfaces. The ectoplasmic segment which binds polymeric Ig is comprised of five Ig-type domains. Chemically and enzymatically modified forms of the ectoplasmic portion of SC (FSC) were produced and tested for their ability to bind to PIgA and PIgM. Deglycosylated FSC bound specifically to PIg, indicating that N-linked carbohydrate moieties on FSC are not required for binding. Denatured, reduced, and alkylated FSC did not bind to PIgA, and bound to PIgM with significantly reduced affinity, suggesting that native conformation of the polypeptide backbone of SC was important to binding. Tryptic fragments of FSC which bound to PIg were isolated and identified to be derived from domain I of SC. Synthetic peptides comprising overlapping portions of domain I bound to PIg to varying degrees. The strongest affinity was demonstrated by a peptide comprised of residues 15 to 37 of SC. A comparison of the amino acid sequences of human, rabbit, and rat SC indicated that this region contained a high degree of residue identity (78%) and may represent a consensus sequence for binding of FSC to PIg. Importantly, the peptide comprised of residues 15 to 37 was also recognized by a monoclonal antibody, 6G11, which inhibited the binding of FSC to PIgA. These results demonstrate that the binding of human SC to PIg is critically dependent on a highly conserved peptide region within the first domain of SC centering at residues 15-37.     

Probing the topography of free and polymeric Ig-bound human secretory component with monoclonal antibodies.

Secretory component (SC), an integral membrane protein expressed on basolateral surfaces of secretory epithelial cells, mediates the transport of polymeric Ig (PIg) into external secretions. The ectoplasmic segment of SC is released into secretions either in a free form (FSC) or bound to PIg as secretory IgA or IgM. The topography of human SC in its free and PIgA-bound form was studied by using mAb directed against each form of SC. Competition experiments identified a minimum of nine SC epitopes, one of which was dependent on an N-glycosidic moiety. Three of the polypeptide-derived epitopes were displayed on denatured, reduced, and alkylated SC, whereas the others were fully or partially dependent on the native conformation of SC. Epitopes recognized by the latter class of antibodies were mapped to discrete domains of SC, based on amino acid sequence and antibody-binding analysis of limited proteolytic fragments. One of the mAb (6G11), which was directed against an epitope on domain I of SC, inhibited the binding of FSC to PIgA. Overall, our results provide evidence that a region within domain I, as well as protease-sensitive interdomain regions of FSC, become masked or altered when SC binds to PIgA. Furthermore, the binding of SC to PIgA results in conformational changes, or formation of combinatorial epitopes, involving regions within domains II and III of SC but not domain V.     

In vitro refolding of recombinant human free secretory component using equilibrium gradient dialysis

Human secretory component (SC) is associated with secretory immunoglobulins (IgA and IgM) and serves to protect the immunoglobulin in the harsh mucosal environment. SC is derived from the polymeric immunoglobulin receptor (pIgR) which transports polymeric immunoglobulins across epithelial cells into secretions. In this present study, we describe the first cloning, expression, in vitro refolding and purification of a free form of human secretory component (rSC) containing the five functional ligand binding domains using Escherichia coli BL21 (DE3). Free rSC was refolded from inclusion bodies by equilibrium dialysis after purification by nickel affinity chromatography under denaturing conditions. Refolded rSC was purified by gel filtration chromatography. Surface plasmon resonance and dot blot association analysis have shown that purified rSC binds IgM with a physiological equilibrium dissociation constant (KD) of 4.6x10(-8) M and shares structural similarity to native SC. This provides an important step in the elucidation of the structure of this immunologically important receptor.     

Direct interaction between Rab3D and the polymeric immunoglobulin receptor and trafficking through regulated secretory vesicles in lacrimal gland acinar cells

The lacrimal gland is responsible for tear production, and a major protein found in tears is secretory component (SC), the proteolytically cleaved fragment of the extracellular domain of the polymeric Ig receptor (pIgR), which is the receptor mediating the basal-to-apical transcytosis of polymeric immunoglobulins across epithelial cells. Immunofluorescent labeling of rabbit lacrimal gland acinar cells (LGACs) revealed that the small GTPase Rab3D, a regulated secretory vesicle marker, and the pIgR are colocalized in subapical membrane vesicles. In addition, the secretion of SC from primary cultures of LGACs was stimulated by the cholinergic agonist carbachol (CCH), and its release rate was very similar to that of other regulated secretory proteins in LGACs. In pull-down assays from resting LGACs, recombinant wild-type Rab3D (Rab3DWT) or the GDP-locked mutant Rab3DT36N both pulled down pIgR, but the GTP-locked mutant Rab3DQ81L did not. When the pull-down assays were performed in the presence of guanosine-5'-(gamma-thio)-triphosphate, GTP, or guanosine-5'-O-(2-thiodiphosphate), binding of Rab3DWT to pIgR was inhibited. In blot overlays, recombinant Rab3DWT bound to immunoprecipitated pIgR, suggesting that Rab3D and pIgR may interact directly. Adenovirus-mediated overexpression of mutant Rab3DT36N in LGACs inhibited CCH-stimulated SC release, and, in CCH-stimulated LGACs, pull down of pIgR with Rab3DWT and colocalization of pIgR with endogenous Rab3D were decreased relative to resting cells, suggesting that the pIgR-Rab3D interaction may be modulated by secretagogues. These data suggest that the novel localization of pIgR to the regulated secretory pathway of LGACs and its secretion therefrom may be affected by its novel interaction with Rab3D.     

Functional expression of the polymeric immunoglobulin receptor from cloned cDNA in fibroblasts

The polymeric immunoglobulin receptor, a transmembrane protein, is made by a variety of polarized epithelial cells. After synthesis, the receptor is sent to the basolateral surface where it binds polymeric IgA and IgM. The receptor-ligand complex is endocytosed, transported across the cell in vesicles, and re-exocytosed at the apical surface. At some point the receptor is proteolytically cleaved so that its extracellular ligand binding portion (known as secretory component) is severed from the membrane and released together with the polymeric immunoglobulin at the apical surface. We have used a cDNA clone coding for the rabbit receptor and a retroviral expression system to express the receptor in a nonpolarized mouse fibroblast cell line, psi 2, that normally does not synthesize the receptor. The receptor is glycosylated and sent to the cell surface. The cell cleaves the receptor to a group of polypeptides that are released into the medium and co-migrate with authentic rabbit secretory component. Cleavage and release of secretory component do not depend on the presence of ligand. The cells express on their surface 9,600 binding sites for the ligand, dimeric IgA. The ligand can be rapidly endocytosed and then re-exocytosed, all within approximately 10 min. Very little ligand is degraded. At least some of the ligand that is released from the cells is bound to secretory component. The results presented indicate that we have established a powerful new system for analyzing the complex steps in the transport of poly-Ig and the general problem of membrane protein sorting.     

The J chain is essential for polymeric Ig receptor-mediated epithelial transport of IgA.

Local production of secretory (S)IgA provides adaptive immunologic protection of mucosal surfaces, but SIgA is also protective when administered passively, such as in breast milk. Therefore, SIgA is a potential candidate for therapeutic administration, but its complex structure with four different polypeptide chains produced by two distinct cell types complicates recombinant production. The J chain is critical in the structure of SIgA because it is required for efficient polymerization of IgA and for the affinity of such polymers to the secretory component (SC)/polymeric (p)IgR. To better understand the role of the J chain in SIgA production, we have generated various mutant forms of the human J chain and analyzed the function of these mutants when coexpressed with IgA. We found that the C terminus of the J chain was not required for the formation of IgA polymers, but was essential for the binding of pIgA to SC. Likewise, we found that two of the intrachain disulfide bridges (Cys(13):Cys(101) and Cys(109):Cys(134)) were also required for the binding of pIgA to SC but, interestingly, not for IgA polymerization. Conversely, the last intrachain disulfide bridge (Cys(72):Cys(92)) was not essential for either of these two J chain functions. Finally, we demonstrated that the presence of only Cys(15) or Cys(69) was sufficient to support polymerization of IgA, but that these polymers were mostly noncovalently stabilized. Nevertheless, these polymers bound free SC with nearly the same affinity as pIgA containing wild-type J chain, but were transcytosed by pIgR-expressing polarized epithelial cells at a reduced efficiency.     

Effect of a disulfide-interchange enzyme on the assembly of human secretory immunoglobulin A from immunoglobulin A and free secretory component.

A disulfide-interchange enzyme from rat liver microsomes was found to promote binding in vitro of human free secretory component (SC) to dimeric serum-type IgA containing J chain, as assessed by immune precipitation and gel filtration. This effect was greater withe native than with partially reduced SC. Most of the bound SC was covalently linked, as determined by electrophoresis in polyacrylamide gels in detergent. The enzyme did not promote binding of native or partially reduce SC to IgG, IgA monomer, IgA dimer without J chain, or IgM. In the case of IgM, the enzyme did, however, promote covalent bonding of previously non-covalently linked SC. The results overall suggest that a disulfide-interchange enzyme could play a role in vivo in the cell-associated assembly of secretory IgA by promoting the covalent attachment of SC to a dimer of serum-type IgA and that the J chain in the IgA dimer contributes to the enzyme effect.     

Differences in fragmentation between bound and unbound bovine secretory component suggest a model for its interaction with polymeric immunoglobulin.

Unbound bovine secretory component was cleaved into two-domain and one-domain fragments by trypsin within 1 h. Bovine secretory component covalently bound to bovine IgA dimer, as in secretory IgA, was much more resistant to fragmentation, which did not proceed beyond the three-domain stage even after 5 h. Bovine secretory component non-covalently bound to bovine IgM or to human IgM or IgA polymer was also relatively resistant to fragmentation, which again was largely arrested at the three-domain stage. A model for the binding of secretory component to polymeric immunoglobulin is proposed.     

Erythrocyte membrane-associated immunoglobulins during malaria infection of mice.

Immunoglobulin (Ig) is associated with erythrocyte membranes during infection of A/J mice with Plasmodium berghei. It was detected by agglutination of the cells with rabbit antibodies to mouse IgG and by a radioimmunoassay with 125I-labelled rabbit antibodies to mouse IgG. As shown by the degree of agglutination and of binding of radiolabeled antibodies to the erythrocyte membranes, the amount of Ig increased with time after infection and paralleled parasitemia and reticulocytosis. The erythrocyte-associated immunoglobulins are mainly IgG but IgM was also present on the cells of some mice. A large proportion of the Ig could be eluted at 37 degrees C and was analyzed by immunoprecipitation and acrylamide gel electrophoresis. A radioautographic study with 125I-labeled anti-mouse IgG revealed that both parasitized and nonparasitized reticulocytes of infected mice had much larger amounts of membrane-bound immunoglobulin than did mature, nonparasitized erythrocytes. The nature of the bonds between the Ig and the surface membrane of the reticulocytes is not known. The Ig could be part of immune complexes nonspecifically bound to the cell surface. However, since we have not detected Fc or C3d receptors on reticulocytes, it is possible that the Ig constitutes autoantibodies against reticulocytes or antibodies against parasitic antigens present on the cell membrane.     

IgM expressed by leukemic CD5(+) B cells binds mouse immunoglobulin light chain

Mouse immunoglobulin (Ig) molecules have previously been shown to bind to the surface of CD5(+) B cells from patients with B-cell chronic lymphocytic leukemia (B-CLL). The results indicated that surface IgM was involved in the interaction and suggested the phenomenon was an example of the polyreactive binding capacity of the surface Ig (sIg) expressed by these malignant cells. This article describes the further characterization of the interaction between human IgM and mouse Ig molecules and subunits. Mouse Ig molecules of both kappa and lambda light chain classes bound to the B-CLL cell surface. The dissociation constant for the interaction of mouse IgG1 (K121) with the B-CLL cell surface was 3.6 x 10(-7) M. To confirm the involvement of the human IgM expressed by the B-CLL cells in the interaction, the malignant cells were stimulated in vitro to induce secretion of human IgM. Enzyme immunoassay was used to show that secreted human IgM bound to intact mouse Ig, as occurred with the cell surface analysis. The mouse Ig epitope recognized by the purified secreted human IgM was shown by Western blot analysis to be located on the light chain of the mouse Ig molecule and to be conformationally dependent. K121 light chain was cloned and expressed in E. coli and the recombinant light chain bound to the surface of CLL B cells. The results confirm that human IgM is the reactive ligand in the interaction with mouse Ig and indicate that the interaction of polyreactive IgM with mouse IgG occurs via the light chain component of IgG.     

Ectodomains 3 and 4 of human polymeric Immunoglobulin receptor (hpIgR) mediate invasion of Streptococcus pneumoniae into the epithelium

Streptococcus pneumoniae binds to the ectodomain of the human polymeric Ig receptor (pIgR), also known as secretory component (SC), via a hexapeptide motif in the choline-binding protein SpsA. The SpsA-pIgR interaction mediates adherence and internalization of the human pathogen into epithelial cells. In this study the results of SpsA binding to human, mouse, and chimeric SC strongly supported the human specificity of this unique interaction and suggested that binding sites in the third and fourth Ig-like domain of human SC (D3 and D4, respectively) are involved in SpsA-pIgR complex formation. Binding of SpsA to SC-derived synthetic peptides indicated surface-located potential binding motifs in D3 and D4. Adherence and uptake of pneumococci or SpsA-coated latex beads depended on the SpsA hexapeptide motif as well as SpsA-binding sites in D3 and D4 of human pIgR. The involvement of D3 and D4 in adherence and invasion was demonstrated by the lack of binding of SpsA-coated latex beads to transfected epithelial cells expressing mutated pIgR. Finally, blocking experiments with chimeric human-mouse SC as well as synthetic peptides indicated the participation of D3 and a key role of D4 in pneumococcal invasion.     

猕猴和小鼠分泌片的分离纯化及某些生化特性

为了深入了解分泌片和ＩｇＡ在粘膜免疫系统中的作用,自猕猴和小鼠胆汁中提取和纯化分泌片,ＳＤＳ－ＰＡＧＥ结果表明猕猴和小鼠游离分泌片的分子量均约为６０ｋＤａ,但在非解聚型聚丙烯酰胺梯度凝胶电泳时分子量分别为７４ｋＤａ和６２ｋＤａ。采用ＬＫＢ－８１００等电聚焦柱测定其ＰＩ范围,猕猴分泌片为４．３－５．９,小鼠分泌为３．９－５．４。免疫双扩散证明,猕猴和小鼠胆汁中的分泌片均能与兔抗人分泌片免疫血清发生交     

A novel IgA receptor expressed on a murine B cell lymphoma.

The specificity and properties of a novel IgA receptor expressed on the surface of a tissue culture-adapted B cell lymphoma, T560, that originated in murine gut-associated lymphoid tissue, have been explored. Like the IgA receptors of murine T and splenic B cells studied by others, the T560 IgA receptor is trypsin sensitive and neuraminidase resistant and is up-regulated on T560 cells by exposing them overnight to high concentrations of polymeric IgA. Unlike them, the T560 IgA receptor is inhibited by low concentrations of IgM and high concentrations of IgG2a and IgG2b, binds at pH 4.0 but not at pH 8.0, is down-regulated by activation of protein kinase C and is sensitive to phosphatidylinositol-specific phospholipase C, indicating that it is glycosyl phosphatidylinositol-linked to the cell membrane. It is not a cell-bound form of galactosyl transferase, does not appear to bind to Ig through carbohydrate residues and does not react specifically with antibody to secretory component. It may be a completely new, cross-reactive receptor, perhaps related in some way to the polymeric Ig receptor or to the receptor for IgA expressed on the apical surface of Peyer's patch M cells, which is known to cross-react with IgG. Alternatively, it may be homologous to the highly IgA-specific Fc alpha R of T cells but, perhaps because of its glycosyl phosphatidylinositol linker, may have an ability to move and interact with other Ig receptors on the cell surface such that Ig bound to them are cross-inhibitory.     

Inhibition of natural killer cell activity by IgA

The in vitro effect of IgA on natural killer (NK) activities of human peripheral blood mononuclear cells was investigated. Purified myeloma polymeric IgA2 (pIgA2) and secretory IgA (S-IgA) from human colostrum inhibited NK activity, while myeloma polymeric IgA1 (pIgA1), monomeric IgA1 (mIgA1), IgG, and IgM were ineffective. Inhibition was proportional to the concentration of pIgA2 (0.125-1 mg/ml) and was observed after as little as 1 hr of incubation at various effector to K562 target cell ratios. pIgA2 and S-IgA also inhibited NK activity of NK cell-enriched lymphoid cells and gamma-interferon-treated effector cells, but did not interfere with effector-target cell binding. The inhibitory effect was slightly diminished after 24 hr culture in pIgA2-free medium. Inhibition of cytotoxicity was not due to direct toxicity on lymphoid cells by IgA because PBL treated with pokeweed mitogen in the presence of pIgA2 or S-IgA differentiated into immunoglobulin-producing cells. Viability after 24 hr of preculture with pIgA2 and S-IgA was comparable to that of untreated control cells. Morphological examination of effector cells cultured with pIgA2 or S-IgA showed a decrease in the number of granules, and the formation of cytoplasmic vacuoles. These morphological changes appeared to coincide with the depressed cytotoxicity of NK cells. The results demonstrate that purified pIgA2 and S-IgA have significant immunomodulatory effects on human NK activity.