alpha3-galactosylated glycoproteins can bind to the hepatic asialoglycoprotein receptor.

In mammals, clearance of desialylated serum glycoproteins to the liver is mediated by a galactose-specific hepatic lectin, the 'asialoglycoprotein receptor'. In humans, serum glycoprotein glycans are usually capped with sialic acid, which protects these proteins against hepatic uptake. However, in most other species, an additional noncharged terminal element with the structure Galalpha1-->3Galbeta1-->4R is present on glycoprotein glycans. To investigate if alpha3-galactosylated glycoproteins, just like desialylated glycoproteins, could be cleared by the hepatic lectin, the affinities of alpha3-galactosylated compounds towards this lectin were determined using an in vitro inhibition assay, and were compared with those of the parent compounds terminating in Galbeta1-->4R. Diantennary, triantennary and tetraantennary oligosaccharides that form part of N-glycans were alpha3-galactosylated to completion by use of recombinant bovine alpha3-galactosyltransferase. Similarly, desialylated alpha1-acid glycoprotein (orosomucoid) was alpha3-galactosylated in vitro. The alpha3-galactosylation of a branched, Galbeta1-->4-terminated oligosaccharide lowered its affinity for the membrane-bound lectin on whole rat hepatocytes 50-250-fold, and for the detergent-solubilized hepatic lectin 7-50-fold. In contrast, alpha3-galactosylation of asialo-alpha1-acid glycoprotein caused only a minor decrease in affinity, increasing the IC50 from 5 to 15 nM. Fully alpha3-galactosylated alpha1-acid glycoprotein, intravenously injected into the mouse, was rapidly cleared from the circulation, with a clearance rate close to that of asialo-alpha1-acid glycoprotein (t1/2 of 0.42 min vs. 0.95 min). Its uptake was efficiently inhibited by pre-injection of an excess asialo-fetuin. Organ distribution analysis showed that the injected alpha1-acid glycoprotein accumulated predominantly in the liver. Taken together, these observations suggest that serum glycoproteins that are heavily alpha3-galactosylated will be rapidly cleared from the bloodstream via the hepatic lectin. It is suggested that glycosyltransferase expression in murine hepatocytes is tightly regulated in order to prevent undesired uptake of hepatocyte-derived, circulating glycoproteins.     

Asialoglycoprotein clearance in the chicken: evidence for the lack of a hepatic asialoglycoprotein receptor.

The behaviour of desialylated human and chicken acid alpha1-glycoproteins is reported in chickens. Although desialylation resulted in accelerated disappearance rates from the plasma of both proteins, nevertheless the asialoproteins were eliminated much more slowly than expected on the basis of earlier observations in mammals. Analysis of tissue radioactivities, including kidney, liver, lung and spleen, failed to demonstrate any accumulation of the labeled asialoproteins in the liver, which is contrary to the situation in mammals. The main pathway for the elimination of desialylated human acid alpha1-glycoprotein in the chicken is via the kidney (tubular catabolism and/or urinary excretion). The clearance of desialylated chicken acid alpha1-glycoprotein is more complex as it involves the kidney as well as the reticuloendothelial system. These findings indicate that, unlike mammals, chickens do not possess a hepatic plasma membrane receptor for asialoglycoproteins. This raises the possibility that the presence or absence of this specific receptor may constitute a fundamental biological difference between mammals and birds.     

Tyrosine phosphorylation of the asialoglycoprotein receptor

The asialoglycoprotein (ASGP) receptor undergoes constitutive endocytosis through the coated pit/coated vesicle pathway in hepatocytes. Studies on HepG2 cells have shown that the receptor is phosphorylated at serine under control conditions and following protein kinase C stimulation. This study examined whether the ASGP receptor could also serve as a substrate for a tyrosine kinase in HepG2 cells. 32P labeling was performed in membrane preparations, in permeabilized cells at 4 degrees C, and in intact cells at 37 degrees C. The phosphorylated ASGP receptor was isolated by immunoprecipitation, hydrolyzed in 6 N HCl at 110 degrees C, and analyzed by two-dimensional high voltage electrophoresis. The receptor isolated from a membrane preparation incubated in vitro with [gamma-32P]ATP incorporated radiolabel predominantly (greater than 90%) into phosphotyrosine. ASGP receptor phosphorylation at both tyrosine and serine was detected in intact cells incubated with phosphatase inhibitors for 60 min at 37 degrees C. The presence of both phenylarsine oxide (20 microM) and sodium orthovanadate (200 microM) was required for tyrosine phosphorylation. Use of these inhibitors together resulted in a 16.4-fold increase in phosphorylation of the immunoprecipitated ASGP receptor, whereas phosphorylation of total HepG2 membrane proteins was not significantly augmented by this procedure. Selective proteolytic digestion of ASGP receptors in isolated vesicles demonstrated that the phosphorylation site identified in these studies is located at tyrosine 5 in the cytoplasmic tail.     

Biosynthesis of the human asialoglycoprotein receptor

The asialoglycoprotein receptor (ASGP-R) isolated from human liver is a single polypeptide of Mr = 46,000. Monospecific polyclonal anti-human ASGP-R antibodies as well as anti-rat ASGP-R antibodies specifically inhibit binding of 125I-asialoorosomucoid to human hepatoma Hep G2 ASGP-R. These anti-ASGP-R antibodies specifically immunoprecipitate the 46,000-Da polypeptide from hepatoma cells labeled biosynthetically with 35S-amino acid. The receptor is initially synthesized as a 40,000-Da precursor which is converted to the mature 46,000-Da species with a t1/2 of approximately 45 min. The precursor species is sensitive to endo-beta-N-acetylglucosaminidase H and becomes resistant coincident with the appearance of the mature 46,000-Da receptor. In addition, the receptor synthesized in the presence of tunicamycin is approximately 34,000 Da. The newly synthesized ASGP-R reaches the cell surface after 45-60 min, where only the mature 46,000-Da species is present. In Hep G2 cells, the ASGP-R has a mean lifetime of approximately 30 h, a value which is unaltered during maximal rates of receptor-mediated endocytosis of ASGP.     

Structures of the sialylated oligosaccharide chains in swine tracheal mucin glycoproteins

The structures of the major sialylated oligosaccharide chains in swine tracheal mucin glycoprotein were established. The oligosaccharide chains were released by treatment with alkaline borohydride and isolated by gel filtration on Bio-Gel P6 columns and chromatography on DEAE-cellulose. The neutral oligosaccharide chains in this glycoprotein have been characterized in previous studies (Rana, S.S., Chandrasekaran, E.V., Kennedy, J., and Mendicino, J. (1984) J. Biol. Chem. 259, 12899-12907; Chandrasekaran, E.V., Rana, S.S., Davila, M., and Mendicino, J. (1984) J. Biol. Chem. 259, 12908-12914). The present study reports the isolation of four monosialylated chains ranging in length from 6 to 14 sugar units, two disialylated chains containing 6 and 12 sugar units, and one trisialylated chain containing 9 sugar units. The structure of the sialylated oligosaccharides was determined by permethylation analysis and sequential hydrolysis with specific exoglycosidases. The following structures (where GalNAcol is N-acetylgalactosaminitol) were assigned to these oligosaccharides.     

Recognition of complex oligosaccharides by the multi-subunit asialoglycoprotein receptor.

The hepatic asialoglycoprotein receptor, a galactose lectin, is an oligomer of two types of similar polypeptide chains, each of which weakly binds galactose. High-affinity binding of complex oligosaccharides requires a precise geometric arrangement of receptor subunits. The two subunits have different functions in receptor assembly, ligand binding and endocytosis.     

The hepatic asialoglycoprotein receptor

Asialo- (i.e., galactose-terminal) glycoproteins are specifically and avidly recognized by a mammalian hepatic parenchymal cell receptor. This receptor, itself a glycoprotein, binds ligand molecules and directs their delivery to lysosomes for catabolism. The receptor is reutilized during this process of receptor-mediated endocytosis. Ligand specificity is conferred by galactose or N-acetyl-galactosamine at the nonreducing termini of the oligosaccharide chains. The receptor appears to be a transmembrane protein and is localized both to the cell surface as well as to several membranous intracellular compartments.     

Identification of sialylated glycoproteins from metabolically oligosaccharide engineered pancreatic cells

In this study, we investigated the use of metabolic oligosaccharide engineering and bio-orthogonal ligation reactions combined with lectin microarray and mass spectrometry to analyze sialoglycoproteins in the SW1990 human pancreatic cancer line. Specifically, cells were treated with the azido N-acetylmannosamine analog, 1,3,4-Bu₃ManNAz, to label sialoglycoproteins with azide-modified sialic acids. The metabolically labeled sialoglyproteins were then biotinylated via the Staudinger ligation, and sialoglycopeptides containing azido-sialic acid glycans were immobilized to a solid support. The peptides linked to metabolically labeled sialylated glycans were then released from sialoglycopeptides and analyzed by mass spectrometry; in parallel, the glycans from azido-sialoglycoproteins were characterized by lectin microarrays. This method identified 75 unique N-glycosite-containing peptides from 55 different metabolically labeled sialoglycoproteins of which 42 were previously linked to cancer in the literature. A comparison of two of these glycoproteins, LAMP1 and ORP150, in histological tumor samples showed overexpression of these proteins in the cancerous tissue demonstrating that our approach constitutes a viable strategy to identify and discover sialoglycoproteins associated with cancer, which can serve as biomarkers for cancer diagnosis or targets for therapy.

Electronic supplementary material

The online version of this article (doi:10.1186/s12014-015-9083-8) contains supplementary material, which is available to authorized users.     

Phosphorylation of the rat hepatocyte asialoglycoprotein receptor

Phosphorylation of asialoglycoprotein receptor was investigated by using rat hepatocytes. Analysis of the purified receptor by SDS-PAGE and autoradiogram revealed that the 64 and 54 Kd polypeptides of the receptor were phosphorylated but the 43 Kd one was not and that phosphorylation took place at the cell surface. These results are compatible with the fact that the 64 and 54 Kd species exist predominantly at the cell surface. The sites of phosphorylation were identified as Ser and Thr with no detectable radioactivity in phosphotyrosine.     

Rapid cell corpse clearance by stabilin-2, a membrane phosphatidylserine receptor

Rapid phagocytic clearance of apoptotic cells is crucial for the prevention of both inflammation and autoimmune responses. Phosphatidylserine (PS) at the external surface of the plasma membrane has been proposed to function as a general 'eat me' signal for apoptotic cells. Although several soluble bridging molecules have been suggested for the recognition of PS, the PS-specific membrane receptor that binds directly to the exposed PS and provides a tickling signal has yet to be definitively identified. In this study, we provide evidence that stabilin-2 is a novel PS receptor, which performs a key function in the rapid clearance of cell corpses. It recognizes PS on aged red blood cells and apoptotic cells, and mediates their engulfment. The downregulation of stabilin-2 expression in macrophages significantly inhibits phagocytosis, and anti-stabilin-2 monoclonal antibody provokes the release of the anti-inflammatory cytokine, transforming growth factor-beta. Furthermore, the results of time-lapse video analyses indicate that stabilin-2 performs a crucial function in the rapid clearance of aged and apoptotic cells. These data indicate that stabilin-2 is the first of the membrane PS receptors to provide tethering and tickling signals, and may also be involved in the resolution of inflammation and the prevention of autoimmunity.     

Phosphorylation of the human asialoglycoprotein receptor.

The human asialoglycoprotein receptor was isolated via immune precipitation from hepatoma Hep G2 cells following incubation with [32P]Pi. Analysis on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis revealed incorporation of 32P into both the 46 000 Da mature form of the receptor as well as the 40 000 Da precursor. The incorporated 32P was associated with phosphoserine. The degree of 32P incorporation was not substantially altered in cells endocytosing asialoglycoprotein ligand at maximal rates nor in cells in which receptor recycling was abolished by incubation with primaquine. That endocytosis and phosphorylation can be dissociated is supported by the observation that 32P is incorporated from [gamma-32P]ATP into the asialoglycoprotein receptor in isolated plasma membranes of Hep G2 cells.     

An improved protocol for N-glycosylation analysis of gel-separated sialylated glycoproteins by MALDI-TOF/TOF

Different glycoforms of some proteins have been identified as differential spots for certain diseases in 2-DE, indicating disease-related glycosylation changes. It is routine to determine the site-specific glycosylation of nonsialylated N-glycoproteins from a single gel spot, but some obstacles still exist in analyzing sialylated glycoproteins due to the lability and higher detection limit of acid glycans in MALDI-TOF/TOF analysis. Thus, we present an improved protocol here. Tryptic glycopeptides were separated and subjected to MALDI-TOF/TOF analysis, resulting in the identification of site-specific glycosylation of high-intensity glycopeptides. Sequential deglycosylation and desialylation were used to improve the identification of glycosylation sites and desialylated glycans. The site-specific glycosylation of large glycopeptides and low-intensity glycopeptides was deduced based on the masses of glycopeptides, deglycosylated peptides and desialylated glycans. By applying it to 2-DE separated human serum, the difference of N-glycosylation was successfully determined for α1-antitrypsin between different gel spots.     

NMR detection and characterization of sialylated glycoproteins and cell surface polysaccharides

Few solution NMR pulse sequences exist that are explicitly designed to characterize carbohydrates (glycans). This is despite the essential role carbohydrate motifs play in cell-cell communication, microbial pathogenesis, autoimmune disease progression and cancer metastasis, and despite that fact that glycans, often shed to extra-cellular fluids, can be diagnostic of disease. Here we present a suite of two dimensional coherence experiments to measure three different correlations (H3-C2, H3-C1, and C1-C2) on sialic acids, a group of nine-carbon carbohydrates found on eukaryotic cell surfaces that often play a key role in disease processes. The chemical shifts of the H3, C2, and C1 nuclei of sialic acids are sensitive to carbohydrate linkage, linkage conformation, and ionization state of the C1 carboxylate. The experiments reported include rigorous filter elements to enable detection and characterization of isotopically labeled sialic acids with high sensitivity in living cells and crude isolates with minimal interference from unwanted signals arising from the ~1% (13)C-natural abundance of cellular metabolites. Application is illustrated with detection of sialic acids on living cells, in unpurified mixtures, and at the terminus of the N-glycan on the 55 kDa immunoglobulin G Fc.     

Differential ligand binding by two subunits of the rat liver asialoglycoprotein receptor

The rat liver asialoglycoprotein receptor consists of two typesof subunits, a predominant polypeptide designated rat hepaticlectin 1 (RHL-1) and a minor polypeptide, RHL-2/3, that comesin two differentially glycosylated forms. The exact stoichiometryand arrangement of the subunits in the RHL oligomer are notknown. The carbohydrate-recognition domain of RHL-2/ has beenprepared by limited proteolysis of the liver receptor so thatits properties can be compared with those of the correspondingdomain of RHL-1 previously produced in a bacterial expressionsystem. Binding studies indicate that while RHL-1 binds N-acetylgalactosaminewith approximately 60-fold higher affinity than it binds galactose,RHL-2/ has only 2-fold selectivity for N-acetylgalactosamine.In general, the pattern of monosaccharide-binding specificityfor RHL-2/ is similar to RHL-1, but the discrimination of varioussugars relative to galactose is reduced substantially. Limitedproteolysis and crosslinking studies demonstrate that RHL- 2/is easily removed from the RHL oligomer in detergent solutionand that RHL-1 remains at least trimeric following removal ofRHL-2/. These studies suggest that RHL-1 forms a ligand-bindingcore while RHL-2/ acts more as an accessory subunit contributingto selective binding of certain oligosaccharide structures. asialoglycoprotein receptor binding carbohydrate recognition lectin proteolysis     

The binding of d-glucosyl-neoglycoproteins to the hepatic asialoglycoprotein receptor

The binding of D-glucosyl-neoglycoproteins and D-galactose-terminated glycoproteins to the hepatic asialoglycoprotein receptor of rabbit liver membranes were characterized and compared. The binding of both types of glycoproteins showed the same dependence on calcium concentration, sensitivity to neuraminidase, and degree of inhibition by various carbohydrate derivatives. These results, along with the observation that the rabbit liver membranes bound both the D-glucosyl- and D-galactosyl-terminated glycoproteins to the same extent, indicated that both types of glycoproteins bound to the same receptor. To confirm this hypothesis, receptors were isolated from rabbit livers by affinity chromatography using D-galactosyl-bovine serum albumin or D-glucosyl-bovine serum albumin immobilized on Sepharose. These receptors were shown to be identical by several chemical and immunological criteria as well as in their ability to bind equal amounts of D-galactosyl- and D-glucosyl-terminated glycoproteins. The conclusion is that the rabbit hepatic asialoglycoprotein receptor cannot discriminate between D-galactosyl and D-glucosyl-terminated glycoproteins and binds both.     

Phosphorylation-dependent interaction of the asialoglycoprotein receptor with molecular chaperones

A membrane protein trafficking mutant (Trf1) of HuH-7 alters the asialoglycoprotein (ASGPR) and transferrin receptor subcellular distribution. Expression cloning of a cDNA complementing the trf1 mutation led to the discovery of a novel casein Kinase 2 catalytic subunit (CK2alpha"). To purify potential CK2alpha" phosphorylation-dependent sorting proteins from cytosol, the ASGPR cytoplasmic domain was expressed as a GST fusion protein and immobilized on glutathione-agarose. In the absence of phosphorylation, only trace amounts of cytosol protein were bound and eluted. When the fusion protein was phosphorylated, a heterocomplex of potential sorting proteins was recovered. Mass spectrometer and immunoblot analysis identified five of these proteins as gp96, HSP70, HSP90, cyclophilin-A, and FKBP18. Treatment of HuH-7 with rapamycin to disrupt the heterocomplex reduced surface ASGPR binding activity by 65 +/- 5.7%. In Trf1 cells, surface-binding activity was 48 +/- 7% of that in HuH-7 and was not further reduced by rapamycin treatment. Immunoanalysis showed significantly fewer surface receptors on rapamycin-treated HuH7 cells than on nontreated cells, with no affect on the level of surface receptors in Trf1 cells. The data presented provide evidence that phosphorylation of the ASGPR cytoplasmic domain is required for the binding of specific molecular chaperones with the potential to regulate receptor trafficking.     

The clearance of glycoproteins in diabetic mice

The clearances of N-acetyl-D-glucosaminyl-bovine serum albumin (GlcNAc-BSA), L-fucosyl-bovine serum albumin (Fuc-BSA) and asialoorosomucoid (ASOR) were evaluated in alloxan-induced diabetic mice and compared to non-diabetic litter mates. The clearances of Fuc-BSA and ASOR were identical in both groups. However, the clearance of GlcNAc-BSA was considerably slower in the diabetic animals. When GlcNAc-BSA was injected into normal mice in the presence of a blood glucose of 25 to 50 mM, competition was observed. No competition was seen with Fuc-BSA or D-galactosyl-BSA (Gal-BSA) in the presence of 50 mM glucose. $\text{In}\text{,}\text{vitro}$ binding studies with purified receptor for GlcNAc/mannose confirm that glucose competes for binding of GlcNAc-BSA to this receptor.