Microtubule and motor-dependent endocytic vesicle sorting in vitro.

Endocytic vesicles undergo fission to sort ligand from receptor. Using quantitative immunofluorescence and video imaging, we provide the first in vitro reconstitution of receptor-ligand sorting in early endocytic vesicles derived from rat liver. We show that to undergo fission, presegregation vesicles must bind to microtubules (MTs) and move upon addition of ATP. Over 13% of motile vesicles elongate and are capable of fission. After fission, one vesicle continues to move, whereas the other remains stationary, resulting in their separation. On average, almost 90% receptor is found in one daughter vesicle, whereas ligand is enriched by approximately 300% with respect to receptor in the other daughter vesicle. Although studies performed on polarity marked MTs showed approximately equal plus and minus end-directed motility, immunofluorescence microscopy revealed that kinesins, but not dynein, were associated with these vesicles. Motility and fission were prevented by addition of 1 mM 5'-adenylylimido-diphosphate (AMP-PNP, an inhibitor of kinesins) or incubation with kinesin antibodies, but were unaffected by addition of 5 microM vanadate (a dynein inhibitor) or dynein antibodies. These studies indicate an essential role of kinesin-based MT motility in endocytic vesicle sorting, providing a system in which factors required for endocytic vesicle processing can be identified and characterized.

Immunocytochemical localization of lysosomal beta-galactosidase in rat liver.

beta-galactosidase is a ubiquitous lysosomal hydrolase that specifically cleaves terminal beta-galactosyl residues from glycoproteins, glycosaminoglycans, oligosaccharides, and glycolipids. To study the intracellular distribution of this enzyme, we prepared a specific polyclonal antibody to lysosomal beta-galactosidase by immunizing rabbits with a highly purified preparation of beta-galactosidase from rat liver. Using this antibody we employed an immunocytochemical technique (protein A coupled to horseradish peroxidase and diaminobenzidine cytochemistry) and showed that beta-galactosidase is present in all hepatocytes of the rat liver. All types of lysosomes, the rough endoplasmic reticulum, and the specialized region of smooth endoplasmic reticulum known as GERL showed immunoreactivity. This in situ distribution suggests that these organelles are involved in the biosynthesis and intracellular sorting of this lysosomal enzyme.

Hepatic binding protein: the protective role of its sialic acid residues.

Removal of sialic acid from a specific hepatic binding protein virtually abolishes its capacity to bind certain asialoglycoproteins. The loss of this capacity is the result of competition for the binding sites by galactosyl residues, of hepatic binding protein, that become terminal after desialylation.

Mammalian hepatic lectin.

A rabbit hepatic protein that specifically binds asialoglycoproteins is also a lectin that agglutinates untreated human and rabbit erythrocytes and neuraminidase-treated erythrocytes from rat, mouse, and guinea pig. Both binding of asialoglycoproteins and agglutination of erythrocytes appear to involve reaction on the same active sites of the hepatic protein.

Unique antigen of cultured Hodgkin's cells. A putative sialyltransferase.

Hodgkin's disease-derived giant cell lines (HD-cells) express high levels of ectosialyltransferase activity presumed to be a galactose-specific lectin recognizing the desialylated 3-fucosyl-N-acetyllactosamine structure (X-hapten). Both the anti-X-hapten monoclonal antibody VIM-D5 and a polyclonal antiserum to another galactose-lectin, the hepatic asialoglycoprotein receptor (HBP), recognize a 55,000-mol wt HD-cell protein (Paietta, E., R. J. Stockert, A. G. Morell, V. Diehl, and P. H. Weirnik. 1986. Proc. Natl. Acad. Sci. USA. 83:3451-3455.) That the expression of the 55,000-mol wt protein is restricted to HD-cells among X-hapten positive cells lines is confirmed in this study. The 55,000-mol wt protein is shown to be present on the cell surface and intracellularly, where an additional immunocrossreactive 150,000-mol wt protein is recognized. Extraction of the 55,000 mol wt protein from HD-cell lysates by affinity chromatography results in the loss of sialyltransferase activity. While evidence for a single protein possessing both the antigenic and the enzymatic activity is not direct, these results suggest that the ectosialyltransferase unique to HD-cells is a 55,000-mol wt membrane glycoprotein possessing the X-hapten oligosaccharide.

Immunological studies of an organic anion-binding protein isolated from rat liver cell plasma membrane.

The mechanism of organic anion uptake by hepatocytes has kinetics that suggest facilitated diffusion, and carrier-mediated membrane transport has been postulated. In previous studies, we purified a 55,000-mol wt organic anion-binding protein (OABP) by affinity chromatography on sulfobromophthalein (BSP)-Sepharose of deoxycholate solubilized liver cell plasma membrane preparations. Using specific goat and rabbit antibodies to OABP, we have now investigated the distribution of this protein in liver fractions and other tissues by an enzyme-linked immunosorbent assay and by the immunoblot (Western blot) procedure. These studies indicated that OABP is present in significant amounts in all tissues examined except for blood. Although OABP has not as yet been isolated from each of these tissues and characterized, OABP in heart retained the ability to bind organic anions, and was purified by affinity chromatography on BSP-sepharose. In liver, OABP was membrane bound and remained so after extraction with 0.9 M NaCl, which suggests that it is an intrinsic membrane protein. OABP did not have a ubiquitous subcellular distribution within the hepatocyte. Preparation of subfractions of liver cell plasma membrane revealed that OABP is present in the sinusoidal and absent from the canalicular membrane. Immunofluorescence studies performed in short-term cultured hepatocytes suggest that OABP is associated with the surface of these cells and does not have a significant intracellular distribution.

Hepatocellular copper toxicity and its attenuation by zinc.

We studied the mechanisms by which excess copper exerts, and zinc mitigates, toxic effects on HepG2 cells. Survival and cell growth were reduced in media containing greater than 500 microM copper chloride for 48 h; LD50 was 750 microM. At 1,000 microM copper for 1 h, there was a general reduction of protein synthesis, and no recognizable changes in cellular ultrastructure. Incubation of cells with 200 microM zinc acetate before exposure to copper, raised the LD50 for confluent cells to 1,250 microM copper chloride, improved protein synthesis, and increased synthesis of a 10-kD protein, apparently metallothionein. The mitigation, by zinc, of copper's toxicity may in part be mediated through induction of this protein in the hepatocyte.

The rat hepatocyte plasma membrane organic anion binding protein is immunologically related to the mitochondrial F1 adenosine triphosphatase beta-subunit.

A 55-kD organic anion binding protein (OABP) was identified previously in liver cell plasma membrane sinusoidal subfractions. Although this protein was localized to the surface of hepatocytes by immunofluorescence, immunoblot analysis revealed reactivity toward both plasma membrane and mitochondrial fractions. To clarify these findings, an immunoreactive clone from a rat liver cDNA expression library was isolated, the 1,500-base pair cDNA insert was sequenced, and the corresponding beta-galactosidase fusion protein was expressed and purified. The resulting sequence corresponded to that of the rat mitochondrial F1-adenosine triphosphatase (F1-ATPase) beta-subunit. This protein and OABP are of similar size and are mutually immunologically cross-reactive. That the antigen was present on the cell surface as well as in mitochondria was suggested from studies of immunoprecipitation after cell-surface iodination, and light- and electron-microscopic immunocytochemistry. Photoaffinity labeling of bovine F1-ATPase with high-specific-activity [35S]sulfobromophthalein revealed binding only to the beta-subunit. Hepatocyte uptake of bilirubin and sulfobromophthalein requires cellular ATP and mitochondria also transport these organic anions, which at high doses inhibit respiration. The presence of an organic anion binding site on the F1-ATPase beta-subunit suggests that it may play a role in these processes.

Diminished hepatic binding protein for desialylated glycoproteins during chemical hepatocarcinogenesis.

The lectin hepatic binding protein has a specific binding capacity for desialylated serum glycoproteins and is limited to hepatocyte membranes. This binding capacity was reduced by approximately 60% in the neoplastic nodules which resulted from exposure of rat livers to N-2-acetylaminofluorene. The binding capacity of the primary hepatocellular carcinomas which resulted from this regimen was reduced by 95%. The loss of binding capacity was found to be proportional to the decreased concentration of immunologically detectable lectin in the altered tissues.

A membrane-bound lectin responsive to monocytic maturation in the promyelocytic leukemia cell line HL-60.

A novel mammalian lectin activity responsive to monocytic differentiation is described in the human promyelocytic leukemia cell line HL-60. Glycoprotein binding indicates that the lectin recognizes both N-acetylneuraminic acid and galactose-terminating biantennary oligosaccharide structures. Lectin activity is independent of calcium and appears to reside in a Mr 17,000 intracellular membrane protein. Induction of wild-type HL-60 cells into their macrophage-like counterparts by 1,25-dihydroxyvitamin D3 markedly enhances lectin activity. Induction of granulocytic differentiation by retinoic acid does not affect expression of the lectin. HL-60 sublines which are resistant to granulocytic differentiation by retinoic acid, dimethylsulfoxide, or 6-thioguanine are largely deficient in orosomucoid-binding activity. Induction of monocyte/macrophage differentiation of these sublines upregulates lectin activity to the level seen in induced wild-type cells.

Hodgkin's cell lectin: an ectosialyltransferase and lymphocyte agglutinant related to the hepatic asialoglycoprotein receptor.

The galactophilic lectin expressed on the surface of cultured Hodgkin's cells, recently described by this laboratory, has binding characteristics similar to those of the hepatic asialoglycoprotein receptor (HBP), and has been recognized as a Mr 55,000 (p55) membrane glycoprotein by a polyclonal antiserum to rat HBP. This study confirms the close structural relationship between the two lectins showing immunological cross-reactivity of monoclonal and polyclonal antibodies recognizing distinct epitopes on rat or human HBP. In support of the suggested dual nature of p55 as lectin and ectosialyltransferase, enzyme activity is inhibited by the monoclonal anti-HBP antibody, anti-HA 116. Cultured Hodgkin's cells, as purified HBP, agglutinate T-lymphocytes expressing hyposialylated membrane glycosyl determinants. This cell-cell interaction mediated by p55 results in the incorporation of sialic acid into lymphocyte surface asialo-glycans. The function of the Hodgkin's lectin as lymphocyte agglutinant in vitro suggests its role as an immunomodulator contributing to the immunodeficiencies associated with Hodgkin's disease.

Growth-dependent expression of a cell surface glycoprotein.

The expression of the hepatocellular membrane receptor for desialylated galactose-termining glycoproteins was studied during different proliferative stages of a human hepatoma cell line. Rapidly growing cells exhibited a reduced endocytotic rate of desialylated orsomucoid as compared to non-growing cells. This reduction was shown to be the consequence of a lower concentration of active cell-surface associated receptor protein in the dividing cells.

Two galactose-specific receptors in the liver with different function.

In the rat liver both hepatocytes and macrophages have been shown to express on the surface lectins with similar binding specificity for galactose residues. Functionally the two lectins differ in the uptake of ligands. Whereas the hepatocytes ingest molecules and small particles (less than 10 nm), the macrophages take up particles only. Antisera raised against hepatic galactose-specific receptor failed to react with the macrophage lectin but blocked ligand binding to the hepatocyte only, indicating either a different antigenic structure or membrane localization of the two lectins.

A marker and putative pathoantigen of Hodgkin's cells.

A galactose-specific lectin, recently described by our laboratory, is immunologically demonstrable on the surface of neoplastic cells derived from patients with Hodgkin's disease. This Hodgkin's lectin is shown to be functionally and antigenically related to the galactose-N-acetylgalactosamine-specific lectin of the hepatocyte (HBP). Poly- and monoclonal antibodies against either the cytoplasmic tail or the cell-surface binding site of HBP recognize the Hodgkin's lectin as a 55 Kd protein. Expression of the 55 Kd antigen appears to be restricted to Hodgkin's disease involved tissues and cells of the monocyte/macrophage lineage. The putative identification of the Hodgkin's lectin as an ectosialyltransferase unique to Hodgkin's cells is supported by inhibition of enzymatic activity by anti-HBP antibodies. Cultured Hodgkin's cells, in analogy to purified HBP, agglutinate T-lymphocytes mediated by the Hodgkin's lectin. This cell-to-cell interaction results in the incorporation of sialic acid into lymphocyte surface asialoglycans as well as in the stimulation of lymphocyte proliferation. The function of the Hodgkin's lectin as lymphocyte agglutinant in vitro suggests its role as an immunomodulator contributing to the immunodeficiencies associated with Hodgkin's disease.

The asialoglycoprotein receptor: relationships between structure, function, and expression.

Transport of macromolecules into the cell by receptor-mediated endocytosis follows a complex series of intracellular transfers, passing through distinct environments. The asialoglycoprotein receptor is a prototype of the class of receptors that constitutively enters cells via coated pits and delivers ligand to these intracellular compartments. In addition to being a model of receptor-mediated endocytosis, the presence of the receptor on hepatocytes provides a membrane-bound active site for cell-to-cell interactions, has made possible the selective targeting of chemotherapeutic agents and foreign genes, and has also been implicated as a site mediating hepatitis B virus uptake. Regulated expression of receptor subunits and their intracellular trafficking during biosynthesis and endocytosis has provided insights into the relationship of receptor structure to its overall function. As a marker of hepatocellular differentiation, its study has uncovered a unique response to intracellular guanosine 3',5'-cyclic monophosphate and translational regulation of the receptor. In this review, an overview of these diverse findings is provided in an attempt to relate the various aspects of structure and function as they impact on receptor expression.

Regulation of the human asialoglycoprotein receptor by cAMP.

The mechanism of regulated expression of the asialoglycoprotein receptor (ASGR) by cAMP was investigated in the human hepatoblastoma cell line, HepG2. Incubation of HepG2 cells with the cell-permeant 8-bromo-cAMP or induction of intracellular cAMP with forskolin reduced receptor expression in confluent HepG2 cultures. Immunoblot analysis established that this reduction of receptor activity was due to a reduction of expression of both ASGR subunit polypeptides H1 and H2. Estimates of the steady-state levels of H1- and H2-related mRNA by Northern blot analysis indicated that reduced ASGR expression was a result of a decrease in gene transcript number. By a combination of run-on and mRNA turnover studies, it was suggested that this reduction of ASGR-related mRNA resulted from its destabilization induced by 8-bromo-cAMP. The effect of 8-bromo-cAMP appears not to be limited to ASGR expression as a rapid reduction in the albumin mRNA was also observed. In contrast, both the beta-actin and glyceraldehyde-3-phosphate dehydrogenase mRNA levels were elevated by exposure to 8-bromo-cAMP.

Second messenger modulation of the asialoglycoprotein receptor.

Post-transcriptional regulation of the asialoglyco-protein receptor (ASGR) in the HepG2 cell line can be mediated by the presence of biotin in the culture medium. To determine if the induction by biotin of intracellular cGMP affects ASGR expression, HepG2 were grown in biotin-depleted medium with the cell-permeant 8-bromo-cGMP (8-Br-cGMP). Both cell-surface and total ASGR binding of iodinated asialoorosomucoid (125I-ASOR) was increased from 30 to 95% of control levels by the addition of increasing concentrations of 8-Br-cGMP. The rate of ASGR-mediated endocytosis of 125I-ASOR also increased with increasing concentrations of 8-Br-cGMP. Estimates of the steady state levels of ASGR by transblot analysis utilizing both antisera to affinity-purified ASGR and to isoform-specific antibodies prepared against synthetic peptides confirmed that the increase in 125I-ASOR binding was due to an increase in ASGR expression. Metabolic labeling of biotin-deprived HepG2 with [35S] cysteine and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of immunoprecipitants revealed an increase of radiolabeled ASGR within 30 min of the addition of 8-Br-cGMP. Induction of cGMP by atrial natriuretic factor also increased the metabolic labeling of ASGR. ASGR expression in a second hepatocellular carcinoma cell line, HuH-7, responded in a similar fashion to the addition of 8-Br-cGMP. In contrast to 8-Br-cGMP, exposure to 8-bromo-cAMP results in a reduction of ASGR expression even in the presence of biotin-containing medium. The antagonistic roles of cGMP and cAMP suggest a balance between cyclic nucleotides is required for the maintenance of differentiated functions by the hepatocyte.

Cytoplasmic protein mRNA interaction mediates cGMP-modulated translational control of the asialoglycoprotein receptor.

Expression of the asialoglycoprotein receptor by the human hepatocellular carcinoma cell line HuH-7 in response to intracellular cGMP concentrations was previously shown to be regulated at the translational level. In a cell-free system, initiation of asialoglycoprotein receptor mRNA translation was dependent on the presence of the 7-methylguanylate cap site and was independent of 8-bromo-cGMP levels in which the cells were grown prior to RNA isolation. Stable transfection of COS-7 cells with deletion constructs of the asialoglycoprotein receptor H2b subunit localized the cGMP-responsive cis-acting element to the mRNA 5'-untranslated region (UTR). Addition of biotin (an activator of guanylate cyclase) induced the expression of beta-galactosidase present as a chimeric plasmid containing the H2b 187-nucleotide 5'-UTR. An RNA gel retardation assay identified a 37-nucleotide cognate sequence within this 187-nucleotide region. Titration of the 5'-UTR with a cytosolic fraction isolated from HuH-7 grown in the presence or absence of 8-bromo-cGMP or biotin provided direct evidence for an RNA-binding protein responsive to intracellular levels of cGMP. Based on these findings, it seems reasonable to propose that reduction of intracellular levels of cGMP by biotin deprivation results in a negative trans-acting factor associating with the 5'-UTR of asialoglycoprotein receptor mRNAs, thereby inhibiting translation.