Biosynthesis of J-Chain in Mouse IgA and IgM

RECENT evidence^1,2 suggests that the polymeric immunoglobulins, IgM, serum IgA and secretory IgA, but not IgG or monomeric serum IgA contain a third polypeptide chain, the J-chain, of molecular weight about 23,000¹ or 26,000². Each polymeric molecule irrespective of the number of monomeric (H₂L₂) subunits, is thought to contain only one molecule of J-chain. It is distinguishable from light chain by its amino-acid composition, tryptic peptide map and fast electrophoretic mobility on alkaline-urea-polyacrylamide gels^1,2, but J-chains isolated from IgM and secretory IgA are indistinguishable by these criteria².     

Human J-chain: isolation and molecular weight studies

J-chain, a polypeptide chain unique to polymeric immunoglobulins (Igs) which has been postulated to be responsible for joining monomeric subunits into the polymeric forms, was isolated from human IgA, secretory IgA, and IgM by 3 different procedures, disc electrophoresis, immunoadsorbent radioimmunoassay, and dialysis against distilled water. Precipitation in water was the simplest and yielded satisfactory results. Molecular weights of the various J-chain isolates were studied by using 2-species plot analysis of sedimentation equilibrium data. 2 populations of molecules were found: 1 had a molecular weight of 13,300-17,700 and the other of 6400-11,500. Variations in these data from those of other investigators are discussed in terms of isolation procedure differences.     

Humoral response of the immunologic secretory system of the small intestine in children infected with Giardia intestinalis

Local humoral response of the intestinal mucosa was determined with secretory IgA levels and secretory component activity in enterocytes and duodenal content of 15 children infected with G. intestinalis. The obtained results were compared to those in 5 children with coeliac disease and 12 children with diarrhoea without lambliasis. Secretory IgA was increased in about 50% of children with lambliasis (in the remaining groups in 25% of children) to the values higher than that in the comparative groups. Secretory component activity was relatively high in the intestinal epithelium. Secretory component activity in the duodenal content was high in about 40% of children independently of the examined group. No correlation between the said variables was noted except positive correlation of secretory IgA levels and secretory component activity in the bile.     

Problem of J-chain of immunoglobulins

Molecules of secretory immunoglobulins (Ig) of classes A and M (sIgA and sIgM) play the main role in protection of mucosae from pathogenic factors. The apparatus of synthesis of these molecules represent the most powerful part of the immune system. One of the key elements of the sIgA and sIgM is J-chain. It represents an acid polypeptide of molecular mass of about 15 kDa composed of 137 amino acid residues including 8 cysteine residues and one site of N-glycosylation. The primary structure of the J-chain is unique: attempts to ascribe it to any family of known proteins so far have failed. The J-chain is inserted into the sIgA and sIgM molecules by forming disulfide bonds with C-terminal sites of α-or μ-chains. It is necessary for formation of IgA dimers and IgM pentamers, for reception of these molecules by epithelial cells, binding of secretory component to them, and for transfer of sIgA and sIgM molecules onto mucosal surfaces and into secrets of exocrine glands. The J-chain has been revealed in the cytoplasm of the early T-and B-lymphocyte precursors not producing Ig. The J-chain is detected in the human embryonic liver cells earlier than the expression of the μ-chain gene begins. Study of mice with knockout of J-chain B-lymphocytes-producers has shown their block of functions of T-helpers providing formation of immunologic memory. Comparison of J-chain genes of mammals, amphibians, reptiles, and cartilaginous fishes has shown the degree of interspecies homology of these proteins to vary from 33% to 70%. The J-chain genes were revealed in representatives of all vertebrate classes except for cyclostomes and bony fishes. In 1996, data were published about the presence of the J-chain genes-homologs in invertebrates, tunicates, and cyclostomes. No papers reproducing or confirming these data have been published. On the contrary, in the literature an opinion appeared that indicate necessity to revise the notion about the presence of J-chain in invertebrates. The main unsolved issues on the J-chain involve the tertiary structure of this protein, its relation to some particular protein family, its functions in cells of the T-and B-lymphocytic differentiation lineages as well as its evolutionary age.     

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.     

Ultrastructural changes in the oviductal mucosa throughout the sexual cycle in Bufo arenarum

In Bufo arenarum the oviduct exhibits conspicuous changes throughout the sexual cycle. In the present study, we analyzed the optical and ultrastructural characteristics of the oviductal pars convoluta mucosa, the portion responsible for jelly secretion, during both the preovulatory and postovulatory periods. Secretory epithelial cells, ciliated cells, basal cells, and glandular cells are described. Secretory epithelial cells are characterized by the presence of secretory granules, the size, shape and electron density of which vary markedly. Their contents are mainly released by exocytosis into the oviductal lumen. Moreover, in the preovulatory period, apocrine, and holocrine secretion processes frequently occur. During the postovulatory period, these cells exhibit a marked diminution of secretory granules. Ciliated cells show a typical ultrastructural organization. Basal cells are distinguished in the lower part of the epithelium by their heterochromatic nuclei and electron‐lucent cytoplasm. These cells, to the best of our knowledge, are reported for the first time in Amphibia. Glandular cells exhibit oval, round, or polyhedric granules, most of them with one or more cores. Our results indicate that the contents of epithelial and glandular secretory cells are partially secreted during the preovulatory period. Additional secretion occurs during the transit of the oocytes. J. Morphol. 239:61–73, 1999. © 1999 Wiley‐Liss, Inc.     

Rat liver membrane secretory component is larger than free secretory component in bile: evidence for proteolytic conversion of membrane form to free form

Secretory component is a receptor for polymeric immunoglobulins on epithelial cells and hepatocytes that facilitates transport of polymeric immunoglobulins into external secretions. Little is known about the transcellular migration of secretory component-polymeric IgA complexes or the membrane forms of secretory component. We therefore examined rat bile and liver membranes to identify and compare the various molecular species of secretory component. Bile or liver membrane proteins were electrophoresed in sodium dodecyl sulfate-polyacrylamide gels and electrophoretically transferred to nitrocellulose membranes. Protein profiles on blots were probed with antisecretory component antiserum, and the immunoreactive bands were visualized by indirect immunoperoxidase staining. Bile collected in the presence of proteolytic inhibitors showed an immunoreactive doublet band (Mr = 82,000 and 78,000) in the molecular weight range of free secretory component. By contrast, free secretory component in bile collected in the absence of proteolytic inhibitors and purified by affinity chromatography migrated as a single protein with an Mr = 70,000. Both components of the free secretory component doublet bound dimeric IgA when blots were probed with human dimeric IgA. Crude liver membranes prepared in the presence of proteolytic inhibitors showed two immunoreactive secretory component-containing bands, Mr = 107,000 and 99,000, whereas membranes prepared without proteolytic inhibitors showed two smaller immunoreactive bands; one of these proteolytically severed proteins comigrated with the 82,000-dalton free secretory component in bile. These results indicate that membrane forms of secretory component are present in rat liver. The observations that the membrane secretory component is larger than biliary free secretory component and yields biliary SC-like forms of secretory component upon proteolysis support the hypothesis that free secretory component in bile is a proteolytic product of larger liver membrane-associated secretory component.     

Cloning and expression of the turtle (<Emphasis Type="Italic">Trachemys scripta</Emphasis>) immunoglobulin joining (J)-chain cDNA

The J-chain protein is a M(r) 15000 polypeptide associated with polymeric IgA and IgM. The complete cDNA sequences of human, mouse, cow, brushtail possum, chicken and frog J chains have been previously reported, but nothing is known about the cDNA and amino acid sequences of reptilian J chain. Here, we determined a turtle J-chain cDNA sequence by RT-PCR and RACE, and examined J-chain mRNA and protein expression by Northern blotting and immunohistochemistry. This turtle J-chain cDNA was 1934 bp and had an open reading frame of 477 nucleotides, encoding 159 amino acids. The mature J-chain protein is composed of 137 amino acids, M(r) approximately 15000. The deduced amino acid sequence of the turtle J chain was highly homologous to that of human (60%), mouse (61%), cow (60%), rabbit (60%), chicken (69%), brushtail possum (65%), Rana catesbeiana (47%) and Xenopus laevis (58%). Eight cysteine residues were located at the same positions as in these other species, with the exception of X. laevis. PROSITE database analysis indicated the presence of two N-glycosylation sites in turtle, one of which was novel. Northern blot analysis revealed that turtle J-chain mRNA was expressed in lung, stomach, spleen and intestine. In addition, immunohistochemistry showed J-chain-positive plasma cells in the intestine and spleen. These results suggest the presence of a mucosal immune system mainly composed of J-chain-containing Ig in the turtle.     

J-chain expression in human cells producing IgG subclasses

Previous studies have demonstrated that J chain is expressed not only in cells that produce polymeric immunoglobulins, but also in those engaged in synthesis of monomers including IgG and IgD. The presence of J chain in these cells suggested that its role may not be restricted to the formation of polymers. For the present study, fluorochrome-labeled polyclonal anti-J-chain and monoclonal antibodies to IgG subclasses were used to determine the distribution of J chain in IgG plasma cells from normal human tissues and from pokeweed mitogen (PWM)-stimulated human peripheral blood lymphocytes. The results indicate that J chain is not equally distributed among cells producing different IgG subclasses. The percentages of PWM-stimulated cells containing J chain were: 22 +/- 5 (SE) for IgG1, 49 +/- 6 for IgG2, 17 +/- 7 for IgG3, and 64 +/- 11 for IgG4. Examination of sections of various human lymphoid tissues revealed that the frequency of IgG cells that coexpressed J chain was lower than that observed in the PWM system and displayed variable distribution among IgG subclasses. The frequency of J-chain expression in IgG-producing cells may be related to the degree of cellular maturation and may differ according to the origin of cells.     

Electron microscope studies of Fasciola hepatica III. Fine structure of the prostate gland.

An ultrastructural study of the prostate gland of Fasciola hepatica shows it to be composed of numerous unicellular glands. These gland cells contain an extensive granular endoplasmic reticulum (GER) system parts of which are intimately associated with septum-like invaginations of the plasma membrane extending almost to the nucleus. Also associated with the GER are many Golgi complexes which secrete large electron-lucid carbohydrate-rich secretory vesicles. The secretion passes up the gland ducts along with a very dense granular and fibrillar material. The ducts have a peripheral microtubular skeleton and are tightly bound to the epithelium of the ejaculatory duct by septate desmosomes. Secretory vesicles are stored in the expanded ends of the ducts where they pass through the ejaculatory epithelium and their content is discharged by the bursting of their limiting membrane.     

Cloning and expression of the chicken immunoglobulin joining (J)-chain cDNA

 The J chain is a component of polymeric immunoglobulin (Ig) molecules and may play an important role in their polymerization and the transport of polymeric Ig across epithelial cells. In this study, the primary structure of the chicken J chain was determined by sequencing cDNA clones. The cDNA had an open reading frame of 476 nucleotides encoding a putative protein of 158 amino acid residues including the signal sequence. The 3′ untranslated region consisted of 1216 nucleotides and a poly(A) tail. The deduced amino acid sequence of the chicken J chain had a high degree of homology to that of human, cow, rabbit, mouse, frog, and earthworm, with eight conserved Cys residues identical to the mammalian J chains. Northern blot hybridization performed with total RNA from various chicken tissues revealed high levels of J-chain mRNA expression in spleen, intestine, Harderian gland, and bursa of Fabricius, and low levels in the thymus. The J chain was expressed in the bursa as early as day 15 of embryogenesis. These data indicated that the chicken J-chain gene displays a high degree of homology with that of other species, and is expressed at an early stage of development of the chicken immune system. Received: 21 May 1999 / Revised: 30 August 1999     

The polymeric immunoglobulin receptor translocates pneumococci across human nasopharyngeal epithelial cells

The polymeric immunoglobulin receptor (pIgR) plays a crucial role in mucosal immunity against microbial infection by transporting polymeric immunoglobulins (pIg) across the mucosal epithelium. We report here that the human pIgR (hpIgR) can bind to a major pneumococcal adhesin, CbpA. Expression of hpIgR in human nasopharyngeal cells and MDCK cells greatly enhanced pneumococcal adherence and invasion. The hpIgR-mediated bacterial adherence and invasion were abolished by either insertional knockout of cbpA or antibodies against either hpIgR or CbpA. In contrast, rabbit pIgR (rpIgR) did not bind to CbpA and its expression in MDCK cells did not enhance pneumococcal adherence and invasion. These results suggest that pneumococci are a novel example of a pathogen co-opting the pIg transcytosis machinery to promote translocation across a mucosal barrier.     

Endoplasmic reticulum resident, immunoglobulin joining chain, can be secreted by perturbation of the calcium concentration in the endoplasmic reticulum

We established a transient human joining (J)-chain gene expression system in the baby hamster kidney (BHK) cell. The J-chain was detected as a 29-kDa single band on Western blotting. Immunofluorescent staining of the transfectant revealed an exclusive localization of the J-chain in the endoplasmic reticulum (ER). Intracellular transport experiment revealed that incubating conditions favorable for vesicular stomatitis virus glycoprotein (VSV-G) transport did not allow the J-chain to exit from the ER. Analysis of glycosylation status of the J-chain in the transfectant was examined by tunicamycin treatment, endoglycosidase H digestion, and also by treatment with brefeldin A. It was found that an N-glycosylation consensus site of the J-chain was functional, and intracellular J-chain was endoglycosidase H sensitive. These results indicate that, in the absence of any immunoglobulin molecules, J-chain localizes exclusively in the ER. We also tested whether the J-chain could be exported from the ER by perturbing the Ca2+ concentration in the ER. Cultivation of the J-chain transfectant in the presence of ionomycin resulted in the time-dependent secretion of the J-chain. The secreted J-chain was modified by the Golgi resident glycosylation enzymes, indicating that the secreted J-chain passed through the normal exocytic pathway.     

Localization of the human neonatal Fc receptor (FcRn) in human nasal epithelium

The airway epithelium is a central player in the defense against pathogens including efficient mucociliary clearance and secretion of immunoglobulins, mainly polymeric IgA, but also IgG. Pulmonary administration of therapeutic antibodies on one hand, and intranasal immunization on the other, are powerful tools to treat airway infections. In either case, the airway epithelium is the primary site of antibody transfer. In various epithelia, bi-polar transcytosis of IgG and IgG immune complexes is mediated by the human neonatal Fc receptor, FcRn, but FcRn expression in the nasal epithelium had not been demonstrated, so far. We prepared affinity-purified antibodies against FcRn α-chain and confirmed their specificity by Western blotting and immunofluorescence microscopy. These antibodies were used to study the localization of FcRn α-chain in fixed nasal tissue. We here demonstrate for the first time that ciliated epithelial cells, basal cells, gland cells, and endothelial cells in the underlying connective tissue express the receptor. A predominant basolateral steady state distribution of the receptor was observed in ciliated epithelial as well as in gland cells. Co-localization of FcRn α-chain with IgG or with early sorting endosomes (EEA1-positive) but not with late endosomes/lysosomes (LAMP-2-positive) in ciliated cells was observed. This is indicative for the presence of the receptor in the recycling/transcytotic pathway but not in compartments involved in lysosomal degradation supporting the role of FcRn in IgG transcytosis in the nasal epithelium.     

Distribution and processing of the polymeric immunoglobulin receptor in the rat hepatocyte: morphological and biochemical characterization of subcellular fractions

The transepithelial transport of polymeric immunoglobulins is an essential process in the mucosal immune system. Transport across the epithelial cells of mucous or exocrine glands is affected by an integral membrane glycoprotein receptor known as membrane secretory component (SCm) or as polymeric immunoglobulin receptor (pIgR). This receptor binds polymeric immunoglobulins at the basolateral cell surface and mediates their transcellular translocation and their release from the apical plasma membrane into external secretions. Release depends on cleavage of the membrane-anchoring domain of the receptor, resulting in liberation of polymeric immunoglobulin bound to the ectoplasmic domain of the receptor (secreted SC or SCs) into extracellular secretions. Using a monoclonal antibody directed against the cytoplasmic tail of the receptor and a polyclonal antibody directed against the secreted ectoplasmic domain, we have combined cell fractionation and Western blotting techniques to examine the fate of these receptor domains in the hepatocyte. In this study, we characterize biochemically and morphologically the various subcellular components separated by our fractionation scheme, and correlate this with biochemical analysis of the receptor in each fraction.     

Ultrastructure of sperm storage and male genital ducts in a male holocephalan, the elephant fish, Callorhynchus milii.

In chondrichthyes, the process of spermatogenesis produces a spermatocyst composed of Sertoli cells and their cohort of associated spermatozoa linearly arrayed and embedded in the apical end of the Sertoli cell. The extratesticular ducts consist of paired epididymis, ductus deferens, isthmus, and seminal vesicles. In transit through the ducts, spermatozoa undergo modification by secretions of the extratesticular ducts and associated glands, i.e., Leydig gland. In mature animals, the anterior portion of the mesonephros is specialized as the Leydig gland that connects to both the epididymis and ductus deferens and elaborates seminal fluid and matrix that contribute to the spermatophore or spermatozeugmata, depending on the species. Leydig gland epithelium is simple columnar with secretory and ciliated cells. Secretory cells have periodic acid-Schiff positive (PAS+) apical secretory granules. In the holocephalan elephant fish, Callorhynchus milii, sperm and Sertoli cell fragments enter the first major extratesticular duct, the epididymis. In the epididymis, spermatozoa are initially present as individual sperm but soon begin to laterally associate so that they are aligned head-to-head. The epididymis is a highly convoluted tubule with a small bore lumen and an epithelium consisting of scant ciliated and relatively more secretory cells. Secretory activity of both the Leydig gland and epididymis contribute to the nascent spermatophores, which begin as gel-like aggregations of secretory product in which sperm are embedded. Fully formed spermatophores occur in the ductus. The simple columnar epithelium has both ciliated and secretory cells. The spermatophore is regionalized into a PAS+ and Alcian-blue-positive (AB+) cortex and a distinctively PAS+, and less AB+ medulla. Laterally aligned sperm occupy the medulla and are surrounded by a clear zone separate from the spermatophore matrix. Grossly, the seminal vesicles are characterized by spiral partitions of the epithelium that project into the lumen, much like a spiral staircase. Each partition is staggered with respect to adjacent partitions while the aperture is eccentric. The generally nonsecretory epithelium of the seminal vesicle is simple columnar with both microvillar and ciliated cells.     

Vitamin D, calcium-binding protein and calcium absorption in the intestines

The author presents his own and literature data concerning the action of vitamin D and its analogs on the biosynthesis of calcium-binding protein (CaBP) in the chick intestinal mucosa. The mechanism of involvement of this protein in calcium transport across the intestinal epithelium is discussed. CaCP is considered as a specific test-substance for vitamin D. A close correlation between the CaCP content in the intestinal mucosa and the vitamin antirachitic effect is demonstrated. The quantitative estimates of the biological activity of various forms of vitamin D, their metabolites and synthetic analogs with respect to the CaCP content are given.     

The first normal oral mucosa epithelium in which γ-glutamyl transpeptidase activity has been detected

Summary A portion of consistently -glutamyl transpeptidase-positive epithelium in the normal oral mucosa of rats is described. This is the first normal oral mucosa epithelium reported to express activity of the transpeptidase. This enzyme has been used as a marker of malignant transformation in tissues, such as epidermis and oral mucosa epithelium. Complementary studies of the enzyme-positive portion of oral mucosa and a neighbouring negative portion, suggest that, in this model, expression of -glutamyl transpeptidase is linked to a terminally-differentiated epithelium.     

Oral epithelial stem cells—Implications in normal development and cancer metastasis

Oral mucosa is continuously exposed to environmental forces and has to be constantly renewed. Accordingly, the oral mucosa epithelium contains a large reservoir of epithelial stem cells necessary for tissue homeostasis. Despite considerable scientific advances in stem cell behavior in a number of tissues, fewer studies have been devoted to the stem cells in the oral epithelium. Most of oral mucosa stem cells studies are focused on identifying cancer stem cells (CSC) in oral squamous cell carcinomas (OSCCs) among other head and neck cancers. OSCCs are the most prevalent epithelial tumors of the head and neck region, marked by their aggressiveness and invasiveness. Due to their highly tumorigenic properties, it has been suggested that CSC may be the critical population of cancer cells in the development of OSCC metastasis. This review presents a brief overview of epithelium stem cells with implications in oral health, and the clinical implications of the CSC concept in OSCC metastatic dissemination.