The canine renal parathyroid hormone receptor is a glycoprotein: characterization and partial purification

Covalent labeling of the canine renal parathyroid hormone receptor with [125I]bPTH(1-34) reveals several major binding components that display characteristics consistent with a physiologically relevant adenylate cyclase linked receptor. Through the use of the specific glycosidases neuraminidase and endoglycosidase F and affinity chromatography on lectin-agarose gels, we show here that the receptor is a glycoprotein that contains several complex N-linked carbohydrate chains consisting of terminal sialic acid and penultimate galactose in a beta 1,4 linkage to N-acetyl-D-glucosamine. No high mannose chains or O-linked glycans appear to be present. The peptide molecular weight of the deglycosylated labeled receptor is 62,000 [or 58,000 if the mass of bPTH(1-34) is excluded]. The binding of [125I]bPTH(1-34) to the receptor is inhibited in a dose-dependent fashion by wheat-germ agglutinin, but not by either succinylated wheat-germ agglutinin or Ricinus communis lectin, suggesting that terminal sialic acid may be involved in agonist binding. A combination of lectin affinity chromatography and immunoaffinity chromatography affords a 200-fold purification of the covalently labeled receptor.     

Mammalian beta-adrenergic receptors. Distinct glycoprotein populations containing high mannose or complex type carbohydrate chains

Mammalian beta-adrenergic receptor binding peptides can be visualized by covalently labeling them with the photoaffinity reagent p-azido-m-[125I]iodobenzylcarazolol followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. The receptor peptides migrate as broad bands of Mr approximately equal to 62,000. In the present study, we examined the carbohydrate composition of the mammalian beta receptor through the use of specific exo- and endoglycosidases and lectin affinity chromatography. Treatment of p-azido-m-[125I]iodobenzylcarazolol-labeled beta2-adrenergic receptors from hamster lung or rat erythrocyte with the exoglycosidases neuraminidase and alpha-mannosidase provided evidence for the existence of both high mannose and complex type carbohydrate chains on beta 2-adrenergic receptors. The nonadditivity of the effect of sequential treatments with these enzymes suggested discrete populations of beta-adrenergic receptors containing either complex or high mannose type chains. Deglycosylation of receptor with endoglycosidase F results in a single labeled polypeptide at Mr = 49,000 for both systems. The same two populations of the beta receptors (high mannose or complex type chain) could also be fractionated by lectin affinity chromatography of solubilized p-azido-m-[125I]iodobenzylcarazolol-labeled receptors. The high mannose-containing receptors could be absorbed to and specifically eluted from concanavalin A-agarose. Those containing complex type carbohydrates could be adsorbed to and eluted from wheat germ agglutinin-agarose. Taken together, these data suggest that mammalian beta-adrenergic receptors contain both complex and high mannose type carbohydrate chains and that microheterogeneity of these chains likely explains the broad band pattern typically obtained on sodium dodecyl sulfate-polyacrylamide gel electrophoresis.     

The Mammalian β-Adrenergic Receptor: Structural and Functional Characterization of the Carbohydrate Moiety

Abstract

Mammalian β-adrenergic receptors are glycoproteins consisting of a single polypeptide chain of M_r ～64,000. Treatment of purified [¹²⁵I]-labeled hamster lung β-adrenergic receptor with α-mannosi-dase reveals two discrete populations of receptor consistent with previous studies using membrane bound photoaffinity-labeled receptor. Treatment of the [¹²⁵I]-labeled receptor with endo-glycosidase F results initially in the formation of a M_r ～57,000 peptide which is further converted to M_r ～49,000 suggesting that there are two N-linked carbohydrate chains per receptor polypeptide. Exoglycosidase treatments and lectin chromatography of the [¹²⁵I]-labeled receptor reveals the presence of two complex type carbohydrate chains (～10% of which are fucosylated) on ～45% of the receptors. The remaining ～55% of the receptors appear to contain a mixture of carbohydrate chains (possibly high mannose, hybrid and complex type chains). Deglycosylation of the receptor by endoglycosidase F does not appear to alter the binding affinity of the receptor for a variety of β-adrenergic agonists and antagonists. Moreover, the ability of control, α-mannosidase sensitive or insensitive (fractionated on immobilized wheat germ agglutinin) and neuraminidase, α-mannosidase or endoglycosidase F treated receptors to interact with the stimulatory guanine nucleo-tide regulatory protein in a reconstituted system were virtually identical. The deglycosylated receptor was also unaltered in its heat lability as well as its susceptibility to a variety of proteases. These findings demonstrate that the carbohydrate portion of the β-receptor does not contribute to determining either its specificity of ligand binding or coupling to the adenylate cyclase system.     

The sea urchin egg receptor for sperm: isolation and characterization of the intact, biologically active receptor

The species-specific binding of sea urchin sperm to the egg is mediated by an egg cell surface receptor. Although earlier studies have resulted in the cloning and sequencing of the receptor, structure/function studies require knowledge of the structure of the mature cell surface protein. In this study, we report the purification of this glycoprotein to homogeneity from a cell surface complex of Strongylocentrotus purpuratus eggs using lectin and ion exchange chromatography. Based on the yield of receptor it can be calculated that each egg contains approximately 1.25 x 10(6) receptor molecules on its surface. The receptor, which has an apparent M(r) of 350 kD, is a highly glycosylated transmembrane protein composed of approximately 70% carbohydrate. Because earlier studies on the partially purified receptor and on a pure, extracellular fragment of the receptor indicated that the carbohydrate chains were important in sperm binding, we undertook compositional analysis of the carbohydrate in the intact receptor. These analyses and lectin binding studies revealed that the oligosaccharide chains of the receptor are sulfated and that both N- and O-linked chains are present. Functional analyses revealed that the purified receptor retained biological activity; it inhibited fertilization in a species-specific and dose-dependent manner, and polystyrene beads coated with it bound to acrosome-reacted sperm in a species-specific manner. The availability of biochemical quantities of this novel cell recognition molecule opens new avenues to studying the interaction of complementary cell surface ligands in fertilization.     

Carbohydrate structures of acetylcholine receptor from Torpedo californica and distribution of oligosaccharides among the subunits

The structure of carbohydrates in acetylcholine receptor (AChR) from Torpedo californica is reported. Oligosaccharides released quantitatively from the whole molecule by N-oligosaccharide glycopeptidase digestion were fractionated by thin-layer chromatography and further purified by high-performance liquid chromatography. We show that more than 70% of the total oligosaccharide chains in Torpedo AChR are of the high-mannose type with the structures (Man)8(GlcNAc)2 and (Man)9(GlcNAc)2. The structure of these oligosaccharides were determined by proton nuclear magnetic resonance spectroscopy. These two types of oligosaccharides were shown to be distributed different proportions in all subunits of Torpedo AChR. We also show that several kinds of complex-type oligosaccharides comprising the rest of the carbohydrate in the protein exist mainly in the gamma and delta subunits. The structure of the carbohydrate moiety that is distributed on the four subunits of AChR was also examined by susceptibility to endo-beta-N-acetylglucosaminidase and sialidase and by binding affinity to lectins, e.g. concanavalin A, leucoagglutinating phytohemagglutinin, and wheat germ agglutinin.     

Changes in binding activity of luteinizing hormone receptors by site directed mutagenesis of potential glycosylation sites.

Site directed mutagenesis of the rat ovarian luteinizing hormone (LH) receptor cDNA was performed at each of the six potential N-linked glycosylation sites to determine the effect of putative carbohydrate chains on the activity of the membrane receptor. The conversion of Asn173 to Gln resulted in the total loss of hormone binding to the surface of the transfected cell. Mutant receptors synthesized with substitutions at the remaining potential N-linked glycosylation positions of 77, 152, 269, 277 and 291 revealed no significant change in the hormone affinity. However Asn77Gln and Asn152Gln exhibited significant decreases (approximately 80%) in the number of high affinity hormone binding sites. The changes in hormone binding activity upon elimination of the potential glycosylation sites at 77, 152 and 173 indicate the presence of functional carbohydrate chains at these positions in the rat ovarian LH/hCG receptor.     

Mannose receptor-mediated uptake of ricin toxin and ricin A chain by macrophages. Multiple intracellular pathways for a chain translocation

The role of the high mannose carbohydrate chains in the mechanism of action of ricin toxin was investigated. Ricin is taken up by two routes in macrophages, by binding to cell surface mannose receptors, or by binding of the ricin galactose receptor to cell surface glycoproteins. Removal of carbohydrate from ricin by periodate oxidation led to a large loss in toxicity via both routes of uptake by an effect on the B chain not due to a loss of galactose binding affinity. These data suggest that the carbohydrate chains of ricin B chain may be required for full toxicity. The pathway of uptake of ricin by the macrophage mannose receptor was found to differ in several respects from uptake via the galactose-specific pathway. Analysis of intoxication of macrophages by ricin in the presence of ammonium chloride suggested that mannose receptor bound ligand passes through acidic vesicles prior to translocation, unlike galactose bound ligand. Intoxication by ricin via galactose-specific uptake was potentiated by swainsonine but not by castanospermine, suggesting that ricin may be attacked by an endogenous mannosidase within the cell, and that ricin passes through either a lysosomal or a Golgi compartment prior to translocation.     

Characterization of the binding of ferritin to the rat liver ferritin receptor

The binding characteristics and specificity of the rat hepatic ferritin receptor were investigated using ferritins prepared from rat liver, heart, spleen, kidney and serum, human liver and serum, guinea pig liver and horse spleen as well as ferritins enriched with respect to either H- or L-type subunit composition, prepared by chromatofocusing of rat liver ferritin on Mono-P or by reverse-phase chromatography of ferritin subunits on ProRPC 5/10. No significant difference was apparent in the binding of any of the tissue ferritins, or of ferritins of predominantly acidic or basic subunit composition. However, serum ferritin bound with a lower affinity. The effect of carbohydrate on the ferritin-receptor binding was examined by glycosidase treatment of tissue and serum ferritins. Tissue ferritin binding was unaffected, while serum ferritin binding affinity was increased to that of the tissue ferritins. Inhibition of ferritin binding by lactoferrin was not due to common carbohydrate moieties as previously suggested but was due to direct binding of lactoferrin to ferritin. Therefore, carbohydrate residues do not appear to facilitate receptor-ferritin binding, and sialic acid residues present on serum ferritin may in fact interfere with binding. The results indicate that the hepatic ferritin receptor acts preferentially to remove tissue ferritins from the circulation. The lower binding affinity of serum ferritin for the ferritin receptor explains its slower in vivo clearance relative to tissue ferritins.     

Characterization of the binding of ferritin to the rat liver ferritin receptor

The binding characteristics and specificity of the rat hepatic ferritin receptor were investigated using ferritins prepared from rat liver, heart, spleen, kidney and serum, human liver and serum, guinea pig liver and horse spleen as well as ferritins enriched with respect to either H- or L-type subunit composition, prepared by chromatofocusing of rat liver ferritin on Mono-P or by reverse-phase chromatography of ferritin subunits on ProRPC 5/10. No significant difference was apparent in the binding of any of the tissue ferritins, or of ferritins of predominantly acidic or basic subunit composition. However, serum ferritin bound with a lower affinity. The effect of carbohydrate on the ferritin-receptor binding was examined by glycosidase treatment of tissue and serum ferritins. Tissue ferritin binding was unaffected, while serum ferritin binding affinity was increased to that of the tissue ferritins. Inhibition of ferritin binding by lactoferrin was not due to common carbohydrate moieties as previously suggested but was due to direct binding of lactoferrin to ferritin. Therefore, carbohydrate residues do not appear to facilitate receptor-ferritin binding, and sialic acid residues present on serum ferritin may in fact interfere with binding. The results indicate that the hepatic ferritin receptor acts preferentially to remove tissue ferritins from the circulation. The lower binding affinity of serum ferritin for the ferritin receptor explains its slower in vivo clearance relative to tissue ferritins.     

Preparation of Recombinant Carbohydrate Deficient Active Analogs of Human Chorionic Gonadotropin from Insect Cells

Abstract

Human chorionic gonadotropin (hCG) has four N-glycosyl chains, two in each subunit. Several analogs lacking one or more specific N-linked carbohydrate chains have been purified from insect cells by immunoaffinity chromatography on a monoclonal antibody, B17, column Traces of the hCGβ mutant present, if any, were removed by a second immunoaffinity chromatography on a column of hCGβ specific monoclonal antibody, B158. N-glycosylation was inhibited by the replacement of either Asn or Thr to Gln in the consensus sequence, -Asn × Ser/Thr-, for N-glycosylation. All analogs were overexpressed in High-Five insect cells with the expression levels ranging between 1.5 to 15 μg/ml and were found homogeneous by SDS-PAGE under nonreducing and reducing conditions. Their molecular sizes ranged between 34k to 44k. The receptor binding affinity of all the analogs was unaltered as determined by radio receptor assay using rat ovarian membranes. The availability of these analogs should facilitate studies on the effect of a specific carbohydrate chain on the conformation and in vivo properties of hCG.     

Preparation of eight ovalbumin subfractions by combined lectin affinity chromatography

Ovalbumin was fractionated by successive lectin affinity chromatography using concanavalin A/Sepharose and wheat germ agglutinin/Sepharose. Eight glycoprotein fractions, all behaving as ovalbumin on polyacrylamide gel electrophoresis, were obtained. To characterize the carbohydrate chains, the asparaginyl-carbohydrates were prepared from the Pronase digests of the ovalbumin fractions and their dansyl derivatives were analyzed by high-performance liquid chromatography. The elution profile of the dansylated asparaginyl-carbohydrates from each subfraction was compared with that from the unfractionated ovalbumin. The results indicated that the above eight subfractions could be separated from each other according to their carbohydrate chains and that three of the subfractions were homogeneous with respect to their carbohydrate chains.     

Affinity purification and chemical analysis of the interleukin-1 receptor

Interleukins-1 alpha and -1 beta regulate the metabolism of cells through a common plasma membrane receptor protein. In this study, it is demonstrated that the interleukin-1 (IL-1) receptor from detergent solutions of EL-4 cells can be stably adsorbed to nitrocellulose with full retention of IL-1 binding activity. This assay system was used to monitor the purification of the IL-1 receptor and to investigate the effects of several chemical modifications on receptor binding activity. IL-1 receptors extracted from EL-4 6.1 C10 cells can be bound to and specifically eluted from IL-1 alpha coupled to Sepharose. The affinity chromatography method resulted in the identification by polyacrylamide gel electrophoresis and silver staining of a protein of Mr 82,000 that was present in fractions exhibiting IL-1 binding activity. Experiments in which the cell-surface proteins of EL-4 cells were radiolabeled and 125I-labeled receptor was purified by affinity chromatography suggested that the Mr 82,000 protein was expressed on the plasma membrane. N-Glycanase treatment of this material showed that 23-35% of the total Mr (82,000) of the receptor is N-linked carbohydrate.     

Structural analysis and functional role of the carbohydrate component of somatostatin receptors.

SRIF receptors are membrane-bound glycoproteins. To structurally identify the carbohydrate components of SRIF receptors, solubilized rat brain SRIF receptors were subjected to lectin affinity chromatography. Solubilized SRIF receptors specifically bound to wheat germ agglutinin-lectin affinity columns but not to succinylated wheat germ agglutinin. This finding, as well as the ability of the solubilized receptor to interact with a Sambucus nigra L. lectin affinity column suggested that sialic acid residues are associated with SRIF receptors. The inability of the receptor to bind to concanavalin A, Dolichus biflorus agglutinin, Ulex europeaus I, and Jacalin lectin affinity columns suggests that high mannose, N-acetylgalactosamine, fucose, and O-linked carbohydrates are not associated with receptor. To investigate the functional role of the carbohydrate groups in brain SRIF receptors, specific sugars were selectively cleaved from SRIF receptors and the subsequent effect on the specific high affinity binding of the agonist [125I]MK 678 to SRIF receptors was determined. Treatment of the receptor with endoglycosidase D did not affect the specific binding of [125I] MK 678 to the solubilized SRIF receptors, consistent with the finding from lectin affinity chromatography that high mannose-type carbohydrate structures were not associated with SRIF receptors. Treatment of solubilized SRIF receptors with peptide-N-glycosidase F and endoglycosidases H and F reduced [125I]MK 678 binding to SRIF receptors indicating that either hybrid, or a combination of hybrid and complex N-linked carbohydrate structures, have a role in maintaining the receptor in a high affinity state for agonists. Treatment of solubilized SRIF receptors with neuraminidase from Vibrio cholera abolished high affinity agonist binding to the receptors, whereas treatment of the receptor with neuraminidase from Newcastle disease virus did not affect [125I]MK 678 binding to the receptor. These findings suggest that sialic acid residues in an alpha 2,6-configuration have a role in maintaining the SRIF receptor in a high affinity conformation for agonists. This is further indicated by studies on SRIF receptors in the pituitary tumor cell line, AtT-20. Treatment of AtT-20 cells in culture with neuraminidase (V. cholera) greatly reduces high affinity [125I] MK 678 binding sites, but did not alter the maximal ability of SRIF to inhibit forskolin-stimulated cAMP accumulation in intact AtT-20 cells. This finding suggests that the desialylated SRIF receptor is functionally active and remains coupled to GTP-binding proteins, but exhibits a reduced affinity for agonists. Treatment of AtT-20 cell membranes with neuraminidase from V. cholera was also able to greatly reduce the affinity of SRIF receptors for [125I]MK 678.(ABSTRACT TRUNCATED AT 400 WORDS)     

Structures of the carbohydrate chains of membrane glycoproteins IIb and IIIa of human platelets

Human platelet membrane glycoproteins IIb (GPIIb) and IIIa (GPIIIa), which have been proposed to be subunits of a receptor for fibrinogen, were purified from Triton X-100-solubilized platelet membranes by affinity chromatography on a concanavalin A (Con A)-Sepharose column followed by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Compositional analyses of the purified glycoproteins showed that GPIIb and GPIIIa contain 15% and 18% carbohydrate by weight, respectively, which consists of galactose, mannose, glucosamine, fucose, and sialic acid. This suggested that these glycoproteins contained N-linked carbohydrate chains. The carbohydrate chains were released from each glycoprotein by hydrazinolysis and then fractionated by ion-exchange chromatography on a Mono Q column. From each glycoprotein, mono-, di-, and trisialylated and neutral oligosaccharide fractions were obtained. The structures of these oligosaccharides were investigated by means of compositional and methylation analyses and digestion by exoglycosidase, and their reactivities to immobilized lectins were also examined. The neutral oligosaccharides, which comprised about 14% of the total oligosaccharides released from GPIIb and about 52% of that from GPIIIa, were found to be of the high mannose-type, in that they contained 5 or 6 mannose residues. On the other hand, a major part of the acidic oligosaccharides was found to consist of typical bi- and triantennary complex-type sugar chains, and much smaller amounts of tetraantennary complex-type sugar chains, and complex-type sugar chains with a fucosyl residue at a N-acetylglucosamine residue in the peripheral portion or a bisecting N-acetylglucosamine at a beta-mannosyl residue in the core portion were also detected. In conclusion, we found that GPIIb contained mainly complex-type sugar chains, whereas high mannose-type sugar chains were the predominant carbohydrate units in GPIIIa, and that the detected differences in the carbohydrate moieties of GPIIb and GPIIIa were quantitative but not qualitative.     

Rabbit liver growth hormone receptor and serum binding protein. Purification, characterization, and sequence

A putative growth hormone receptor from detergent-solubilized rabbit liver membranes and the growth hormone binding protein from rabbit serum have been purified 59,000- and 400,000-fold, respectively, primarily by affinity chromatography. Both purified proteins exhibit high affinity binding for human growth hormone; K alpha = 9-30 x 10(9) M-1 for the liver receptor and K alpha = 6 x 10(9) M-1 for the binding protein. The apparent molecular weight of the liver receptor is 130,000 by reduced sodium dodecyl sulfate gel electrophoresis, while that of the binding protein is 51,000. Both contain N-linked carbohydrate. The amino-terminal sequences of the liver growth hormone receptor and the serum binding protein were found to be the same, indicating that the binding protein corresponds to the extracellular domain of the liver receptor. Ubiquitin was found covalently linked to the liver receptor but not to the serum binding protein. The amino acid sequences of several peptides from the liver receptor were also determined after tryptic and V8 protease digestion.     

Mannoprotein of the yeast cell wall as primary receptor for the killer toxin of Saccharomyces cerevisiae strain 28

The killer toxin KT 28 of Saccharomyces cerevisiae strain 28 is primarily bound to the mannoprotein of the cell wall of sensitive yeasts. The mannoprotein of S. cerevisiae X 2180 was purified; gel filtration and SDS-PAGE indicated an estimated Mr of 185,000. The ability to bind killer toxin KT 28 increased during purification of the mannoprotein. Removing the protein part of the mannoprotein by enzymic digestion or removing the alkali-labile oligosaccharide chains by beta-elimination did not destroy the ability to bind killer toxin KT 28. However, binding activity was lost when the 1,6-alpha-linkages of the outer carbohydrate backbone were hydrolysed by acetolysis. The separated oligomannosides of the side chains also failed to bind toxin, indicating that the main mannoside chains were essential for the receptor activity. The reversible adsorption of killer toxin to mannoprotein was demonstrated by linking it covalently to Sepharose and using this material for affinity chromatography. A 90-fold increase in the specific activity of a preparation of killer toxin KT 28 was achieved in this way.     

Glycosylation of the human interferon-gamma receptor. N-linked carbohydrates contribute to structural heterogeneity and are required for ligand binding

The contribution of N-linked carbohydrates to human interferon-gamma receptor (hIFN-gamma-R) structure and function was investigated in four tumor cell lines of various tissue origin. Western and ligand blotting of native and deglycosylated, affinity-purified hIFN-gamma-R of the monocytic cell line U937 and the lymphoid cell line Raji revealed that the different sizes of hIFN-gamma-R from U937 (103 kDa) and Raji (90 kDa) cells are reduced upon either metabolic inhibition or enzymatic deglycosylation of N-linked carbohydrates to a common size of the receptor molecule with an apparent molecular mass of 73 kDa for both cell lines, indicating that heterogeneity in hIFN-gamma-R size is largely due to differential glycosylation. In all cell lines investigated, inhibition of N-linked glycosylation or modulation of carbohydrate processing did not prevent receptor transport to the cell membrane, but blocked hIFN-gamma binding capacity of membrane-expressed receptor molecules, as revealed by specific binding of hIFN-gamma-R-specific monoclonal antibody and specific binding of 125I-labeled hIFN-gamma. These data suggest that a lack of complex-type N-linked carbohydrates is associated with a complete loss of receptor function, i.e. high affinity binding capacity. Recovery of hIFN-gamma binding of deglycosylated receptors was achieved upon affinity purification and adsorption to nitrocellulose membranes, indicating that the carbohydrate side chains themselves do not directly contribute to the ligand binding epitope but seem to be essential for appropriate conformation of the receptor protein in the cell membrane.     

O-linked protein glycosylation structure and function

There has been a recent resurgence of interest in the post-translational modification of serine and threonine hydroxyl groups by glycosylation, because the resulting O-linked oligosaccharide chains tend to be clustered over short stretches of peptide and hence they can present multivalent carbohydrate antigenic or functional determinants for antibody recognition, mammalian cell adhesion and microorganism binding. Co-operativity can greatly increase the affinity of interactions with antibodies or carbohydrate binding proteins. Thus, in addition to their known importance in bearing tumour associated antigens in the gastrointestinal and respiratory tracts, glycoproteins with O-linked chains have been implicated as ligands or co-receptors for selectins (mammalian carbohydrate binding proteins). Microorganisms may have adopted similar mechanisms for interactions with mammalian cells in infection, by having relatively low affinity ligands (adhesins) for carbohydrate binding, which may bind with higher affinity due to the multivalency of the host ligand and which are complemented by other virulence factors such as interactions with integrin-type molecules. In addition to specific adhesion signals from O-linked carbohydrate chains, multivalent O-glycosylation is involved in determining protein conformation and forming conjugate oligosaccharide-protein antigenic, and possible functional determinants.     

Isolation and characterization of the two glycosylation isoforms of low molecular weight mannose 6-phosphate receptor from bovine testis. Effect of carbohydrate components on ligand binding.

Low molecular weight mannose 6-phosphate receptor from bovine testis exhibits two isoforms on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with Mr values of 45,000 (MPR-2A) and 41,000 (MPR-2B), respectively. Each isoform was purified to near homogeneity by the sequential application of differential centrifugation and affinity chromatography. The isoforms contain a common polypeptide core, but differ in their carbohydrate content. Treatment with specific endoglycosidases demonstrated that each isoform contains two high mannose and/or hybrid and two complex N-linked oligosaccharide chains. The results obtained from treatment of each isoform with endo-beta-galactosidase and neuraminidases and from lectin affinity chromatography reveal that MPR-2A contains a linear polylactosamine sequence(s) comprised of approximately 5 lactosamine units. A majority of the outer branches of the complex chains associated with MPR-2A are terminated with sialic acid residues. In contrast, MPR-2B lacks a polylactosamine sequence and a majority of the outer branches of the complex chains are terminated with galactose residues. MPR-2A exhibited a lower affinity than MPR-2B for mannose 6-phosphate-containing ligands. Treatment of MPR-2A with endo-beta-galactosidase and/or neuraminidases followed by affinity chromatography revealed that polylactosamine and sialic acid residues impair the ability of MPR-2A to bind ligands.     

The beta 2-adrenergic receptors of human epidermoid carcinoma cells bear two different types of oligosaccharides which influence expression on the cell surface.

The beta 2-adrenergic receptors of the human epidermoid carcinoma A431 cells reside on two polypeptide chains revealed by photoaffinity labelling with [125I]iodocyanopindolol-diazirine. These proteins correspond to two distinct populations of N-asparagine-linked glycoproteins: the 55-52 kDa molecules are associated with complex carbohydrate chain(s), the 65-63 kDa component with polymannosidic carbohydrate chain(s). Both types of receptors are present in preconfluent cells, but only the polymannosidic type is found in the postconfluent cells. Moreover, complex chains appear to be associated with the receptors with the highest affinity for (-)-isoproterenol and polymannosidic chains with the receptors with the lowest affinity for this agonist. the carbohydrate moiety of the beta-adrenergic receptor is involved in the expression and function of the beta 2-adrenergic receptors at the surface of the A431 cells, since tunicamycin and monensin, complete and partial inhibitors of glycosylation respectively, diminish the number of binding sites at the cell surface and increase the total number of sites in the cell. In these conditions a diminution of cyclic AMP accumulation is also observed.