Receptor-mediated endocytosis of polymeric IgA and galactosylated serum albumin in rat liver. Evidence for intracellular ligand sorting and identification of distinct endosomal compartments

Rat polymeric IgA (pIgA) and galactosylated bovine serum albumin (GalBSA), once injected to rats, are avidly taken up by hepatocytes via receptor-mediated endocytosis. Of injected pIgA, 64% was transferred undigested into bile within 3 h, with a peak at 30-45 min. GalBSA was essentially digested in lysosomes. By electron microscopy using ligand-peroxidase conjugates, both ligands were internalized through coated pits/coated vesicles into similar electron-lucent vesicles and tubules. Subsequently, pIgA remained mostly associated with small vesicles clustering around and fusing with bile canaliculi, while GalBSA was predominantly found in large, heterogeneous endocytic structures and in lysosomes. By subcellular fractionation, they were associated at 3 min after injection with structures that similarly sedimented in the P fraction (250 000 - 3 X 10(6) X g X min) and equilibrated at densities of about 1.13 g/ml in sucrose gradients. At 10 min and 20 min, pIgA distribution remained mostly in the P fraction at the same equilibrium density. A minor component of the pIgA distribution was found at the density of lysosomes, but contrary to lysosomal enzymes, its distribution was not affected by Triton WR 1339. In contrast to pIgA, GalBSA was progressively recovered in the L fraction (33 000 - 250 000 X g X min) with organelles equilibrating around 1.11 g/ml, and, by 20-45 min, was found in the ML fraction (10 000 - 250 000 X g X min), around 1.20 g/ml, i.e. in lysosomes. Chloroquine did not reduce the efficiency but delayed the secretion of pIgA into bile. Similarly, it did not affect the uptake of GalBSA but apparently delayed GalBSA transfer along successive populations of host organelles. The low density, GalBSA-containing structures were devoid of proteolytic activity. Anti-secretory components IgG and F(ab')2 were selectively excreted into bile, partially or totally as compounds of lower molecular mass. These antibody fragments probably result from a disulfide reduction activity along the pIgA pathway. In conclusion, our data (a) strongly suggest that pIgA and GalBSA are sorted between 3 min and 10 min after injection in non-lysosomal acidic organelles, (b) identify two successive and physically distinct endosomal populations containing GalBSA, and (c) provide the first evidence for a disulfide reduction activity along the transcytotic pathway of rat hepatocytes.     

Receptor mediated endocytosis of formaldehyde treated albumin, yeast invertase and chondroitin sulfate in suspensions of rat liver endothelial cells

Isolated rat liver endothelial cells take up and degrade formaldehyde serum albumin (FSA), invertase and chondroitin sulfate (CS) efficiently. Degradation products start to appear in the medium after 5-30 min. Calcium was necessary for binding of invertase to the cells, but not for the two other ligands. Ammonia and monensin inhibited uptake as well as degradation of all three ligands, whereas leupeptin only inhibited the degradation of FSA and invertase. Uptake of CS was strongly inhibited in the presence of 1 microM FSA. The possibility that these two ligands bind to a common receptor is discussed.     

Endocytic vesicles in liver carry polymeric IgA from serum to bile

The distributions both of endogenous IgA and of injected 125I-labelled IgA were determined amongst the components of a liver homogenate. Rate zonal sedimentation, under conditions where separation was principally determined by particle size, showed that IgA was tightly bound to material which sedimented in the size range of the larger endoplasmic reticulum fragments. Further fractionation of the components within this size range according to their densities, by isopycnic centrifugation, showed that the IgA was associated with small vesicles with a density range of 1.12--1.17 g/ml, quite distinct from endoplasmic reticulum fragments. We therefore conclude that the IgA is present in liver cells in a distinct class of vesicles, which are, presumably, responsible for the transport of IgA from blood to bile.     

Receptor mediated endocytosis of N-acetylglucosamine and mannose exposing molecules by cultured chick embryo hepatocytes.

We studied the receptor mediated endocytosis of a modified glycoprotein (N-acetylglucosamine-BSA) and mannan in cultured hepatocytes isolated from 19-days-old embryos. The binding sites for molecules exposing terminal N-acetylglucosamine (GlcNac) and mannose residues were localized and quantified at the ultrastructural level by means of protein-gold complexes. The binding sites were found to be randomly distributed as single gold particles on cultured hepatocyte cell surfaces not restricted to specialized areas of the plasma membrane. The gold ligands were internalized following a receptor mediated pathway, which was studied at different interval times (15, 30 and 60 min.) after incubating the cells with the electron dense markers.     

Extremely rapid endocytosis mediated by the mannose receptor of sinusoidal endothelial rat liver cells.

Isolated sinusoidal endothelial rat liver cells (EC) in suspension bound and internalized ovalbumin, a mannose-terminated glycoprotein, in a saturable manner. The binding and uptake were Ca2+-dependent and were effectively inhibited by alpha-methyl mannoside and yeast mannan, but not by galactose or asialoglycoproteins. This corresponds to the binding specificity described for the mannose receptor of macrophages and non-parenchymal liver cells. Binding studies indicated a surface pool of 20,000-25,000 mannose receptors per cell, with a dissociation constant of 6 x 10(-8) M. Uptake and degradation of ovalbumin by isolated EC were inhibited by weak bases and ionophores which inhibit acidification of endocytic vesicles and dissociation of receptor-ligand complexes. Cycloheximide had no effect on uptake or degradation. Degradation, but not uptake, was inhibited by leupeptin. We conclude that ovalbumin dissociates from the mannose receptors in the endosomal compartment and the receptors are recycled to the cell surface, while the ovalbumin is directed to the lysosomes for degradation. A fraction of the internalized ovalbumin was recycled intact to the cell surface and escaped degradation (retroendocytosis). The rate of internalization of ovalbumin by isolated EC was very fast, with a Ke (endocytotic rate constant) of 4.12 min-1, which corresponds to a half-life of 10 s for the surface pool of receptor-ligand complexes. To our knowledge, this is the highest Ke reported for a receptor-mediated endocytosis system.     

High sensitivity towards monensin of receptor-mediated endocytosis of formaldehyde treated albumin by liver endothelial cells

Endocytosis of formaldehyde-treated serum albumin (f-albumin) in isolated liver sinusoidal endothelial cells was studied. Uptake occurs via the scavenger receptor and was found to be very sensitive to the ionophore monensin. Binding at 4 degrees C of f-albumin was reduced to 50% of control values by preincubation for 2 min with 2 microM monensin. Both uptake and degradation of f-albumin were more sensitive to monensin. No lag-phase in the inhibitory effect on uptake and degradation was detected. A concentration of 0.1 microM monensin reduced uptake of f-albumin by 50%. Degradation of internalized f-albumin was reduced by 50% in the presence of 0.2 microM monensin. Since uptake and degradation of f-albumin were very sensitive to monensin, the effect of introducing the drug during endocytosis of the ligand was tested. All processing of f-albumin stopped instantly upon addition of monensin; hence, there seems to be no step in the endocytic process beyond which monensin is ineffective. The data suggest that the scavenger receptor of liver endothelial cells is internalized and recycled very rapidly.     

Dimeric and polymeric IgA, but not monomeric IgA, enhance the production of IL-6 by human renal mesangial cells

Depositions of IgA in the renal glomerular mesangial area are a hallmark of IgA nephropathy, and are thought to be crucial for the onset of inflammation processes in IgA nephropathy. In this report we show that human mesangial cells (MC) in vitro bind IgA and that binding of IgA enhances the production of IL-6 by MC. Furthermore we show that the size of IgA is crucial in its capability to enhance IL-6 production. Monomeric IgA does not affect basic IL-6 production, whereas dimeric and polymeric IgA enhance IL-6 production up to 3- to 9-fold respectively. Additional studies demonstrate that enhanced IL-6 production by MC is not accompanied by increased proliferation of human mesangial cells, a finding which is distinct from that found with rat mesangial cells. Taken together, these fmdings suggest that deposition of dimeric and polymeric IgA in the mesangial area of human kidneys in IgA nephropathy may amplify local inflammation.     

Paraoxon hydrolysis in human serum mediated by a genetically variable arylesterase and albumin.

Gel filtration chromatography resolves human serum paraoxonase into two fractions: (1) a high molecular weight fraction that is completely inhibited by EDTA and coelutes with arylesterase (E.C.3.1.1.2); and (2) a second fraction that is closely associated with albumin, is only partially inhibited by EDTA, and has relatively little arylesterase activity under the assay conditions used. The activity of the high molecular weight fraction is stimulated by NaCl, whereas the albumin associated activity is partially inhibited by NaCl and is not present in serum derived from an analbuminemic individual. Our data suggest that albumin itself, rather than a protein bound to or cofractionating with albumin, mediates paraoxonase activity. The variation in levels of the activity of the nonalbumin, high molecular weight enzyme is responsible for the observed polymorphism of paraoxonase activity in human serum or plasma. An optimal assay of polymorphic paraoxonase activity should be based on activity measurements of the nonalbumin fraction. It is considered likely that only the nonalbumin fraction is responsible for in vivo hydrolysis of paraoxon.     

Antiport mediated rounding and endocytosis are enhanced by sulphate

Rapid cell detachment concomitant with the flat-to-round (FTR) change that is mediated by an upshifted Na+/H+ antiporter via HCO3(-)-dependent H+ pumping, is significantly enhanced by the addition of Na2SO4 (FTR + SO4): (1) a faster and greater reduction in cell surface area and perimeter, and (2) a higher level of macromolecular internalization which is also amiloride sensitive. At a fixed 1 mg/ml extracellular FITC-dextran (FDx) concentration, the intracellular FDx load is similar irrespective of the particle size, in the range from 4400 to 2 million mol.wt which is a 455-fold diversity. This is inconsistent with entry via limited sized portals which would discriminate against the larger molecular weight species, such as the 2 million mol.wt species that measures up to 5 microns in width. Two million mol.wt FDx loads linearly in direct proportion to the extracellular FDx concentration, simulating simple diffusion. Large-channel endocytosis is considered to be a characteristic of specialized cell types such as phagocytes and macrophages. However, the antiporter mediated endocytosis (AME) shown here is demonstrated in two different cell types which are not known for their endocytic prowess, viz. epitheloid human Chang liver cells (ATCC CCL 13) and human lung fibroblasts (ATCC CCL 202). The rounded cells with internalized FDx start reverting back to their flat and protracted form upon flooding with warm growth medium, a round-to-flat (RTF) change. However the cell surface reversion is not associated with efflux of FDx which are sorted out into 'granular patches', the later stage endosomes without membrane outlines in AME. FDx-loaded cells grow as well as trypsinized cells without FDx loaing and they maintain a significant FDx load even after nearly 4 cell divisions. Toad sperms internalized into Chang cells via antiporter activation are also sorted into granular patches. AME provides (a) distinctive access to large particles, simulating small ion influx, and (b) an alternate membrane recycling capability where granular patches are instrumental in sorting. It appears to be not a simple endocytosis-exocytosis pathway.     

Proteolytic degradation of hemoglobin-haptoglobin complex by lysosomal enzymes from rat liver.

The catabolic degradation of hemoglobin and of its complex with haptoglobin by lysosomal enzymes from rat liver was studied with special emphasis on the action of cathepsins D and E. The digestion of free hemoglobin can be mainly attributed to the action of cathepsin D [EC 3.4.23.5], while the digestion of the complex in the pH rand 2-3 is due more to the action of cathepsin E than that of cathepsin D. The enzymic activities of both cathepsins were strongly inhibited by pepstatin, and 4M urea inactivated cathepsin E. Measurements of the peroxidase activity and optical rotatory dispersion of the hemoglobin-haptoglobin complex showed that the complex suffered rapid denaturation below pH 2.9.     

Receptor mediated endocytosis 8 is a novel PI(3)P binding protein regulated by myotubularin-related 2

Myotubularin related protein 2 (MTMR2) is a member of the myotubularin family of phosphoinositide lipid phosphatases. Although MTMR2 dephosphorylates the phosphoinositides PI(3)P and PI(3,5)P2, the phosphoinositide binding proteins that are regulated by MTMR2 are poorly characterized. In this study, phosphoinositide affinity chromatography coupled to mass spectrometry identified receptor mediated endocytosis 8 (RME-8) as a novel PI(3)P binding protein. RME-8 co-localized with the PI(3)P marker DsRed-FYVE, while the N-terminal region of RME-8 is required for PI(3)P and PI(3,5)P(2) binding in vitro. Depletion of PI(3)P by MTMR2 S58A or wortmannin treatment attenuated RME-8 endosomal localization and co-localization with EGFR on early endosomes. Our results suggest a model in which the localization of RME-8 to endosomal compartments is spatially mediated by PI(3)P binding and temporally regulated by MTMR2 activity.     

Interaction of ergosterol with bovine serum albumin and human serum albumin by spectroscopic analysis

This study was designed to examine the interactions of ergosterol with bovine serum albumin (BSA) and human serum albumin (HSA) under physiological conditions with the drug concentrations in the range of 2.99-105.88?μM and the concentration of proteins was fixed at 5.0?μM. The analysis of emission spectra quenching at different temperatures revealed that the quenching mechanism of HSA/BSA by ergosterol was the static quenching. The number of binding sites n and the binding constants K were obtained at various temperatures. The distance r between ergosterol and HSA/BSA was evaluated according to F?ster non-radioactive energy transfer theory. The results of synchronous fluorescence, 3D fluorescence, FT-IR, CD and UV-Vis absorption spectra showed that the conformations of HSA/BSA altered in the presence of ergosterol. The thermodynamic parameters, free energy change (ΔG), enthalpy change (ΔH) and entropy change (ΔS) for BSA-ergosterol and HSA-ergosterol systems were calculated by the van't Hoff equation and discussed. Besides, with the aid of three site markers (for example, phenylbutazone, ibuprofen and digitoxin), we have reported that ergosterol primarily binds to the tryptophan residues of BSA/HSA within site I (subdomain II A).     

The separation of intracellular serum albumin from rat liver

1. Antibody precipitation of serum albumin from rat liver extracts yields impure preparations of the protein. 2. When rat liver is labelled with l-[1-(14)C]leucine, antibody precipitation of albumin leads to material that is contaminated with a protein or proteins of very high specific radioactivity. Only 10-25% of the radioactivity of the antibody precipitate is associated with serum albumin. 3. A chromatographic procedure is described that can be used to separate radiochemically pure serum albumin from antibody precipitates obtained from extracts of rat liver. 4. Extracellular albumin secreted by liver slices yields a precipitate with antibody which contains much less radioactive impurity. About 70-90% of the radioactivity is associated with serum albumin. Serum albumin separated by antibody precipitation from rat serum labelled in vivo was not contaminated with the radiochemical impurities associated with intracellular albumin. 5. A simple method is described of obtaining the content of serum albumin in rat liver extracts by the technique of isotope dilution and ion-exchange chromatography.     

Resistance of normal serum IgA and secretory IgA to bacterial IgA proteases: evidence for the presence of enzyme-neutralizing antibodies in both serum and secretory IgA, and also in serum IgG

Normal serum IgA and secretory IgA (sIgA) of subclass IgA1 were isolated from pooled human serum and milk, respectively. They were tested for their susceptibility to bacterial IgA proteases from Haemophilus influenzae, Streptococcus pneumoniae, Neisseria gonorrhoeae, and Neisseria meningitidis that cleave IgA of only the IgA1 subclass. They were also tested for susceptibility to a novel IgA-protease from Clostridium ramosum that cleaves IgA of the IgA1 as well as the IgA2 subclass of the A2m(1) allotype. Both normal serum IgA1 and sIgA1 exhibited resistance to most IgA proteases. The one exception was the IgA protease from C. ramosum which readily cleaved both the serum IgA1 and sIgA1 into Fab and Fc fragments. Secretory component (SC) had nothing to do with the resistance of these IgAs. The resistance of these IgAs to most of the IgA proteases was found to be due to their enzyme-neutralizing antibody activity, since the Fab but not the Fc fragment of sIgA1 showed enzyme-inhibitory activity against these IgA proteases. Similar enzyme-neutralizing antibody activity was found in the pepsin-digested normal serum IgG-(Fab')2 fragment. These results indicate that the induction of the enzyme-neutralizing antibodies against the bacterial IgA proteases took place not only in mucosal sIgA but also in serum IgA and IgG. No enzyme-neutralizing antibody activity against the novel IgA-protease of C. ramosum was detected in any immunoglobulin preparations used in the present study or in the serum of a patient who carries the IgA protease-producing strain of C. ramosum in his feces.