Polymeric IgA Research Articles

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.

Differential measurement of monomeric and polymeric IgA by one-directional immunoelectrodiffusion

A technique to specifically quantify monomeric IgA and total IgA in colostrum has been developed using a modified one-dimensional immunoelectrophoretic assay. This method employed electrophoresis in antibody-containing polyacrylamide-agarose gel in the presence of a gel barrier which blocks polymeric IgA. The addition of PEG (polyethylene-glycol 6000) to the anodic gel increased the sharpness of the peaks, the height of which was proportional to the antigen concentration. This method proved to be sufficiently simple, precise, reproducible ( CV < 3%) and linear (from 20–300 mg/l) to measure the monomeric IgA : total IgA ratio rapidly (14 ± 4.5% for 20 samples in duplicate). Immunoelectrodiffusion studies confirmed that human colostral and serum IgA standards could be used to determine directly monomeric IgA, total IgA and polymeric IgA levels (by difference) rather than to apply correction factors to estimate these IgA levels.

Secretory component as the mucosal transport receptor: Separation of physicochemically analogous human iga fractions with different receptor-binding capacities

This paper describes the separation and characterization of several IgA fractions from the same human monoclonal source, based on their ability to bind secretory component (SC). The study was undertaken to elucidate features of the immunoglobulin-binding site for SC, and to examine the dependence of mucosal transport on IgA-SC interaction. Enrichment or depletion of SC-binding activity was accomplished on an affinity adsorbant made with SC from human colostral whey. The affinity-purified human IgA fractions contained IgA polymers and were 77% active in rebinding to the adsorbant; this activity was diminished significantly by direct radioiodination. The non-adherent IgA fractions contained both polymer and monomer, and were only 8% active in rebinding to the adsorbant. When the polymer and monomer components were separated from each other, the non-adherent polymer was found to resemble the affinity-purified fraction by all criteria examined including J-chain content, except that the SC-binding capacity was > five-fold lower. These findings have two implications for the SC-binding site on human IgA: first, the presence of J-chain is insufficient to bestow IgA with SC-binding activity; second, a critical tyrosine participates in maintaining the SC-binding region, possibly on the IgA heavy chain. The relationship between SC binding and mucosal transport was tested in the rat hepatobiliary model. All radiolabeled human IgA fractions were captured rapidly from blood by the rat liver, but only the SC-binding fractions underwent substantial intact transport to bile (> 70% of the injected dose). Even though a nominal proportion of the SC-non-adherent IgA appeared in bile (4–15% of the dose), most IgA in these fractions was rapidly degraded within the liver. Thus, only a small amount of monomeric and polymeric IgA can use alternative receptors to get to bile by diversion from the degradative pathway. Polymeric IgA can undergo efficient transport across the cell, strictly conditional on a high binding capacity for SC. This demonstrates that membrane SC is the receptor conferring specificity on the mucosal-transport pathway.

Serum IgA to enterobacteria in ankylosing spondylitis.

The aetiology of ankylosing spondylitis (AS) may involve certain enterobacteria. It is therefore interesting that serum polymeric IgA, a precursor of secretory IgA, was statistically elevated in active AS (n = 35) and that levels were comparable to those found in yersiniosis (n = 12); this might indicate antigenic stimulation by bacteria which are present in the intestines of AS patients. However, specific serum IgA to the incriminated enterobacteria Klebsiella, Shigella and Yersinia, as determined by ELISA, was not raised in the above AS patients. Nor were these titres raised in patients with idiopathic reactive arthritis (n = 21). In contrast, yersiniosis (n = 12) and shigellosis (n = 96) patients displayed marked increases in specific serum IgA titres to the respective infectants. It is proposed that AS may involve a set of enterobacteria rather than a few suspected species. Thus, despite the lack of raised group averages, screening of individual patients for specific IgA to several indicated bacteria might disclose whether or not raised serum IgA is related to enterobacterial activity. Apart from this, the above supports other reports indicating that serum IgA may be a useful parameter to assist in monitoring of disease activity in AS. Finally, it is suggested that study of a homogeneous group of reactive arthritis patients might facilitate aetiological research of seronegative arthropathies such as AS.

Transcellular transport of polymeric IgA in the rat hepatocyte: biochemical and morphological characterization of the transport pathway.

Polymeric IgA (pIgA) is transported by liver parenchymal cells (hepatocytes) from blood to bile via a receptor-mediated process. We have studied the intracellular pathway taken by a TEPC15 mouse myeloma pIgA. When from 1 microgram to 1 mg 125I-pIgA was injected into the saphenous vein of a rat, 36% was transported as intact protein into the bile over a 3-h period. The concentration of transported 125I-pIgA was maximal in bile 30-60 min after injection, and approximately 80% of the total 125I-pIgA ultimately transported had been secreted into bile by 90 min. A horseradish peroxidase-pIgA conjugate (125I-pIgA-HRP) was transported to a similar extent and with kinetics similar to that of unconjugated 125I-pIgA and was therefore used to visualize the transport pathway. Peroxidase cytochemistry of livers fixed in situ 2.5 to 10 min after 125I-pIgA-HRP injection demonstrated a progressive redistribution of labeled structures from the sinusoidal area to intermediate and bile canalicular regions of the hepatocyte cytoplasm. Although conjugate-containing structures began accumulating in the bile canalicular region at these early times, no conjugate was present in bile until 20 min. From 7.5 to 45 min after injection approximately 30% of the labeled structures were in regions that contained Golgi complexes and lysosomes; however, we found no evidence that either organelle contained 125I-pIgA-HRP. At least 85% of all positive structures in the hepatocyte were vesicles of 110-160-nm median diameters, with the remaining structures accounted for by tubules and multivesicular bodies. Vesicles in the bile canalicular region tended to be larger than those in the sinusoidal region. Serial sectioning showed that the 125I-pIgA-HRP-containing structures were relatively simple (predominantly vesicular) and that extensive interconnections did not exist between structures in the sinusoidal and bile canalicular regions.

IgA-associated glomerular deposits in liver disease

IgA-associated immunopathologic renal injury has been reported in patients with cirrhosis. In an effort to elucidate the pathogenesis of this phenomenon, the livers and kidneys obtained at autopsy from a group of patients with cystic fibrosis were studied; these patients were selected because of their broad spectrum of liver abnormalities. On the basis of histologic examination of sections of liver, the patients were divided into two groups: Group I (20 patients) included patients with focal biliary cirrhosis and multilobular biliary cirrhosis; all had anatomic distortion of the biliary system, and many had cholestasis. Group II (28 patients) showed no bile duct anomalies. Immunofluorescence studies of the corresponding kidneys for immunoglobulin, complement, and free secretory component (FSC) revealed significantly more numerous IgA-containing glomerular deposits in group I (P less than 0.02). Although FSC was virtually absent in these deposits, significant in vitro binding of this protein revealed the polymeric nature of the glomerular IgA. This is consistent with previous observations of elevated serum levels of polymeric IgA, which forms the dominant component of glomerular deposits in cirrhotic patients. Since IgA glomerular deposition occurred in patients with focal biliary and no hepatocellular dysfunction, it seems that the source of this polymeric IgA is related to its impaired serum clearance by a distorted and stagnant bile duct system. However, the mechanism that leads to the deposition of this immunoglobulin in the glomeruli and other tissues remains conjectural.

Intracellular Processing of Human Vs. Rat Immunoglobulin A in the Rat Liver

It is well established that in the rat, rat dimeric IgA is transported from blood to bile across rat liver parenchymal cells via a series of minute smooth membrane-limited vesicles. This pathway is unique from that taken by a number of other ligands, which are internalized for degradation, in that there appears to be little involvement of coated vesicles, multivesicular bodies and lysosomes. The transmembrane receptor for IgA, secretory component, is not recycled but is secreted in part with the ligand into bile and must be produced continuously within the liver cell. Several recent studies have suggested that the receptor for asialoglycoproteins, as well as the structures involved in its processing, may play an important role in IgA processing. It was noted, however, that in all of these studies human polymeric IgA1 was used in the rat model. Using purified rat and human IgA preparations, we have demonstrated by light and quantitative electron microscopic autoradiography, as well as by certain biochemical procedures, that the two ligands are processed quite differently from one another in the rat. Human IgA disappears from the plasma at a slower rate and is much less efficiently transported into the bile. In addition, up to as much as 30% of the human IgA is diverted to the lysosomal pathway. This diversion of human polymeric IgA may be related to either the association of a serine-linked oligosaccharide at the hinge region of the human polymeric IgA1 or that large polymers, often found in human IgA preparations may initiate secretory component receptor aggregation, which in turn, interferes with the normal physiological processing of the IgA molecule.

Charge and size of mesangial IgA in IgA nephropathy

To characterize the physicochemical properties of the mesangial IgA in primary IgA nephropathy, acid-eluates from percutaneous renal biopsies of 20 patients were examined. The acid-eluates were obtained from 1287 +/- 498 glomerular sections. The IgA content (mean 15 +/- 10 ng) represented 0.4% of the total eluted proteins. To analyze the molecular weight and the charge of eluted IgA, 11 eluates were subjected to high pressure liquid chromatography (at pH 6.8 and/or pH 3.5) and five eluates to isoelectric focusing on agarose. IgA was detected in the fractions by an IgA-RIA. Comparison of the elution profiles at different pH showed a statistically significant decrease of the excluded IgA peak (greater than or equal to 1,000,000 daltons), and a significant increase of polymeric IgA peaks (1,000,000-320,000 and 320,000 daltons) in acidic chromatography, as compared to non-dissociating conditions. Under acidic conditions, polymeric IgA represent 64% of total eluted IgA. Secretory component binding to polymeric IgA was demonstrated in four out of eight eluates tested. The isoelectric point (pI) of eluted IgA ranged from 4.5 to 5.6, contrasting with the broader and more neutral pI of normal serum IgA (4.5 to 6.8). This study shows that the multimeric nature of IgA, the formation of IgA complexes, and the anionic charge of IgA are likely to be involved in the mesangial IgA deposition in idiopathic IgA nephropathy.

Molecular forms of tear IgA and distribution of IgA subclasses in human lacrimal glands

Tears from subjects without conjunctival inflammation contained 93% polymeric IgA (pIgA) and of secretory IgA standard for tear samples and monomeric IgA standard for serum samples. Tears from subjects without conjunctival inflammation contained 93% polymeric IgA (pIgA) and 7% monomeric IgA; tears from subjects with conjunctival inflammation had less pIgA (79%). Biopsy specimens of 11 lacrimal glands and one accessory lacrimal gland were obtained from 12 additional subjects. Tissues were stained with polyclonal antisera for immunoglobulins, J chain, and secretory component. In addition, tissues were stained for IgA subclasses with monoclonal reagents specific for IgA1 and IgA2. IgA plasma cells predominated over other types of plasma cells, and most were J chain positive. Secretory component, although absent from the interstitium of the gland, was found in most acinar and ductal cells. The average proportion of IgA1 to IgA2 cells was 56% to 44%. We concluded that the lacrimal system of the human resembles other exocrine systems in the production of predominantly pIgA and the nearly equal occurrence of IgA1 and IgA2 plasma cells.

Urinary IgA in IgA nephropathy and Henoch-Schoenlein purpura.

To determine the concentrations and molecular forms of urinary IgA in IgA nephropathy and Henoch-Schoenlein purpura, we studied 29 patients with these IgA-associated renal diseases (IgAN). Control groups comprised 10 patients with other diverse renal disease and 11 healthy volunteers. Urinary IgA and IgG concentrations were higher in IgAN than in either control group and correlated positively with the serum creatinine concentration as well as the urinary protein excretion (P less than 0.01). However, IgA/IgG ratios did not differ among the three groups. Polymeric IgA (p-IgA) in the urine predominated only in normals; in IgAN and patients with other renal diseases, monomeric IgA (m-IgA) occurred almost exclusively. Serum IgA concentrations were generally normal in IgAN; four patients had concentrations greater than 500 mg/dl. Although the fraction of p-IgA in serum (median, 18%) was increased above normal (5-10%) in 13 of 16 (81%) subjects, neither the concentration of IgA or IgG nor the amount of p-IgA correlated with the serum creatinine concentration. These data suggest that the molecular form and concentration of urinary IgA are not discriminating for IgAN and are independent of these characteristics of serum IgA.

The wasted mutant mouse. I. An animal model of secretory IgA deficiency with normal serum IgA.

The wasted (wst) mutation was recently described as a spontaneous, recessive mutation leading to pathologic changes affecting both the neurologic and the immune systems of wst/wst homozygotes, which presented symptoms analogous to those observed in patients with ataxia-telangiectasia (A.T.). We studied the IgA system of wst/wst mutants and their normal littermates to determine whether IgA deficiency commonly found in A.T. patients was also affecting these mutants. Interestingly, although IgA plasma cells were totally absent from their entire (small and large) intestine, their serum contained a normal level of IgA with a normal ratio of monomeric vs polymeric IgA. The absence of gut IgA plasma cells was not due to malnutrition and was not compensated by the appearance of cells secreting any other isotypes. Studies at the precursor cell level showed the absence of IgA-specific B cell precursors in the Peyer's patches, whereas sIgA B cells and IgA plasma cells were found in normal numbers in the spleen of wasted mice. These data suggest that secretory and serum IgA may comprise distinct systems and that the wasted mutant mouse is a potential model for the study of the physiology and regulation of IgA production.

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.

An immunochemical study of the combining site specificities of C57BL/6J monoclonal antibodies to alpha (1----6)-linked dextran B512.

This is the first report of an immunochemical study of the combining site specificities of a set of monoclonal antibodies to dextran B512 from C57BL/6J mice. The results confirm previous observations on antidextran combining sites and reveal specificities not seen earlier extending the observed repertoire of antibody combining sites to the single alpha (1----6)-linked glucosyl antigenic determinant. Eight C57BL/6J anti-dextran B512 hybridomas, four IgM,kappa and four IgA,kappa, were produced by PEG fusion of immune spleen cells with the nonproducer myeloma cell line P3X63Ag8 6.5.3. Antibody combining site specificities were determined by quantitative precipitin assays with 14 dextrans. Native dextrans with high percentages of linear alpha (1----6)-linked glucoses, similar to the immunogen B512, were the best precipitinogens; dextrans with alternating alpha (1----3), alpha (1----6) linkages, and highly branched dextrans were less effective. All antibodies precipitated with a synthetic, unbranched alpha (1----6)-linked dextran, suggesting their combining sites were "groove-like" and directed toward internal sequences of alpha (1----6)-linked residues, rather than "cavity-like" and directed toward a nonreducing terminal glucose. Two of the IgA hybridomas gave biphasic precipitin curves with dextran B512; this was shown to be due to differences in the precipitability of IgA monomers and polymers. Differences were observed in the reactivities of several dextrans considered previously to be structurally similar, and a newly proposed structural model of dextran B1299S was assessed. Quantitative precipitin inhibition studies with alpha (1----6)-linked isomaltosyl (IM) oligosaccharides, IM2 to IM9, showed that maximum inhibition was reached with IM6 or IM7, consistent with earlier estimates of the upper limit for the sizes of anti-B512 combining sites. Two IgM hybridomas showed a unique pattern, with inhibition being obtained only with IM5 or larger IM oligosaccharides. Association constants of the antidextrans for dextran B512 and for IM7, determined by affinity gel electrophoresis, ranged from 10(2) to 10(4) ml/g, comparable to earlier findings with antidextrans and other anticarbohydrate antibodies.

Carbohydrate-mediated clearance of secretory IgA from the circulation

Radioiodinated human secretory IgA (sIgA) injected intravenously into mice was rapidly cleared from the circulation by the liver. A portion of the sIgA was transported as an intact molecule into the bile. However, this transport was less efficient than that of human serum polymeric IgA (pIgA). The clearance of sIgA from the circulation was inhibited by prior injection of asialofetuin, suggesting that its uptake is mediated by the hepatic binding protein (HBP) specific for asialoglycoproteins. Mouse pIgA did not inhibit the hepatic clearance of sIgA. Results of in vivo studies were confirmed by in vitro experiments. The binding of 125I-asialoorosomucoid to either the paniculate fraction (2000 g pellet of the homogenate) or the plasma membrane fraction of mouse liver was inhibited by sIgA. When polypeptide components of sIgA were used as inhibitors, significant inhibition was obtained with secretory component (SC), while inhibition with light and J-chains was not statistically significant. Examination of the inhibitory activity of IgA1 and IgA2 myeloma proteins and heavy chains isolated from these proteins revealed that binding of polymeric IgA1 and α1 heavy chains can also be mediated by HBP. However, these interactions appear to be of lower avidity than those with SC. The inhibitory activity of human IgA2 and α2 heavy chains was not significant. The involvement of HBP in binding of sIgA was also confirmed by measuring the inhibition of binding of 125I-sIgA. The binding of this protein by the particulate fraction of the mouse liver homogenate was inhibited by asialoglycoproteins and SC while inhibition with IgA1 and α1 heavy chains was not significant. These results suggest that the carbohydrate moieties recognized by HBP reside primarily in the SC portion of sIgA.

Chronic Giardia muris infection in anti-IgM-treated mice. I. Analysis of immunoglobulin and parasite-specific antibody in normal and immunoglobulin-deficient animals.

To investigate the role of B cells and antibody in the immune response of mice to the murine intestinal parasite Giardia muris, we used mice treated from birth with rabbit anti-IgM antisera (aIgM). Such mice developed in serum and in gut secretions extreme Ig deficiency (IgM, IgA, and IgG) relative to control animals. The aIgM-treated mice showed no anti-G. muris antibody in serum or in gut wash material. Infections of G. muris in these mice were chronic, with a high load of parasite present in the small bowel, as reflected by prolonged cyst excretion (greater than 11 wk) and high trophozoite counts. In contrast, normal, untreated mice or NRS-treated animals developed anti-parasite IgA and IgG antibody in serum, demonstrated IgA antibody against the parasite in gut washings, and expelled the parasite within 9 wk. These effects of aIgM treatment on the murine response to primary infection with G. muris were demonstrated in two strains of mice: BALB/c and (C57BL/6 X C3H/He) F1. It was also observed that the response to G. muris infection in untreated animals was characterized by higher than normal total secretion of IgA into the gut and a concomitant increase in the serum polymeric IgA level. Mice treated with aIgM had a marked decrease of both monomeric and polymeric IgA in serum, and little detectable IgA in the intestinal lumen. These experiments provide the first demonstration that anti-IgM treatment suppresses a specific intestinal antibody response to antigen, and provide evidence that B cells and antibody play a role in the development of an effective response to a primary infection with G. muris in mice.

Degradative intracellular transport of antisecretory component in cultured hepatocytes

Abstract The liver efficiently transports dimeric immunoglobulin A (dIgA) from blood to bile in a direct, nonlysosomal pathway involving smooth-surfaced vesicles. Secretory component (SC), the plasma membrane receptor for dIgA, is released into bile still bound to its ligand by disulfide bridges, Rabbit IgG antirat SC binds specifically to plasma membrane SC, yet the biliary secretion of anti-SC is markedly lower than that of dIgA, suggesting that the IgG antibodies utilize an alternate transhepatocellular pathway. Uptake of commercially available antihuman SC conjugated to horseradish peroxidase was examined by quantitative electron microscopic immunocytochemistry using primary rat hepatocyte monolayer cultures. Coincubation with human polymeric IgA, rabbit antiserum to rat SC, free human SC, human secretory IgA, and rat bile, all significantly suppressed uptake of anti-SC-horseradish peroxidase, thus demonstrating the specificity of the labeled antibody. Coated vesicles accounted for >70% of the total uptake of either the anti-SC-horseradish peroxidase preparation or colloidal gold-labeled IgG antirat SC. Both compounds could also be observed in other structures associated with the degradative pathway, i.e., multivesicular bodies and lysosomes. Moreover, the extent to which 125 I-anti-SC was degraded was significantly greater than that of 125 IdIgA. These data demonstrate that dIgA and anti-SC utilize different intracellular pathways, with anti-SC undergoing lysosompl degradation.

Degradative intracellular transport of antisecretory component in cultured hepatocytes: An alternate pathway for the immunoglobulin A receptor

The liver efficiently transports dimeric immunoglobulin A (dIgA) from blood to bile in a direct, nonlysosomal pathway involving smooth-surfaced vesicles. Secretory component (SC), the plasma membrane receptor for dIgA, is released into bile still bound to its ligand by disulfide bridges, Rabbit IgG antirat SC binds specifically to plasma membrane SC, yet the biliary secretion of anti-SC is markedly lower than that of dIgA, suggesting that the IgG antibodies utilize an alternate transhepatocellular pathway. Uptake of commercially available antihuman SC conjugated to horseradish peroxidase was examined by quantitative electron microscopic immunocytochemistry using primary rat hepatocyte monolayer cultures. Coincubation with human polymeric IgA, rabbit antiserum to rat SC, free human SC, human secretory IgA, and rat bile, all significantly suppressed uptake of anti-SC-horseradish peroxidase, thus demonstrating the specificity of the labeled antibody. Coated vesicles accounted for >70% of the total uptake of either the anti-SC-horseradish peroxidase preparation or colloidal gold-labeled IgG antirat SC. Both compounds could also be observed in other structures associated with the degradative pathway, i.e., multivesicular bodies and lysosomes. Moreover, the extent to which 125I-anti-SC was degraded was significantly greater than that of 125IdIgA. These data demonstrate that dIgA and anti-SC utilize different intracellular pathways, with anti-SC undergoing lysosompl degradation.

GLOMERULAR DEPOSITION OF IgA IN EXPERIMENTAL HEPATIC CIRRHOSIS

Wistar rats rendered cirrhotic with carbon tetrachloride excreted significant proteinuria and hematuria. Serum levels of IgA and IgG were significantly elevated in cirrhotic animals. They showed mild mesangial proliferation and immunofluorescent studies revealed deposits of IgA and IgG predominantly in mesangial areas and along capillary walls. These findings were very similar to those seen in patients with hepatic cirrhosis or IgA nephropathy. The deposits of IgA were also found in hepatic tissue from cirrhotic animals. The intensity and distribution of glomerular IgA deposits were not diminished after treatment with acid buffer. These results suggest that glomerular IgA are IgA polymers and decreased hepatic clearance of hepatic IgA polymers may be responsible for the glomerular deposition of IgA.

Hepatic uptake and disposition of human polymeric IgA1 in perfused rat liver: evidence for incomplete biliary excretion and intrahepatic degradation.

The hepatic uptake of polymeric immunoglobulin A (IgA) is mediated by secretory component; the resulting secretory IgA is excreted intact into bile. To define the hepatic metabolism of polymeric IgA, we quantitated the uptake and transport of human polymeric IgA1 after a single pass through the perfused rat liver. Uptake of polymeric IgA1 was compared with that of asialoorosomucoid, a glycoprotein whose uptake is mediated by the asialoglycoprotein receptor. Single-pass hepatic uptake of 125I-polymeric IgA1 and of 125I-asialoorosomucoid averaged 18.0 +/- 3.1% (SE) and 71.8 +/- 2.8%, respectively. The uptake of 125I-polymeric IgA1 was inhibited by excess unlabeled polymeric IgA1 but not by asialoorosomucoid. Only 13.0 +/- 1.6% of the 125I-polymeric IgA1 extracted by the liver was excreted into bile, whereas three-fourths was released into the hepatic venous effluent in degraded form. Thus, both the uptake and biliary excretion of polymeric IgA1 by the rat liver are inefficient processes. Polymeric IgA1 follows two distinct pathways after uptake by the liver: a small proportion is excreted intact into bile, while the majority is degraded and released back into the circulation.

Biogenesis of the polymeric IgA receptor in rat hepatocytes. II. Localization of its intracellular forms by cell fractionation studies.

In the companion paper (Sztul, E. S., K. E. Howell, and G. E. Palade, J. Cell Biol., 100:1248-1254), we have shown that pulse labeling of hepatic proteins with [35S]cysteine can be obtained in vivo in intact rats. Soluble label clears the plasma in approximately 5 min, and incorporated label reaches peak values in the liver approximately 20 min after injection. In the present study, we show that the 105,000-mol-wt protein (105K), kinetically the earliest intracellular form of secretory component (SC), is the predominant form found, between 5 and 20 min postinjection, in homogeneous rough microsomal fractions. The second kinetically defined form, i.e., 116K, is the predominant species present in relatively homogeneous, light Golgi fractions in which it appears at approximately 15 min, and peaks at approximately 25 min, postinjection. The third kinetically defined form, 120K, is found 30 min after injection as the major SC species (albeit still accompanied by its immediate precursor, 116K), in a sinusoidal plasmalemmal fraction isolated by immunoadsorption to anti-SC-coated Sepharose beads. These findings lead to the following conclusions: (a) SC is synthesized on polysomes attached to the rough endoplasmic reticulum (ER) membrane; (b) it is partially translocated across the ER membrane and core glycosylated co-translationally to give a 105K peptide; (c) 105K moves from the ER to the Golgi complex where it is terminally glycosylated to give the 116K form; (d) the latter moves to the sinusoidal plasmalemma where it appears together with the final mature form, 120K. Kinetic evidence indicates that the vesicular carriers involved in the transport of SC from the Golgi complex to the sinusoidal plasmalemma, and from the latter to the biliary front of the hepatocytes, are present in a Golgi heavy fraction and a crude carrier vesicle fraction from which they remain to be isolated, purified, and characterized.