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.     

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.     

Genetic mapping of the human polymeric immunoglobulin receptor gene to chromosome region 1q31----q41

Polymeric immunoglobulin receptor (PIGR) is a transmembrane glycoprotein which is expressed by epithelial cells and is involved in the transcellular transport of polymeric immunoglobulins into secretions. We cloned the human gene for PIGR and used the clone to obtain probes to determine the chromosomal localization of PIGR. Analysis of somatic cell hybrids and in situ chromosomal hybridization localized the human PIGR gene locus to 1q31----q41.     

IgA:IgM and IgA:IgA hybrid hybridomas secrete heteropolymeric immunoglobulins that are polyvalent and bispecific

Polyvalent bispecific antibodies were secreted by hybrid hybridoma cells when both parental clones expressed a naturally polymerizing immunoglobulin. Hybrid hybridomas made from IgA lambda 2 anti-trinitrophenyl (TNP) and IgA kappa anti-phosphocholine (PC) parental cells secreted polymeric IgA antibodies that bound both TNP and PC. Some of the TNP binding was dissociated from the PC binding under conditions of mild reduction and alkylation suggesting that the bispecific polymeric IgA contained disulfide-linked parental monomers as well as bispecific hybrid monomers. Hybrid hybridomas constructed from IgA lambda 2 anti-TNP and IgM kappa anti-ox erythrocyte parental cells secreted bispecific, polymeric immunoglobulin that contained mu-, alpha-, kappa-, and lambda 2-chains. The mu and kappa-chains dissociated from the alpha- and lambda 2-chains under conditions of mild reduction and alkylation, indicating that both parental monomers had been incorporated into the same polymeric immunoglobulin to form a heteropolymeric antibody molecule. Heterologous pairing of alpha and mu heavy chains in monomers was not detected. Hybrid hybridomas constructed from IgA lambda 2 and IgG3 lambda 2 or IgA lambda 2 and IgG1 kappa parents co-secreted both parental immunoglobulins, but the antibodies secreted by these clones did not form heteropolymers or exhibit heterologous heavy chain pairing. These findings establish that polyvalent, bispecific, polymeric immunoglobulin molecules can be produced by hybrid hybridomas when both parents express a naturally polymerizing class of heavy chain but not when only one parent does. Hybrid hybridomas that produce heteropolymeric immunoglobulins are sources of high avidity bispecific antibodies that may find a number of basic and practical applications. The hybridoma cells that produce these antibodies may provide useful tools for investigating the in situ determinants of immunoglobulin chain association and the regulation of antibody assembly and secretion.     

Intracellular routing of wild-type and mutated polymeric immunoglobulin receptor in hippocampal neurons in culture

Certain epithelial cells synthesize the polymeric immunoglobulin receptor (pIgR) to transport immunoglobulins (Igs) A and M into external secretions. In polarized epithelia, newly synthesized receptor is first delivered to the basolateral plasma membrane and is then, after binding the Ig, transcytosed to the apical plasma membrane, where the receptor-ligand complex is released by proteolytic cleavage. In a previous work (Ikonen et al., 1993), we implied the existence of a dendro-axonal transcytotic pathway for the rabbit pIgR expressed in hippocampal neurons via the Semliki Forest Virus (SFV) expression system. By labeling surface-exposed pIgR in live neuronal cells, we now show (a) internalization of the receptor from the dendritic plasma membrane to the dendritic early endosomes, (b) redistribution of the receptor from the dendritic to the axonal domain, (c) inhibition of this movement by brefeldin A (BFA) and (d) stimulation by the activation of protein kinase C (PKC) via phorbol myristate acetate (PMA). In addition, we show that a mutant form of the receptor lacking the epithelial basolateral sorting signal is directly delivered to the axonal domain of hippocampal neurons. Although this mutant is internalized into early endosomes, no transcytosis to the dendrites could be observed. In epithelial Madin-Darby Canine Kidney (MDCK) cells, the mutant receptor could also be internalized into basolaterally derived early endosomes. These results suggest the existence of a dendro-axonal transcytotic pathway in neuronal cells which shares similarities with the basolateral to apical transcytosis in epithelial cells and constitute the basis for the future analysis of its physiological role.     

Metabolic fate of cell surface glycoproteins during immunoglobulin-induced internalization

Goat antibodies directed against a subset of the externally oriented plasma membrane glycoproteins of hepatoma tissue culture (HTC) cells were used to follow the metabolic fate of the membrane antigens and the specifically bound immunoglobulin molecules in this cell type in cultures. Analyses of the immunoprecipitates from cells labeled in situ with neuraminidase and galactose oxidase, followed by reduction with tritiated sodium borohydride, indicate that about 40% of the galactose-labeled plasma membrane glycoproteins are recognized by the antiserum. Fluorescent microscopic analyses of cells treated with fluorescein-conjugated immunoglobulins and analyses of trypsin accessibility indicate that probably all of the antibodies bound to the cell surface are patched and internalized within about 4 hr when the cells are subsequently cultured at 37 degrees C in the presence of rabbit anti-goat immunoglobulins. At the same time, the antigens are also interiorized. Analyses of the cellular localization of the interiorized antigens and antibodies by cell fractionation on Percoll gradients show that the immunoglobulins to the cell surface antigens and the antigens themselves migrate to the same region of the Percoll gradient as lysosomal hydrolases. Although the antibodies bind to the cell surface glycoproteins and bring about patching and interiorization, there is no effect on the degradation of the plasma membrane antigens labeled via the galactose oxidase/borohydride reduction method. Furthermore, the iodinated antibodies directed against these membrane glycoproteins behave in their turnover properties like membrane antigens; the cell-bound specific immunoglobulins have the same half-life as the membrane glycoproteins. When the cells that had been reacted with the goat antibodies to membrane glycoprotein were cultured in the presence of rabbit anti-goat immunoglobulins, degradation of the former antibodies was effectively decreased. Similar results were obtained with concanavalin A and antibodies directed against this plant lectin.     

抗禽流感病毒H5N1 分泌型IgA 在中国仓鼠卵巢细胞中的表达

分泌型IgA (SIgA) 在机体的粘膜免疫中具有重要作用,在外分泌道中比单体IgA和IgG抗体具有更好的抗感染活性。为了表达抗禽流感病毒H5N1人-鼠嵌合分泌型IgA抗体,首先以本室先前构建的稳定表达IgA的中国仓鼠卵巢细胞 (CHO) 细胞系为基础,共转染分泌片和J链表达质粒,然后用抗生素Zeocin选择阳性克隆细胞,利用倍比稀释的方法筛选分泌SIgA的单克隆细胞,通过Western blotting分析培养上清中SIgA的表达情况。结果表明,在CHO细胞中成功表达了SIgA抗体,上述研究为研制分泌型     

Antibodies recognizing different domains of the polymeric immunoglobulin receptor

The receptor responsible for the transepithelial transport of IgA dimer antibodies is a transmembrane glycoprotein known as membrane secretory component (SCm). During transport, the membrane anchoring domain is cleaved and the ectoplasmic domain of the receptor (SCs) remains tightly bound to the IgA dimer in exosecretions. We have produced monoclonal antibodies with distinct specificities against both cytoplasmic and ectoplasmic epitopes of rabbit SCm. One antibody (anti-SC303) reacted both with SCm and free SCs but not with SCs bound to IgA dimer (SIgA). Therefore, it recognized an epitope close to the IgA dimer binding site. The other monoclonal antibody (anti-SC166), which was unable to react with SCs, bound to the 15-kDa cytoplasmic extension of the membrane-spanning domain of the receptor. A polyclonal antibody (GaR-SC), raised in a goat against rabbit milk SCs, reacted with a subpopulation of SCs not recognized by the anti-SC303 monoclonal antibody and in addition also reacted with covalently bound sIgA. The three antibodies cross-reacted with rat SCm. We demonstrate the ability of the anti-SC166 monoclonal antibody to immunoadsorb subcellular organelles as a result of the cytoplasmic orientation of its epitope. Our data indicate that there are functional differences between the high- and low-molecular-weight families of SC in terms of IgA dimer binding.     

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.     

This little pIgR went to the mucosa

In this issue, we report the structure of the terminal domain of the polymeric immunoglobulin receptor (pIgR), which mediates the "suicide" transcytosis of multimeric immunoglobulins (IgA, IgM). This assists in reconciling decades of biochemistry, revealing a long-puzzling interaction.     

The carboxyl-terminal domains of IgA and IgM direct isotype-specific polymerization and interaction with the polymeric immunoglobulin receptor

Mucosal surfaces are protected by polymeric immunoglobulins that are transported across the epithelium by the polymeric immunoglobulin receptor (pIgR). Only polymeric IgA and IgM containing a small polypeptide called the "joining" (J) chain can bind to the pIgR. J chain-positive IgA consists of dimers, and some larger polymers, whereas only IgM pentamers incorporate the J chain. We made domain swap chimeras between human IgA1 and IgM and found that the COOH-terminal domains of the heavy chains (Calpha3 and Cmu4, respectively) dictated the size of the polymers formed and also which polymers incorporated the J chain. We also showed that chimeric IgM molecules engineered to contain Calpha3 were able to bind the rabbit pIgR. Since the rabbit pIgR normally does not bind IgM, these results suggest that the COOH-terminal domain of the polymeric immunoglobulins is primarily responsible for interaction with the pIgR. Finally, we made a novel chimeric IgA immunoglobulin, containing the terminal domain from IgM. This recombinant molecule formed J chain-containing pentamers that could, like IgA, efficiently form covalent complexes with the human pIgR ectodomain, known as secretory component.     

Crystal structure of a polymeric immunoglobulin binding fragment of the human polymeric immunoglobulin receptor

The polymeric immunoglobulin receptor (pIgR) is a type I transmembrane protein that delivers dimeric IgA (dIgA) and pentameric IgM to mucosal secretions. Here, we report the 1.9 A resolution X-ray crystal structure of the N-terminal domain of human pIgR, which binds dIgA in the absence of other pIgR domains with an equilibrium dissociation constant of 300 nM. The structure of pIgR domain 1 reveals a folding topology similar to immunoglobulin variable domains, but with differences in the counterparts of the complementarity determining regions (CDRs), including a helical turn in CDR1 and a CDR3 loop that points away from the other CDRs. The unusual CDR3 loop position prevents dimerization analogous to the pairing of antibody variable heavy and variable light domains. The pIgR domain 1 structure allows interpretation of previous mutagenesis results and structure-based comparisons between pIgR and other IgA receptors.     

Expression in non-lymphoid cells of mouse recombinant immunoglobulin directed against the tumour marker human placental alkaline phosphatase

From a mouse hybridoma cell line secreting a monoclonal antibody directed against the tumour marker human placental alkaline phosphatase, mRNA coding for the H and L chains of this antibody was isolated and cloned as cDNA. Sequence analysis of the H and L chain cDNAs confirmed the IgG2b,kappa subtype previously established. Recloning the H and L chain cDNA information into SV40-based vectors enabled us to obtain expression of functional immunoglobulin upon cotransfection into COS or CHO dhfr- cells. This illustrates that non-lymphoid cells also have the capacity to assemble active immunoglobulins.     

An anchor-minus form of the polymeric immunoglobulin receptor is secreted predominantly apically in Madin-Darby canine kidney cells

The polymeric immunoglobulin receptor is expressed in a variety of polarized epithelial cells. Newly made receptor travels first to the basolateral surface. The receptor is then endocytosed, transported across the cell in vesicles, and exocytosed at the apical surface. We have now deleted the membrane spanning and cytoplasmic portions of the receptor by site-directed mutagenesis, thus converting the receptor to a secretory protein. When expressed in polarized Madin-Darby canine kidney (MDCK) cells the truncated protein is secreted at both surfaces, with a ratio of apical-to-basal secretion of 3.4. In contrast, when the exogenous secretory protein chicken lysozyme is expressed in these cells, it is released at both sides with a ratio of apical-to-basal secretion of 0.43. (Koder-Koch, C., R. Bravo, S. Fuller, D. Cutler, and H. Garoff, 1985, J. Cell Biol., 43:297-306). Lysozyme is thought to lack a signal that targets it to one surface or the other, and so its secretion may represent a default, bulk flow pathway to both surfaces. When compared with lysozyme, the truncated polymeric immunoglobulin receptor is preferentially secreted apically by a factor of 3.4:0.43 or 7.8. We suggest that the lumenal portion of the polymeric immunoglobulin receptor contains a signal that targets it to the apical surface.     

Anchoring a secreted plasmodium antigen on the surface of recombinant vaccinia virus-infected cells increases its immunogenicity.

We show that the subcellular location of foreign antigens expressed in recombinant vaccinia viruses influences their effectiveness as immunogens. Live recombinant viruses induced very poor antibody responses to a secreted repetitive plasmodial antigen (the S-antigen) in rabbits and mice. The poor response accords with epidemiological data suggesting that S-antigens are poorly immunogenic. Appending the transmembrane domain of a membrane immunoglobulin (immunoglobulin G1) to its carboxy terminus produced a hybrid S-antigen that was no longer secreted but was located on the surface of virus-infected cells. This recombinant virus elicited high antibody titers to the S-antigen. This approach will facilitate the use of live virus delivery systems to immunize against a wide range of foreign nonsurface antigens.     

Targeting of a lysosomal membrane protein: a tyrosine-containing endocytosis signal in the cytoplasmic tail of lysosomal acid phosphatase is necessary and sufficient for targeting to lysosomes.

Lysosomal acid phosphatase (LAP) is synthesized as a transmembrane protein with a short carboxy-terminal cytoplasmic tail of 19 amino acids, and processed to a soluble protein after transport to lysosomes. Deletion of the membrane spanning domain and the cytoplasmic tail converts LAP to a secretory protein, while deletion of the cytoplasmic tail as well as substitution of tyrosine 413 within the cytoplasmic tail against phenylalanine causes accumulation at the cell surface. A chimeric polypeptide, in which the cytoplasmic tail of LAP was fused to the ectoplasmic and transmembrane domain of hemagglutinin is rapidly internalized and tyrosine 413 of the LAP tail is essential for internalization of the fusion protein. A chimeric polypeptide, in which the membrane spanning domain and cytoplasmic tail of LAP are fused to the ectoplasmic domain of the Mr 46 kd mannose 6-phosphate receptor, is rapidly transported to lysosomes, whereas wild type receptor is not transported to lysosomes. We conclude that a tyrosine containing endocytosis signal in the cytoplasmic tail of LAP is necessary and sufficient for targeting to lysosomes.     

Stoichiometric binding of low density lipoprotein (LDL) and monoclonal antibodies to LDL receptors in a solid phase assay

The current paper describes a solid phase ligand binding assay for the low density lipoprotein (LDL) receptor that takes advantage of the domain structure of the protein. An antibody directed against one domain, e.g. the cytoplasmic tail, is adsorbed to a microtiter well. A detergent solution containing the LDL receptor is added, and the receptor is allowed to bind to the antibody. The wells are then washed, and one of the following radioiodinated ligands is added: 125I-LDL or an 125I-labeled monoclonal antibody directed against a different domain than the antibody adsorbed to the well. Under these conditions, the human LDL receptor shows high affinity for 125I-LDL and for 125I-IgG-HL1, a monoclonal antipeptide antibody directed against a 10-amino-acid "linker" between repeats 4 and 5 in the ligand binding domain. The binding affinity is the same at 4 degrees C and 37 degrees C. The binding of 125I-LDL and 125I-IgG-HL1 occurs with 1:1 molar stoichiometry, suggesting that the human LDL receptor binds 1 mol of LDL per mol of receptor. The acid-dependent dissociation of 125I-LDL and 125I-labeled monoclonal antibody from LDL receptors that is observed in intact cells was also shown to occur in the solid phase binding assay. We used the solid phase assay to demonstrate the secretion of LDL receptors from monkey cells that have been transfected with a cDNA encoding a truncated form of the human receptor that lacks the membrane-spanning domain. This assay may be useful in measuring the relative amounts of the intact LDL receptor in tissue extracts and the secreted receptor in transfected cells.     

Biosynthesis of immunoglobulin A (IgA) and immunoglobulin M (IgM). Requirement for J chain and a disulphide-exchanging enzyme for polymerization

Mouse myeloma cells secreting 19S IgM (immunoglobulin M) (MOPC 104E and TEPC 183) or monomer and polymer IgA (immunoglobulin A) (MOPC 315) were incubated with radioactive leucine and the intracellular and secreted immunoglobulins and immunoglobulin subunits were prepared by preparative sucrose-density-gradient centrifugation. Samples were reduced in the presence or absence of isolated J chain, passed over Sephadex G-25 and then incubated at 37 degrees C for 30min with or without a source of disulphide-interchange enzyme. The extent of reassembly of reduced subunits was then evaluated by electrophoresis in polyacrylamide gels. Provided that J chain and the disulphide-interchange enzyme were supplied, both IgM and IgA could be assembled from their respective subunits, obtained by reductive cleavage of polymeric forms. Under similar conditions, assembly of polymeric forms from intracellular or secreted 7S monomer subunits also occurred. Under these conditions polymerization was total, there being no residue of the monomeric form. Reassembly did not occur in the absence of either J chain or the enzyme. All of the J chain released from IgM by reductive cleavage was incorporated back into the reassembled polymer. The J chain is therefore likely to be an essential structural requirement for polymeric immunoglobulins. A variety of controls ruled out non-specific interactions, and further suggested that the amino acid sequence of polypeptide chains determines the specificity of polymerization. The fact that intracellular IgA and IgM monomer subunits known to be deficient in galactose and fucose can be completely polymerized suggests that the addition of carbohydrate does not control polymerization.     

Biosynthesis of the IgA antibody receptor: a model for the transepithelial sorting of a membrane glycoprotein

Secretory IgA dimer antibodies in exosecretions provide the primary immunological defense for mucosal surfaces. Transmission of IgA2 across the epithelia of mucous and exocrine glands is mediated by a receptor called secretory component (SC). Using three antibodies directed against different domains of SC, we examine its processing in the lactating rabbit mammary gland. SC is synthesized as a core glycosylated transmembrane glycoprotein on the rough endoplasmic reticulum. Pulse-chase experiments reveal the time course of SC maturation in the Golgi, as demonstrated by the acquisition of Endo H resistance (30-60 min). The subsequent routing of SC to the basolateral plasma membrane, where IgA2 binding and endocytosis occurs, the cleavage of the membrane anchoring domain of SC, and the exocytosis from the apical plasma membrane of IgA, bound to the ectoplasmic domain of SC takes place rapidly (30-60 min). Thus maturation in the Golgi may represent the rate limiting step in SC routing. We also demonstrate that SC exists in several conformational states that are processed at different rates.