Parathyroid hormone receptors are plasma membrane glycoproteins with asparagine-linked oligosaccharides

In the preceding article, we described physicochemical and kinetic properties of parathyroid hormone (PTH) receptors in clonal rat osteosarcoma cells (ROS 17/2.8) using photoaffinity ligand labeling and showed that the physiologically relevant receptor-ligand complex has an apparent Mr = 80,000. In this study, the photoaffinity labeled Mr = 80,000 receptor was localized exclusively on the cell surface plasma membrane and its glycoprotein nature was demonstrated through the use of lectin affinity-chromatography and specific exo- and endoglycosidases. Rinsing ROS cells, preincubated in the dark with 125I-labeled [Nle8, N-epsilon-(4-azido-2-nitrophenyl)Lys13,Nle18,Tyr34]bovine PTH-(1-34)-NH2 (NAP-NlePTH) (4 h, 15 degrees C, equilibrium conditions) with acidic phosphate-buffered saline (pH 2.5, 30 s, 4 degrees C) before photolysis resulted in selective and nearly total disappearance of the labeled Mr = 80,000 receptor. PTH receptor integrity to acid rinsing and photolysis was shown by relabeling the Mr = 80,000 receptor after a second incubation of these cells with 125I-labeled NAP-NlePTH, followed by photolysis. Adsorption of Triton X-100-solubilized, 125I-labeled NAP-NlePTH receptors to wheat germ agglutinin-agarose is nearly complete and highly selective, and elution with N-acetylglucosamine resulted in virtually total recovery of the labeled receptors from the column. The wheat germ agglutinin-retarded PTH receptors show increased electrophoretic mobility upon treatment with neuraminidase which was inhibited by simultaneous addition of 2,3-dehydro-3-desoxy-N-acetylneuraminic acid, a specific neuraminidase inhibitor. Endoglycosidase F treatment of the Mr = 80,000 receptors generated a single, labeled polypeptide with a Mr = 59,000 which migrated as a narrow band. PTH receptors on ROS 17/2.8 cells appear to be monomeric plasma membrane glycoproteins with an apparent Mr of 80,000 which contain a Mr = 59,000 polypeptide backbone and a polymeric arrangement of N-acetylglucosamine with N-acetylneuraminic acid as major terminal sugar residues.     

Characterisation of the asparagine-linked oligosaccharides from Trypanosoma brucei type-I variant surface glycoproteins

The complete primary structures of the Asn-linked oligosaccharides from the conserved glycosylation site of the type-I variant surface glycoproteins of Trypanosoma brucei MITat 1.4 and MITat 1.6 were determined using a combination of exoglycosidase digestions, permethylation analysis, acetolysis and 1H NMR. Both variants contained almost exclusively oligomannose-type oligosaccharides, identical in structure to those of mammalian glycoproteins. The oligosaccharides ranged in size from (Man)9(GlcNAc)2 to (Man)5(GlcNAc)2. The relative abundance of each component was similar in both variants. The major components were (Man)8(GlcNAc)2 and (Man)7(GlcNAc)2 with slightly less (Man)9(GlcNAc)2 and (Man)6(GlcNAc)2 and much less (Man)5(GlcNAc)2. Both variants also contained the same structural isomers. The close similarity of the oligomannose series indicates identical processing at the conserved site in both variants.     

Rapid characterization of asparagine-linked oligosaccharides isolated from glycoproteins using a carbohydrate analyzer

Chromatographic methods were developed for the separation and characterization of acidic (sialylated) and neutral (asialo-complex and high-mannose) oligosaccharides released from glycoproteins with peptide N-glycosidase F. endo-beta-N-acetylglucosaminidase F and endo-beta-N-acetylglucosaminidase H using a carbohydrate analyzer (Dionex BioLC). All the carbohydrate separations were carried out on a polymeric pellicular anion-exchange column HPIC-AS6/CarboPac PA-1 (Dionex) using only two eluants namely, 0.5 M NaOH and 3% acetic acid/NaOH pH 5.5, which were mixed with water to generate various gradients. Developed conditions for quantitative detection of carbohydrates with pulsed amperometry were necessary to obtain steady baselines at 0.1-0.3 microA output with suitable sensitivity (less than 5 pmol) in separations employing a variety of acidic and alkaline sodium acetate gradients. Oligosaccharides released from heat-denatured and trypsin-treated glycoproteins were purified initially from large-scale digestion (greater than 0.1 g) by extraction of peptide material into phenol/chloroform and finally by ion-exchange chromatography of the acqueous phase. Oligosaccharides isolated from the peptide N-glycosidase digests of bovine fetuin, human transferrin and alpha 1-acid glycoprotein gave multiple peaks in each charge group in separations based on the charge content at pH 5.5. Alkaline sodium acetate gradients were developed to obtain oligosaccharide maps of the glycoproteins within 60 min, in which separated oligosaccharides eluted in the order of neutral, mono-, di-, tri- and tetra-sialylated species based on both charge, size and structure. Baseline separations were obtained with neutral oligosaccharide types but mixtures of high-mannose and complex types were poorly resolved. The high-mannose peaks were eliminated specifically from complex oligosaccharides by digesting with alpha-mannosidase. Treatment with beta-galactosidase, beta-N-acetylglucosaminidase and alpha-mannosidase resulted in a decrease of the oligosaccharide elution times corresponding to the number of sugar residues lost, the profile of changes was highly reproducible. In contrast, treatment with alpha-L-fucosidase, endo-beta-N-acetylglucosaminidase F and endo-beta-N-acetylglucosaminidase H resulted in an increase in their corresponding oligosaccharide retention times similar to the presence of an additional sugar residue. Conditions developed for separation of the reduced oligosaccharides and also a mixture of monosaccharide to oligosaccharide containing about 15 sugar residues within 30 min were useful in determining the effect of endo- and exo-glycosidases on porcine thyroglobulin oligosaccharides. Changes in elution time of the oligosaccharides following specific glycosidase digestions combined with methylation analysis provided a rapid and sensitive tool for confirmation of the carbohydrate primary structures present in thyroglobulin.     

Complex-type asparagine-linked oligosaccharides in glycoproteins synthesized by Schistosoma mansoni adult males contain terminal beta-linked N-acetylgalactosamine

Many studies have shown that the human blood fluke Schistosoma mansoni contains glycoproteins whose oligosaccharide side chains are antigenic in infected hosts. We report here that adult male schistosomes synthesize glycoproteins containing complex-type N-linked chains that have structural features not commonly found in mammalian glycoproteins. Adult male worms were incubated in media containing either [3H]mannose, [3H]glucosamine, or [3H]galactose, and the metabolically radiolabeled oligosaccharides on newly synthesized glycoproteins were analyzed. Schistosomes synthesize triantennary- and biantennary-like complex-type asparagine-linked chains that contain mannose, fucose, N-acetylglucosamine, and N-acetylgalactosamine. Interestingly, none of the complex-type chains contain sialic acid, and few of the chains contain galactose. Since N-acetylgalactosamine is not a common constituent of mammalian-derived N-linked chains, we investigated the position and linkage of this residue in the schistosome-derived glycopeptides. Virtually all of the N-acetylgalactosamine was beta-linked and in a terminal position. The unusual features of the S. mansoni glycoprotein oligosaccharides support the possibility that they may be involved in the host immune response to infection.     

Structural characterization of the asparagine-linked oligosaccharides from Trypanosoma brucei type II and type III variant surface glycoproteins

The complete primary structures of the major Asn-linked oligosaccharides from the type II variant surface glycoproteins (VSGs), MITat 1.2 and MITat 1.7, and the type III VSG, MITat 1.5, were determined using a combination of exo- and endoglycosidase digestions, methylation analysis, acetolysis, and 500 MHz 1H NMR spectroscopy. Each variant contained classical branched oligomannose-type and biantennary complex oligosaccharides, a proportion of the latter substituted with terminal alpha(1-3)-linked galactose residues, the first report of the presence of this epitope in Trypanosoma brucei. In addition both the type II variants contained relatively large amounts of the unusual small oligomannose-type oligosaccharides, Man4GlcNAc2 and Man3GlcNAc2, and a diverse array of novel branched poly-N-acetyllactosamine oligosaccharides, similar but not identical to those from mammalian glycoproteins. These latter structures were also partially substituted with terminal alpha(1-3)-linked galactose residues. Glycosylation in the type II variants showed site specificity in that the poly-N-acetyllactosamine and Man(9-5)GlcNAc2 oligosaccharides were located exclusively at Asn-glycosylation site 1 very close to the C terminus, whereas the Man(4-3)GlcNAc2 and biantennary complex oligosaccharides were located exclusively at site 2. This is the first report of the presence of poly-N-acetyllactosamine oligosaccharides in protozoa.     

Biosynthesis of sulfated asparagine-linked oligosaccharides on bovine lutropin

The asparagine-linked oligosaccharides on bovine lutropin (bLH) are unusual, containing GalNAc and sulfate but no galactose or sialic acid. Oligosaccharides from metabolically radiolabeled or purified bLH consist of non- (neutral), mono- (S-1), and di- (S-2) sulfated structures. We have previously shown that S-2 is a complex type oligosaccharide bearing two peripheral branches with the sequence SO4----GalNAc----GlcNAc attached to a typical Man3GlcNAc2 core (Green, E.D., van Halbeek, H., Boime, I., and Baenziger, J.U. (1985) J. Biol. Chem. 260, 15623-15630). We have now characterized the S-1 oligosaccharides on bLH which, in contrast to S-2, consist of several different structures of both the hybrid and complex types. The sulfate on S-1 oligosaccharides is located exclusively within the peripheral sequence SO4----GalNAc----GlcNAc. The GalNAc bearing hybrid structures, either with or without sulfate, cannot be processed to mono- or disulfated complex oligosaccharides due to the inability of either alpha-mannosidase II or GlcNAc-transferase II to act on GalNAc containing oligosaccharides. Since both Gal and GalNAc are added to oligosaccharides on some pituitary hormones, for example bovine and ovine follitropin and human lutropin, the Gal- and GalNAc-transferases appear to be key elements in regulating the synthesis of sulfated oligosaccharides on bLH and the other pituitary glycoprotein hormones.     

Characterization of asparagine-linked oligosaccharides on a mouse submandibular mucin

The asparagine-linked oligosaccharides from an adult femalemouse submandibular gland mucin were released by treatment withpeptide-N⁴-(N-acetyl-ß-glucosaminyl)asparagine amidaseF or endo-ß-N-acetylglucosaminidase H. Endo-ß-N-acetylglucosaminidaseH appeared to be more effective at releasing the asparagine-linkedoligosaccharides from this mucin than was peptide-N⁴-(N-acetyl-ß-glucosaminyl)-asparagineamidase F. After quantitative reductive labelling with the fluorophore,8-aminonaphthalene-1, 3, 6-sulphonic acid, the oligosaccharideswere separated by polyacrylamide gel electrophoresis and isolated.The individual oligosaccharides were sequenced by a batteryof recombinant exoglycosidases. Approximately 50% of the oligosaccharideswere of the high-mannose type. The five-mannose member of thisfamily was the most prevalent. The second group of oligosaccharideswere of the non-bisected hybrid type. No complex asparagine-linkedoligosaccharides were detected. The hybrids exhibited both biantennaryand triantennary branching patterns. The triantennary hybridwas the most common hybrid at >30% of all oligosaccharides.With 98% of the hybrid oligosaccharides sialylated and all lackinga bisecting N-acetylglucosamine, these oligosaccharides as agroup have been only rarely observed in other glycoproteins.The fully sialylated triantennary hybrid may be unique. asparagine-linked oligosaccharides biantennary salivary mucin sialylated hybrid triantennary     

Use of N-glycanase to release asparagine-linked oligosaccharides for structural analysis

An enzymatic procedure for releasing asparagine-linked oligosaccharides from glycoproteins by treatment with N-glycanase (peptide-N4-(N-acetyl-beta-glucosaminyl) asparagine amidase) has been investigated. Ribonuclease B, transferrin, fetuin, and alpha 1-acid glycoprotein were treated with N-glycanase and the released oligosaccharides were radiolabeled with NaB3H4. Lectin staining of the N-glycanase-treated proteins indicated that the deglycosylation reactions had proceeded to completion. The labeled carbohydrate chains were analyzed by HPLC on Micro-Pak AX-5 and AX-10 columns. The proportion of high-mannose and bi-, tri-, and tetraantennary complex chains obtained from each glycoprotein was in agreement with literature values. These results demonstrate that N-glycanase provides a simple method to release all common classes of asparagine-linked oligosaccharides from a glycoprotein in a form that can be radiolabeled directly for structural analysis.     

High-performance anion-exchange chromatography of asparagine-linked oligosaccharides.

Oligosaccharides released enzymatically by N-glycanase from fetuin, alpha-acid glycoprotein, human chorionic gonadotropin, platelet-derived growth factor, and kallikrein were chromatographed on a polymeric pellicular anion-exchange column at pH values of 5 and 13. Separations occurred into groups of peaks containing the same number of sialic acids with an additional separation dependent upon the nature of the antennary structure present. High pH conditions were required for the optimum separation of fetuin oligosaccharides, while low pH conditions significantly improved resolution of oligosaccharides obtained from the other glycoproteins. The analytical separation of oligosaccharides under conditions of low pH has important implications in the development of chromatographic mapping and identification techniques for N-linked oligosaccharides present on recombinant proteins.     

Structures of asparagine-linked oligosaccharides of human placental fibronectin

The asparagine-linked sugar chains of fibronectin purified from human placenta were quantitatively released as oligosaccharides by hydrazinolysis. After N-acetylation, they were converted to radioactive oligosaccharides by NaB3H4 reduction. The radioactive oligosaccharides were fractionated by their charge on an anion-exchange column chromatography. All of the acidic oligosaccharides could be converted to neutral oligosaccharides by sialidase digestion. These oligosaccharides were then fractionated by serial affinity chromatography using immobilized lectin columns. Study of each oligosaccharide by sequential exoglycosidase digestion and methylation analysis revealed the following information as to the structures of the sugar chains of human placental fibronectin: 1) nine sugar chains are included in one molecule; 2) all sialic acid residues are exclusively linked at the C-3 position of the galactose residues; 3) bi-, tri-, and tetraantennary complex-type oligosaccharides with the Man alpha 1----6(Man alpha 1----3)Man beta 1----4GlcNAc beta 1----4 (+/- Fuc alpha 1----6)-GlcNac as their cores were found; 4) the bisecting N-acetylglucosamine residue and the Gal beta 1----4GlcNAc beta 1----repeating groups are included in some of the sugar chains.     

New methods for rapid separation and detection of oligosaccharides from glycoproteins

Ion exchange chromatography at high pH with pulsed amperometric detection of the eluted glycans permitted resolution of the eight major components in the mixture of asparagine-linked glycans derived from the single glycosylation site of ovalbumin. The individual glycans were first partially separated according to size, and were characterized by fast atom bombardment-mass spectrometry and specific enzymatic degradation with beta-galactosidase and endoglycosidase H; subnanomolar quantities of all eight components could subsequently be unequivocally identified in the elution diagram. To ascertain that the chromatographic separation of the ovalbumin glycan mixture was not restricted to the asparagine-linked glycans, it was established that the corresponding mixture of reducing oligosaccharides (asparagine removed) or Asn-oligosaccharides blocked at the alpha-amino group with biotin gave very similar resolution of the eight glycans. In the absence of pure reference compounds, the quantification of the different glycans by the amperometric detection system was evaluated by comparing the electrochemical signal to the molecular ion peak intensity in the mass spectrometer. With one exception, the two methods were in good agreement, which suggests that the amperometric detection system yields a valid quantitative estimate for most of these chemically related compounds.     

Structure of asparagine-linked oligosaccharides on human and rabbit testosterone-binding globulin

We have previously demonstrated, using two-dimensional polyacrylamide gel electrophoresis, that much of the microheterogeneity of human (h) and rabbit (rb) testosterone-binding globulin (TeBG) is due to differential glycosylation of a single protomer. Since glycosylation has been shown to be a physiologically important modification of proteins, we have examined the structure of the oligosaccharide chains attached to hTeBG and rbTeBG to facilitate future studies on the mechanisms of action of the proteins. The structures of the oligosaccharides attached to TeBG were determined by using serial lectin chromatography. About 10% of the TeBG from castrated male rabbits and about 20% of the TeBG from pregnant rabbits and from a human sample were not retained on a column of immobilized concanavalin-A (Con-A). This fraction would consist of TeBG with attached asparagine (Asn)-linked tri- and tetraantennary complex and serine/threonine (O)-linked oligosaccharides as well as non-glycosylated forms. None of the lectins used to subfractionate these species was effective. Forty to 50% of the TeBG applied to Con-A possessed biantennary complex oligosaccharides as indicated by the fact that it could be eluted with 10 mM 1-O-methyl-alpha-D-glucopyranoside and by its retention on wheat germ agglutinin (WGA). About 8% of the biantennary complex oligosaccharides on hTeBG and none of those on rbTeBG were fucosylated on the chitobiose core, as determined by chromatography on Lens culinaris lectin (LcH). Galactosylated oligosaccharides were also present on the TeBG in this fraction as indicated by its interaction with Ricinus communis-I (RCA-I). Thirty to 40% of the TeBG applied to Con-A was retained and could be eluted with 0.5 M methyl-alpha-D-mannopyranoside. This fraction contains TeBG possessing high mannose-type, hybrid-type, and complex galactosylated glycans as determined by chromatography on Con-A, WGA, and RCA-I. Evidence based on the binding of mannoside-eluted TeBG to Con-A, WGA, and RCA-I indicated that at least the TeBG in this fraction contained two glycosylation sites and that the sites were differentially glycosylated.     

The histochemistry of asparagine-linked oligosaccharides of glycoproteins in mammalian and avian tissues as studied by digestion with almond glycopeptidase

Summary In a series of mammalian and avian tissues, asparagine-linked oligosaccharides of glycoproteins have been localized histochemically by mearis of combined periodic acid-Schiff (PAS) and almond glycopeptidase digestion procedures. According to the present results, the particular saccharide residues were found to be localized primarily in glycoproteins of connective and muscular tissue elements but were hardly visualizable or absent in glycoproteins of epithelial tissue elements. In view of the substrate specificity of almond glycopeptidase and the known staining selectivity of the PAS reaction, the majority of connective and muscular tissue elements are thought to be the main sites where plasma types of glycoproteins are involved.     

Structural study of the asparagine-linked oligosaccharides of lipophorin in locusts

The complete structure of oligosaccharides from locust lipophorin was studied. The asparagine-linked oligosaccharides were first liberated from the protein moiety of lipophorin by digestion with almond glycopeptidase (N-oligosaccharide glycopeptidase, EC 3.5.1.52). Two major oligosaccharides (E and F), separated by subsequent thin-layer chromatography, were analyzed by methylation analysis and 1H-NMR. Based on the experimental data, the whole structure of oligosaccharide E was identified as Man alpha 1----2Man alpha 1----6(Man alpha 1----2Man alpha 1----3) Man alpha 1----6(Man alpha 1----2Man alpha 1----2Man alpha 1----3)Man beta 1----4GlcNAc beta 1----4GlcNAc. The data also revealed that oligosaccharide F is identical with oligosaccharide E in the structure, except for one glucose residue that is linked to the nonreducing terminal Man alpha 1----2 residue.     

Histochemical demonstration of asparagine-linked oligosaccharides in glycoproteins of human placenta and umbilical cord tissues by means of almond glycopeptidase digestion

Almond glycopeptidase is an enzyme which cleaves specifically beta-aspartylglucosylamine linkages in glycoproteins with asialo-carbohydrate moieties. With this enzyme, it was possible to demonstrate the localization of asparagine-linked oligosaccharides in glycoproteins of human placenta and umbilical cord tissues. In these tissues, the oligosaccharides were shown to react positively for a series of histochemical procedures for neutral complex carbohydrates such as periodic acid-Schiff (PAS), peroxidase-labelled Ricinus communis agglutinin-I-diaminobenzidine (PO-RCA-DAB) and concanavalin A-peroxidase-diaminobenzidine (Con A-PO-DAB). The asparagine-linked carbohydrates were localized in the placental villi, blood vessels and perivascular tissues and the umbilical cord blood vessels and matrix. The results of previous biochemical analyses performed upon the same tissues (Takahashi et al., 1981) have corroborated the results of the histochemical studies. The present results appear to substantiate the usefulness of almond glycopeptidase for the histochemical demonstration of the particular oligosaccharides of glycoproteins in tissues in general.     

Structural analyses of asparagine-linked oligosaccharides of porcine pancreatic kallikrein

The structures of asparagine-linked oligosaccharides of porcine pancreatic beta-kallikrein are reported. Asparagine-linked neutral oligosaccharides were released by N-oligosaccharide glycopeptidase digestion, and the reducing ends of the oligosaccharides were derivatized with a fluorescent reagent, 2-aminopyridine. The mixture of pyridylamino oligosaccharides was separated by reverse-phase and amide-adsorption high-performance liquid chromatography. The pyridylamino oligosaccharides were separated into more than 50 kinds of oligosaccharides. The structures of 5 kinds of triantennary and 12 kinds of tetraantennary oligosaccharides were determined by the use of high-resolution proton nuclear magnetic resonance spectroscopy and methylation analysis. Furthermore, the structures of five kinds of oligomannose-type oligosaccharides were elucidated by a combination of exoglycosidase digestion and high-performance liquid chromatography. 1H NMR data for 14 out of the 17 kinds of N-acetyllactosamine-type oligosaccharides reported here have not previously been described in the literature. (1) It has been shown that fucose containing tri- and tetraantennary oligosaccharides is predominant in porcine pancreatic beta-kallikrein B. (2) It has also been shown that the heterogeneity of the structure in these types of oligosaccharides is derived from the variety of the positions of galactose residues linked to outer N-acetylglucosamine residues. (3) The distribution of oligosaccharides into two glycosylation sites, asparagine-95 and asparagine-239, of beta-kallikrein B was determined. It has been found that oligomannose-type oligosaccharides are exclusively present at asparagine-239, although N-acetyllactosamine-type oligosaccharides occur at both glycosylation sites.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structures of asparagine-linked oligosaccharides of the glycoprotein fetuin having sialic acid linked to N-acetylglucosamine

In the accompanying paper (Bendiak et al., 1989), the separation of a series of oligosaccharides released from asparagine residues of fetuin was described. A series of NMR experiments, which included one- and two-dimensional nuclear Overhauser enhancement, two-dimensional correlation spectroscopy, and two-dimensional relayed-coherence spectroscopy, as well as permethylation analyses, established a Gal beta 1----3(NeuAc alpha 2----6)GlcNAc beta 1----4Man unit common to a series of purified structures. These oligosaccharides contained either three, four, or five glycosidically linked sialic acid residues. The NeuAc residue in alpha 2----6 linkage to GlcNAc gives rise to diagnostic chemical shift perturbations of particular proton signals in the oligosaccharides.     

Analysis of asparagine-linked oligosaccharides by sequential lectin affinity chromatography

Lectins are proteins that specifically bind to a particular carbohydrate structure. Affinity chromatography with immobilized lectins is a quite effective technique not only for the fractionation of glycoproteins or oligosaccharides but also their structural assessment. In this article, we focus on the separation of glycopeptides and oligosaccharides derived from glycoproteins by affinity chromatography on immobilized lectin columns.