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Biomedical subjects

A G Morell

Publications and source records attributed to A G Morell.

At least 19 recordsLinked to original sources
PubMed1994

Modulation of the asialoglycoprotein receptor in human hepatoma cells: effect of glucose.

The hepatic receptor for asialoglycoproteins was found to be modulated by the glucose concentration in the medium of the human hepatoma cell line HepG2. The surface binding of asialoorosomucoid, a well-documented ligand for this receptor, increased from 20 ng/mg of cellular protein to about 40 ng/mg as the glucose concentration was increased from 10 to 50 mg/dl. The up-modulating effect of glucose was mimicked by pyruvate, a product of glucose metabolism, and abolished by both 2-deoxyglucose, an inhibitor of glucose metabolism, and by cycloheximide, an inhibitor of protein synthesis. Scatchard plot analysis indicated a rise in the number of binding sites and a twofold increase in binding affinity. In contrast, the binding of antibody remained unchanged with respect to alterations in glucose concentration, an indication that the actual number of receptors remained constant in face of an increased number of binding sites. Specificity of the glucose effect was shown by the binding of insulin and transferrin to their respective receptors, which was unaffected by the high glucose concentration that increased asialoorosomucoid binding. The repression of receptor binding seen with cells grown in biotin-deprived medium was reversed by increasing the glucose concentration of the medium. In this case, binding was restored to a level sixfold to sevenfold higher than that of the control cells grown in dialyzed serum. The stimulatory effect of glucose was shown to be independent of and significantly greater than that of cyclic GMP, a known regulator of receptor expression of biotin-deficient HepG2 cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Asialoglycoprotein Receptor↗
PubMed1992

Posttranscriptional regulation of the asialoglycoprotein receptor by cGMP.

The human asialoglycoprotein receptor expressed by the HepG2 cell line is composed of the two homologous polypeptides H1 and H2. Transblot analysis of HepG2 cell lysates indicated that the progressive loss in the steady-state level of asialoglycoprotein receptor (ASGR) when cells were maintained in medium supplemented with dialyzed fetal bovine serum was reversed by the addition of cell-permeant 8-bromo-cGMP. Estimates of the steady-state levels of H1- and H2-related mRNA by Northern blot analysis indicated that the reduction of ASGR was not the result of a concomitant reduction in gene transcript number. No difference in the translatability of the mRNAs derived from cells grown in medium supplemented with fetal bovine serum or its dialyzed counterpart was detected. Resolution of the mRNAs by sucrose gradient centrifugation suggests that cGMP-mediated posttranscriptional regulation of ASGR expression was due to a shift of both H1 and H2 mRNAs from the ribonucleoprotein fraction into a translationally active membrane-associated polysomal pool.

Asialoglycoprotein Receptor↗
PubMed1990

Detection of multiple forms of human ceruloplasmin. A novel Mr 200,000 form.

Three polypeptides with apparent Mr = 200,000, 135,000, and 115,000, reacting with antibody to human ceruloplasmin (Cp), were consistently found in sera of normal adult and newborn subjects, patients with Wilson's disease, as well as in the oxidase-active fraction of purified human Cp, resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The concentrations of the three Cp polypeptides were proportional to the total Cp oxidase activity measured in whole serum. Peptide mapping revealed that the three Cp polypeptides were closely related. Cross-linking of Cp135 resulted in dimers with electrophoretic mobility similar to that of Cp200. A common shift in electrophoretic mobility following N-glycanase treatment indicated that all three polypeptides were N-glycosylated, and that the apparent differences in molecular mass could not be related to the carbohydrate moiety. Immunoprecipitates of cell lysates of [35S]cysteine labeled HepG2 cells revealed the presence of two species of newly synthesized Cp polypeptides, Mr 200,000 and 135,000, which were secreted into the media. Secretion of Cp200 by the human liver appears to be physiologic and may be the result of posttranslational modification of Cp135.

Carcinoma, Hepatocellular↗
PubMed1990

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.

Amino Acid Sequence↗
PubMed1988

Biotin-dependent expression of the asialoglycoprotein receptor in HepG2.

The asialoglycoprotein receptor (AsGR) is characteristic of fully differentiated hepatocytes. AsGR expression in confluent cultures of HepG2 cells grown in minimal essential medium (MEM) requires a 300-350-dalton dialyzable fraction of fetal bovine serum (FBS). Addition to dialyzed FBS (dFBS) of 10(-7) M biotin or biocytin (Mr 372) permitted full expression of AsGR by HepG2. Affinity chromatography of FBS on streptavidin-Sepharose abolished its ability to support AsGR production. The bound material, when released by heat denaturation and resolved by thin layer chromatography, yielded three cinnamaldehyde-positive components, of which the major detectable one migrates with authentic biocytin and reconstitutes dFBS. Sera from several species, which do not support AsGR production by HepG2, contain less than 10% biotin found in FBS as determined by direct enzyme-linked immunosorbent assay. These results indicate that biotin or a derivative is the low molecular weight serum factor of FBS required for expression of AsGR. Isolation of messenger RNA from HepG2 revealed no difference in AsGR transcripts when cells were grown in MEM-10% FBS or MEM-10% dFBS. Thus a biotin-dependent post-transcriptional event permits the ultimate expression of the AsGR by HepG2 cells.

Actins↗
PubMed1986

Lectin activity as a marker for Hodgkin disease cells.

Treatment of cultured Hodgkin disease (HD) cells with neuraminidase results in decreased reactivity of monoclonal antibody VIM-D5 with its antigen, the X hapten, a fucosyl-N-acetyllactosamine. The other feature characteristic of HD cells is the expression of high levels of ectosialyltransferase activity. We present evidence for a cause-effect relationship between these two findings in that VIM-D5 antigenicity can be restored on neuraminidase-treated HD cells by modulating transferase activity. This can be interpreted in terms of a lectin activity of the ectosialyltransferase that binds the X hapten's desialylated galactosyl residues, thereby preventing antigen recognition by VIM-D5 antibody. This proposed mechanism is indistinguishable from the autoinhibition phenomenon described for another galactophilic binding protein, the hepatic binding protein (HBP), which binds its own terminal galactosyl residues following neuraminidase treatment. We establish a close relationship between the HD galactophilic binding site and HBP in that antiserum to HBP (i) inhibits the neuraminidase-induced loss of VIM-D5 antigenicity, (ii) blocks the binding of asialoglycoprotein to hepatocytes after being absorbed by and eluted from HD cells, and (iii) recognizes a single HD protein, which in its high level of expression is unique to HD cells. The presence of lectin activity in its classic sense on the surface of HD cells is confirmed by the erythrocyte-agglutinating ability of these cells. This lectin activity, which appears to be related to an ectosialyltransferase on the surface of HD cells, may serve as a marker for the abnormal cells characteristic of HD.

Animals↗
PubMed1986

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.

Antibodies, Neoplasm↗
PubMed1982

Asialoglycoprotein receptors in hepatic regeneration.

The hepatic binding protein which is responsible for receptor-mediated endocytosis of desialylated glycoproteins undergoes cell-cycle dependent modulation of its expression. Endocytosis of desialylated orosomucoid by hepatocytes isolated from regenerating liver following two-thirds hepatectomy is reduced by 80% and returns to normal when regeneration is substantially complete. This decrease in endocytotic activity is due to selective loss of cell-surface associated receptor during liver regeneration.

Animals↗
PubMed1982

IgA interaction with the asialoglycoprotein receptor.

IgA present in normal human serum reacts with the hepatic receptor specific for asialoglycoproteins as demonstrated by inhibition of receptor-mediated erythroagglutination. Inhibition is reversibly abolished by the oxidation of the galactose or N-acetylgalactosamine residues of IgA with galactose oxidase. The site of receptor recognition appears to be the O-glycosidically linked oligosaccharides present on the hinge region of the IgAI subtype of IgA. The demonstration of a specific binding, in vitro, of IgA by the hepatic receptor suggests that the uptake of polymeric IgA by the liver in vivo may be mediated by this reaction.

Animals↗
PubMed1981

Carbohydrate-specified endocytosis: localization of ligand in the lysosomal compartment.

Carbohydrate-directed endocytosis is mediated by a receptor, the hepatic binding protein; it is responsible for the clearance of galactose-terminated glycoproteins from the circulation. This process was investigated by using lactosaminated ferritin which is recognized by this receptor. Ferritin was seen in elements of an extensive "lysosomal compartment" that includes secretory vacuoles, coated vesicles, and GERL. The compartment marked in hepatocytes by the distribution of ligand is similar to that previously described in Kupffer cells (marked by acid phosphatase reaction product and horseradish peroxidase reaction product).

Animals↗
PubMed1980

Functional segregation of hepatic receptors for asialoglycoproteins during endocytosis.

To examine whether hepatic plasma membrane receptors for asialoglycoproteins are replaced or reutilized during endocytosis, receptors on the surface of isolated hepatocytes were uniquely labeled by neuraminidase treatment. This enzyme abolishes binding of asialo-orosomucoid, a galactose-terminated glycoprotein, and restricts the ligand specificity of the cell surface receptor protein to N-acetylgalactosamine-terminated glycoproteins such as desialylated ovine submaxillary mucin. Although a much larger intracellular pool of apparently identical receptor protein is unaffected by neuraminidase, endocytosis of asialo-orosomucoid does not occur in neuraminidase-treated cells, while endocytosis of desialylated ovine submaxillary mucin is unaffected. Endocytosis of desialylated ovine submaxillary mucin, in amounts far exceeding cell surface binding capacity, is not a consequence of replacement of neuraminidase-treated surface receptor by intracellular receptor. Binding and endocytosis of asialoglycoproteins appear, therefore, to be maintained through a continuous reutilization of plasma membrane receptors rather than through their replacement from an intracellular receptor pool.

Animals↗
PubMed1980

Effects of receptor-specific antibody on the uptake of desialylated glycoproteins in the isolated perfused rat liver.

Removal of the terminal sialic acid residues from most mammalian glycoproteins results in their rapid transfer from the circulation into the liver. In vitro, these desialylated glycoproteins bind to a specific membrane-associated hepatic lectin which has a ubiquitous distribution within the liver cell. In the present study, infusion of a specific antibody to the purified lectin into the portal vein of an isolated perfused rat liver prior to injection of radiolabeled asialoorosomucoid or bilirubin reduced the rate of influx of asialoorosomucoid into the liver by over 80%, while the influx of bilirubin was unchanged. Although uptake of asialoorosomucoid remained blocked for at least 90 min after excess antibody was removed from the perfusion system, the total hepatic content of functional binding protein was nearly normal. These results indicate that interaction with specific cell surface lectin is essential for removal of asialoorosomucoid from the circulation. During the 90 min following infusion of antibody, no functional lectin is restored to the surface of hepatocytes.

Animals↗
PubMed1980

Endocytosis of asialoglycoprotein-enzyme conjugates by hepatocytes.

Desialylated glycoproteins were covalently linked to two cytochemically detectable enzymes, horseradish peroxidase or tyrosinase, and injected intravenously in amounts of approximately 0.5 per cent of total plasma glycoproteins into rats. Comparative studies of the rates of disappearance and distribution of free enzyme and conjugates established that recognition of the conjugates by the plasma membranes of hepatocytes was due to the exposure of the terminal galactose residue of the desialylated glycoproteins. At 1 minute after injection, reaction products of the enzyme markers were seen in coated pits and vesicles, elongated pinocytic channels and pleomorphic vesicles, at or close to the sinusoidal surface of hepatocytes. Vesicles containing reaction products were also observed along the lateral surfaces of hepatocytes. By 10 minutes, reaction products were seen in residual bodies near the biliary poles of hepatocytes. These studies confirm the existence of hepatocellular channels previously seen only with large excess of hemoglobin or following partial hepatectomy. They also indicate that the specific receptor for asialoglycoproteins is not restricted to the sinusoidal surfaces of hepatocytes and that transport to the catabolic sites proceeds via cytoplasmic channels and vesicles.

Animals↗
PubMed1977

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.

Acetylgalactosamine↗
PubMed1974

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.

Agglutination↗