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.     

JAM-A is both essential and inhibitory to development of hepatic polarity in WIF-B cells

Junctional adhesion molecule (JAM) is involved in tight junction (TJ) formation in epithelial cells. Three JAMs (A, B, and C) are expressed in rat hepatocytes, but only rat JAM-A is present in polarized WIF-B cells, a rat-human hepatic line. We used knockdown (KD) and overexpression in WIF-B cells to determine the role of JAM-A in the development of hepatic polarity. Expression of rat JAM-A short hairpin RNA resulted in approximately 50% KD of JAM-A and substantial loss of hepatic polarity, as measured by the absence of apical cysts formed by adjacent cells and sealed by TJ belts. When inhibitory RNA-resistant human JAM-A (huWT) was expressed in KD cells, hepatic polarity was restored. In contrast, expression of JAM-A that either lacked its PDZ-binding motif (huDeltaC-term) or harbored a point mutation (T273A) did not complement, indicating that multiple sites within JAM-A's cytoplasmic tail are required for the development of hepatic polarity. Overexpression of huWT in normal WIF-B cells unexpectedly blocked WIF-B maturation to the hepatic phenotype, as did expression of three huJAM-A constructs with single point mutations in putative phosphorylation sites. In contrast, huDeltaC-term was without effect, and the T273A mutant only partially blocked maturation. Our results show that JAM-A is essential for the development of polarity in cultured hepatic cells via its possible phosphorylation and recruitment of relevant PDZ proteins and that hepatic polarity is achieved within a narrow range of JAM-A expression levels. Importantly, formation/maintenance of TJs and the apical domain in hepatic cells are linked, unlike simple epithelia.     

The Activity of Menkes Disease Protein ATP7A Is Essential for Redox Balance in Mitochondria

Copper-transporting ATPase ATP7A is essential for mammalian copper homeostasis. Loss of ATP7A activity is associated with fatal Menkes disease and various other pathologies. In cells, ATP7A inactivation disrupts copper transport from the cytosol into the secretory pathway. Using fibroblasts from Menkes disease patients and mouse 3T3-L1 cells with a CRISPR/Cas9-inactivated ATP7A, we demonstrate that ATP7A dysfunction is also damaging to mitochondrial redox balance. In these cells, copper accumulates in nuclei, cytosol, and mitochondria, causing distinct changes in their redox environment. Quantitative imaging of live cells using GRX1-roGFP2 and HyPer sensors reveals highest glutathione oxidation and elevation of H₂O₂ in mitochondria, whereas the redox environment of nuclei and the cytosol is much less affected. Decreasing the H₂O₂ levels in mitochondria with MitoQ does not prevent glutathione oxidation; i.e. elevated copper and not H₂O₂ is a primary cause of glutathione oxidation. Redox misbalance does not significantly affect mitochondrion morphology or the activity of respiratory complex IV but markedly increases cell sensitivity to even mild glutathione depletion, resulting in loss of cell viability. Thus, ATP7A activity protects mitochondria from excessive copper entry, which is deleterious to redox buffers. Mitochondrial redox misbalance could significantly contribute to pathologies associated with ATP7A inactivation in tissues with paradoxical accumulation of copper (i.e. renal epithelia).     

Identification of rat hepatocyte plasma membrane proteins using monoclonal antibodies

We have localized and identified five rat hepatocyte plasma membrane proteins using hybridoma technology in combination with morphological and biochemical methods. Three different membrane preparations were used as immunogens: isolated hepatocytes, a preparation of plasma membrane sheets that contained all three recognizable surface domains of the intact hepatocyte (sinusoidal, lateral, and bile canalicular), and a glycoprotein subfraction of that plasma membrane preparation. We selected monoclonal IgGs that were hepatocyte specific and localized them using both immunofluorescence on 0.5-micron sections of frozen liver and immunoperoxidase at the ultrastructural level. One antigen (HA 4) was localized predominantly to the bile canalicular surface, whereas three (CE 9, HA 21, and HA 116) were localized predominantly to the lateral and sinusoidal surfaces. One antigen (HA 16) was present in all three domains. Only one antigen (HA 116) could be detected in intracellular structures both in the periphery of the cell and in the Golgi region. The antigens were all integral membrane proteins as judged by their stability to alkaline extraction and solubility in detergents. The apparent molecular weights of the antigens were established by immunoprecipitation and/or immunoblotting. In a related study (Bartles, J.R., L.T. Braiterman, and A.L. Hubbard, 1985, J. Cell. Biol., 100:1126-1138), we present biochemical confirmation of the domain-specific localizations for two of the antigens, HA 4 and CE 9, and demonstrate their suitability as endogenous domain markers for monitoring the separation of bile canalicular and sinusoidal lateral membrane on sucrose density gradients.     

Endogenous and exogenous domain markers of the rat hepatocyte plasma membrane

We have used a combined biochemical and morphological approach to establish the suitability of certain endogenous and exogenous domain markers for monitoring the separation of rat hepatocyte plasma membrane domains in sucrose density gradients. As endogenous domain markers, we employed two of the integral plasma membrane protein antigens, HA 4 and CE 9, localized to the bile canalicular and sinusoidal/lateral domains, respectively, of the hepatocyte plasma membrane in rat liver tissue (Hubbard, A. L., J. R. Bartles, and L. T. Braiterman, 1985, J. Cell Biol., 100:1115-1125). We used immunoelectron microscopy with a colloidal gold probe to demonstrate that HA 4 and CE 9 retained their domain-specific localizations on isolated hepatocyte plasma membrane sheets. When the plasma membrane sheets were vesiculated by sonication and the resulting vesicles were centrifuged to equilibrium in sucrose density gradients, quantitative immunoblotting revealed that the vesicles containing HA 4 and those containing CE 9 exhibited distinct density profiles. The density profile for the bile canalicular vesicles (marked by HA 4) was characterized by a single peak at a density of 1.10 g/cm3. The density profile for the sinusoidal/lateral vesicles (marked by CE 9) was bimodal, with a peak in the body of the gradient at a density of 1.14 g/cm3 and a smaller amount in the pellet (density greater than or equal to 1.17 g/cm3). We used this sucrose gradient fractionation as a diagnostic procedure to assign domain localizations for several other hepatocyte plasma membrane antigens and enzyme activities. In addition, we used the technique to demonstrate that 125I-wheat germ agglutinin, introduced during isolated liver perfusion at 4 degrees C, can serve as an exogenous domain marker for the sinusoidal domain of the rat hepatocyte plasma membrane.     

Enzooty of non‐Hodgkin's malignant lymphoma of Papio hamadryas in Sukhumi monkey colony. Clinical and morphological signs of pre‐lymphoma

Inoculation of hamadryas baboons with blood of leukemia ill people‐induced malignant non‐Hodgkin's lymphoma in experimental animals for a very considerable latency period. At close contact of inoculated baboons with healthy non‐inoculated animals, the lymphoma spread between them. The epidemiological analysis, postmortem examination, histological analysis, tissue culturing, and PCR were used for the diagnostics of lymphoma and pre‐lymphoma, purification, identification of STLV‐1, and HVP viruses. Characteristic clinical and morphological signs designated by us as pre‐lymphoma often precede the lymphoma development. In some cases, pre‐lymphoma does not develop in lymphoma because animals die from various diseases and do not reach the point of the lymphoma development. The horizontal transmission of lymphoma arising with the participation of T‐lymphotropic retrovirus STLV‐1 is shown.     

The major subunit of the rat asialoglycoprotein receptor can function alone as a receptor

Mammalian hepatic asialoglycoprotein receptors (ASGP-R) are composed of two unique, but closely related polypeptides, which in the rat are designated rat hepatic lectins 1 and 2/3 (RHL 1, RHL 2/3). Despite numerous studies, the composition of a functional ASGP-R has remained unclear. We examined this question in rat hepatoma tissue culture (HTC) cells (which lack endogenous ASGP-R) that were co-transfected with cDNAs for both RHL 1 and RHL 2/3. The original population was cloned, but derivatives were unstable. We therefore used fluorescence-activated cell sorting to separate a subpopulation of cells (positive) that specifically endocytosed fluoresceinated asialoorosomucoid (ASOR) from one that did not (negative). We then used indirect immunofluorescence with polypeptide-specific ASGP-R antibodies, immunoanalysis, and binding and uptake studies with two Gal ligands (ASOR and NAc-galactosylated poly-L-lysine (Gal-Lys] to further define the ASGP-R status in these two populations. As reported by others, we found that expression of both RHL 1 and RHL 2/3 in the positive cells resulted in binding, uptake and degradation of ASOR, the most commonly used ASGP-R ligand. The negative cells expressed only RHL 1 and neither bound nor processed ASOR. However, the presence of RHL 1 was sufficient for specific high affinity binding and processing of the synthetic ligand, Gal-Lys, by negative cells. These results show that RHL 1 can function as an ASGP-R, given a highly galactosylated ligand, and that RHL 2/3 must play an important role in the organization of native ASGP-R in the membrane.     

Cell-Specific Trafficking Suggests a new role for Renal ATP7B in the Intracellular Copper Storage

Human Cu-ATPases ATP7A and ATP7B maintain copper homeostasis through regulated trafficking between intracellular compartments. Inactivation of these transporters causes Menkes disease and Wilson disease, respectively. In Menkes disease, copper accumulates in kidneys and causes tubular damage, indicating that the renal ATP7B does not compensate for the loss of ATP7A function. We show that this is likely due to a kidney-specific regulation of ATP7B. Unlike ATP7A (or hepatic ATP7B) which traffics from the TGN to export copper, renal ATP7B does not traffic and therefore is unlikely to mediate copper export. The lack of ATP7B trafficking is not on account of the loss of a kinase-mediated phosphorylation or simultaneous presence of ATP7A in renal cells. Rather, the renal ATP7B appears 2–3 kDa smaller than hepatic ATP7B. Recombinant ATP7B expressed in renal cells is similar to hepatic protein in size and trafficking. The analysis of ATP7B mRNA revealed a complex behavior of exon 1 upon amplification, suggesting that it could be inefficiently translated. Recombinant ATP7B lacking exon 1 traffics differently in renal and hepatic cells, but does not fully recapitulate the endogenous phenotype. We discuss factors that may contribute to cell-specific behavior of ATP7B and propose a role for renal ATP7B in intracellular copper storage.     

Disruption of a yeast very-long-chain acyl-CoA synthetase gene simulates the cellular phenotype of X-linked adrenoleukodystrophy

X-linked adrenoleukodystrophy (X-ALD) is characterized biochemically by elevated levels of saturated very long-chain fatty acids (VLCFAs) in plasma and tissues. In X-ALD, peroxisomal very-long-chain acyl-CoA synthetase (VLCS) fails to activate VLCFAs, preventing their degradation via β-oxidation. However, the product of the defective XALD gene (ALDP) is not a VLCS, but rather a peroxisomal membrane protein (PMP). Disruption of either or both of two yeast PMP genes related to the XALD gene did not produce a biochemical phenotype resembling that found in X-ALD fibroblasts. The authors identified a candidate yeast VLCS gene (the FAT1 locus) by its homology to rat liver VLCS. Disruption of this gene decreased VLCS activity, but had no effect on long-chain acyl-CoA synthetase activity. In FAT1-disruption strains, VLCS activity was reduced to 30–40% of wild-type in both a microsome-rich 27,000g supernatant fraction and a peroxisome- and mitochondria-rich pellet fraction of yeast spheroplast homogenates. Separation of the latter organelles by density gradient centrifugation revealed that VLCS activity was peroxisomal and not mitochondrial. VLCS gene-disruption strains had increased cellular VLCFA levels, compared to wild-type yeast. The extent of both the decrease in peroxisomal VLCS activity and the VLCFA accumulation in this yeast model resembles that observed in cells from X-ALD patients. Characterization of the gene(s) responsible for the residual peroxisomal VLCS activity may suggest new therapeutic approaches in X-ALD.     

Vesicular distribution of Secretory Pathway Ca<Superscript>2+</Superscript>-ATPase isoform 1 and a role in manganese detoxification in liver-derived polarized cells

Manganese is a trace element that is an essential co-factor in many enzymes critical to diverse biological pathways. However, excess Mn²⁺ leads to neurotoxicity, with psychiatric and motor dysfunction resembling parkinsonism. The liver is the main organ for Mn²⁺ detoxification by excretion into bile. Although many pathways of cellular Mn²⁺ uptake have been established, efflux mechanisms remain essentially undefined. In this study, we evaluated a potential role in Mn²⁺ detoxification by the Secretory Pathway Ca²⁺, Mn²⁺-ATPase in rat liver and a liver-derived cell model WIF-B that polarizes to distinct bile canalicular and sinusoidal domains in culture. Of two known isoforms, only secretory pathway Ca²⁺-ATPase isoform 1 (SPCA1) was expressed in liver and WIF-B cells. As previously observed in non-polarized cells, SPCA1 showed overlapping distribution with TGN38, consistent with Golgi/TGN localization. However, a prominent novel localization of SPCA1 to an endosomal population close to, but not on the basolateral membrane was also observed. This was confirmed by fractionation of rat liver homogenates which revealed dual distribution of SPCA1 to the Golgi/TGN and a fraction that included the early endosomal marker, EEA1. We suggest that this novel pool of endosomes may serve to sequester Mn²⁺ as it enters from the sinusoidal/basolateral domains. Isoform-specific partial knockdown of SPCA1 delayed cell growth and formation of canalicular domain by about 30% and diminished viability upon exposure to Mn²⁺. Conversely, overexpression of SPCA1 in HEK 293T cells conferred tolerance to Mn²⁺ toxicity. Taken together, our findings suggest a role for SPCA1 in Mn²⁺ detoxification in liver.