Pituitary glycoprotein hormone oligosaccharides: structure, synthesis and function of the asparagine-linked oligosaccharides on lutropin, follitropin and thyrotropin

Luteinizing hormone (LH), follicle-stimulating hormone (FSH) and thyroid-stimulating hormone (TSH) from pituitary and chorionic gonadotropin (CG) from placenta are a family of closely related glycoproteins. Each hormone is a heterodimer, consisting of an alpha- and a beta-subunit. Within an animal species, the alpha-subunits of all four glyco-protein hormones have an identical amino acid sequence, whereas each beta-subunit is distinct and confers hormone-specific features to the heterodimer. LH and FSH are synthesized within the same cell, the gonadotroph of the anterior pituitary, but are predominantly stored in separate secretory granules. We have characterized the asparagine-linked oligosaccharides on bovine, ovine and human LH, FSH and TSH. The various pituitary hormones were found to contain unique sulfated oligosaccharides with the terminal sequence SO4-4GalNAc beta 1----4GlcNAc beta 1----2Man alpha, sialylated oligosaccharides with the terminal sequence SA alpha Gal beta GlcNAc beta Man alpha, or both sulfated and sialylated structures. Despite synthesis of LH and FSH in the same pituitary cell, sulfated oligosaccharides predominate on LH while sialylated oligosaccharides predominate on FSH for all three animal species. We have examined the reactions leading to synthesis of the sulfated oligosaccharides to determine which steps are hormone specific. The sulfotransferase is oligosaccharide specific, requiring only the sequence GalNAc beta 1----4GlcNAc beta 1----2Man alpha. In contrast, the GalNAc-transferase appears to be protein specific, accounting for the preferential addition of GalNAc to LH, TSH, and free (uncombined) alpha-subunits compared with FSH and other pituitary glycoproteins. The predominance of sulfated oligosaccharide structures on LH may account for sorting of LH and FSH into separate secretory granules. Differences in sulfation and sialylation of LH, FSH and TSH may also play a role in the regulation of hormone bioactivity.     

The sialylated oligosaccharides of recombinant bovine lutropin modulate hormone bioactivity

The Asn-linked oligosaccharides from bovine lutropin (bLH(Pit] are predominantly dibranched complex-type structures with the terminal sequence SO4-4GalNAc beta 1,4GlcNAc beta 1,2Man alpha. Recombinant bLH expressed in Chinese hamster ovary cells (bLH(CHO] bears di- (60%) and tribranched (30%) complex-type oligosaccharides; however, these terminate in the sequence Sia alpha 2,3Gal beta 1,4GlcNAc beta 1,2Man alpha. In contrast to the limited spectrum of oligosaccharide structures present on recombinant bLH(CHO), the endogenous glycoproteins synthesized by CHO cells bear a heterogeneous array of Asn-linked oligosaccharides with 0, 1, 2, 3, or 4 sialic acid moieties. The sialic acid moieties on the Asn-linked oligosaccharides of both endogenous glycoproteins and recombinant bLH(CHO) are exclusively alpha 2,3-linked, suggesting that the alpha 2,6-sialyl-transferase is not active in CHO cells. The bioactivities of bLH(Pit) and bLH(CHO) were compared using MA-10 cells following sequential digestion with neuraminidase and beta-galactosidase. Neither the ED50 (dose producing 50% of the maximum response) for progesterone production (7.2 ng/ml) nor the Pmax (maximum level of progesterone produced) (470 ng/ml) was altered for bLH(Pit) by these treatments, consistent with the absence of either sialic acid or Gal on bLH(Pit). The ED50 for progesterone production by recombinant bLH(CHO) (16.4 ng/ml) was significantly greater than for bLH(Pit) but was reduced to 5.3 ng/ml following removal of terminal sialic acid. Removal of the subterminal Gal was without further effect. The Pmax for bLH(CHO) (180 ng/ml) was not altered by these treatments. The reduction in bLH(CHO) bioactivity caused by the presence of terminal sialic acid suggests that the presence of terminal sulfate on bLH(Pit) oligosaccharides may also reduce its bioactivity and may play a modulatory role in regulating hormone bioactivity.     

Cell-free sulfation of human and bovine pituitary hormones. Comparison of the sulfated oligosaccharides of lutropin, follitropin, and thyrotropin

Lutropin (LH), follitropin (FSH), and thyrotropin (TSH) from pituitary and human chorionic gonadotropin (hCG) from placenta are a family of glycoprotein hormones, each with an alpha and beta subunit. The alpha subunits of all four hormones have the same amino acid sequence, whereas biological specificity is determined by their unique beta subunits. The carbohydrate compositions of these hormones indicate the structures of their Asn-linked oligosaccharides are not identical. Sulfate is present on most, but not all, of these hormones, and for bovine LH is attached to GalNAc (Green, E.D., van Halbeek, H., Boime, I., and Baenziger, J.U. (1985) J. Biol. Chem. 260, 15623-15630). We used a reconstituted cell-free system to study sulfation of bovine (b) and human (h) glycoprotein hormones and its relationship to glycosylation. Exogenously added bLH, bTSH, bFSH, hLH, and hTSH are sulfated exclusively on the oligosaccharides of both alpha and beta subunits. The distribution of sulfated oligosaccharide structures varies among the hormones and appears to result from differences in the extent and/or pathway of oligosaccharide processing. Significant amounts of disulfated, dibranched complex oligosaccharides are present on all the sulfated hormones. Human FSH is not susceptible to sulfation unless first treated with neuraminidase. The sulfated oligosaccharides obtained from bovine FSH and desialylated human FSH are unlike those of the other hormones. Therefore, there is differential processing of the oligosaccharides on pituitary hormones. For FSH and LH, which are believed to be synthesized in the same cell, we would suggest that the unique beta subunits may regulate processing of all oligosaccharides present on the alpha-beta dimers.     

Asparagine-linked oligosaccharides on lutropin, follitropin, and thyrotropin. II. Distributions of sulfated and sialylated oligosaccharides on bovine, ovine, and human pituitary glycoprotein hormones

The asparagine-linked oligosaccharides on the pituitary glycoprotein hormones lutropin (LH), follitropin (FSH), and thyrotropin (TSH) consist of a heterogeneous array of neutral, sulfated, sialylated, and sulfated/sialylated structures. In the accompanying paper (Green, E.D., and Baenziger, J.U. (1987) J. Biol. Chem. 262, 25-35), we elucidated the structures of the anionic asparagine-linked oligosaccharides found on the bovine, ovine, and human pituitary glycoprotein hormones. In this study, we determined the relative quantities of the various asparagine-linked oligosaccharides on LH, FSH, and TSH from these three animal species. The proportions of sulfated versus sialylated oligosaccharides varied markedly among the different hormones. Both hormone- and animal species-specific differences in the types and distributions of sulfated, sialylated, and sulfated/sialylated structures were evident. In particular, LH and FSH, which are synthesized in the same pituitary cell and bear alpha-subunits with the identical amino acid sequence, contained significantly different distributions of sulfated and sialylated oligosaccharides. For all three animal species, the ratio of sialylated to sulfated oligosaccharides differed by greater than 10-fold for LH and FSH, with sulfated structures dominating on LH and sialylated structures on FSH. Sialylated oligosaccharides were also heterogeneous with respect to sialic acid linkage (alpha 2,3 versus alpha 2,6). In addition to differences in the proportion of sulfated and sialylated structures on LH and FSH, there were site-specific variations in the amount of mono- and disulfated oligosaccharides at different glycosylation sites on LH alpha-beta dimers. The differences in oligosaccharide structures among the various pituitary glycoprotein hormones as well as among the various glycosylation sites within a single hormone support the hypothesis that glycosylation may serve important functional roles in the expression and/or regulation of hormone bioactivity.     

Asparagine-linked oligosaccharides on lutropin, follitropin, and thyrotropin. I. Structural elucidation of the sulfated and sialylated oligosaccharides on bovine, ovine, and human pituitary glycoprotein hormones

We have elucidated the structures of the anionic asparagine-linked oligosaccharides present on the glycoprotein hormones lutropin (luteinizing hormone), follitropin (follicle-stimulating hormone), and thyrotropin (thyroid-stimulating hormone). Purified hormones, isolated from bovine, ovine, and human pituitaries, were digested with N-glycanase, and the released oligosaccharides were reduced with NaB[3H]4. The 3H-labeled oligosaccharides from each hormone were then fractionated by anion-exchange high performance liquid chromatography (HPLC) into populations differing in the number of sulfate and/or sialic acid moieties. The anionic oligosaccharides were further purified as well as structurally characterized using a variety of preparative and analytical techniques, including HPLC, endo- and exoglycosidase digestions, and lectin affinity chromatography. The sulfated, sialylated, and sulfated/sialylated structures, which together comprised 67-90% of the asparagine-linked oligosaccharides on the pituitary glycoprotein hormones, were highly heterogeneous and displayed hormone- as well as animal species-specific features. The sulfated oligosaccharides consisted of hybrid and complex type oligosaccharides with one or two branches terminating in SO4-4GalNAc beta 1,4. In contrast, the sialylated oligosaccharides consisted of a wide array of differing structures containing two or three peripheral branches as well as one, two, or three sialic acid moieties. A previously uncharacterized dibranched oligosaccharide, bearing one residue each of sulfate and sialic acid, was found on all of the hormones except bovine lutropin. In this study, we describe the purification and detailed structural characterizations of the sulfated, sialylated, and sulfated/sialylated oligosaccharides found on lutropin, follitropin, and thyrotropin from several animal species. In the accompanying paper (Green, E.D., and Baenziger, J.U.(1987) J. Biol. Chem. 262, 36-44) we demonstrate the marked quantitative differences among the pituitary glycoprotein hormones in terms of sulfation, sialylation, and underlying oligosaccharide structures, as well as provide evidence for site-specific synthesis of oligosaccharides on individual hormones.     

The asparagine-linked oligosaccharides on bovine fetuin. Structural analysis of N-glycanase-released oligosaccharides by 500-megahertz 1H NMR spectroscopy

The structures of the entire population of sialylated asparagine-linked oligosaccharides present on bovine fetuin were elucidated. Asparagine-linked oligosaccharides were released from fetuin with N-glycanase, radiolabeled by reduction with NaB[3H]4, and fractionated by anion-exchange high performance liquid chromatography (HPLC), ion-suppression amine adsorption HPLC, and concanavalin A affinity chromatography. The 3H-labeled oligosaccharide fractions obtained were analyzed by 500-MHz 1H nuclear magnetic resonance spectroscopy, revealing the presence of 23 distinct oligosaccharide structures. These oligosaccharides differed in extent of sialylation (3% mono-, 35% di-, 54% tri-, and 8% tetrasialylated), number of peripheral branches (17% di- and 83% tribranched), linkage (alpha 2,3 versus alpha 2,6) and location of sialic acid moieties, and linkage (beta 1,4 versus beta 1,3) of galactose residues. This represents the first time that the asparagine-linked oligosaccharides of fetuin have been successfully fractionated and characterized as sialylated species. The sialylated oligosaccharides derived from fetuin were also used to further define the specificities of the lectins leukoagglutinating phytohemagglutinin and Ricinus communis agglutinin I. The behavior of these oligosaccharides during lectin affinity HPLC further establishes the structural features which predominate in the interaction of oligosaccharides with leukoagglutinating phytohemagglutinin and R. communis agglutinin I.     

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     

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.     

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.     

Complex asparagine-linked oligosaccharides in Mgat1-null embryos

To investigate the developmental role of complex N-linked oligosaccharides,we previously inactivated the mouse Mgat1 gene which encodesUDP-N-acetylglucosamine:     

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)     

The structures of the asparagine-linked sugar chains of bovine interphotoreceptor retinol-binding protein. Occurrence of fucosylated hybrid-type oligosaccharides

The sugar chains of interphotoreceptor retinol-binding protein purified from the interphotoreceptor matrix of bovine eyes were liberated from the polypeptide portion by hydrazinolysis followed by N-acetylation and NaB[3H]4 reduction. The oligosaccharide fraction thus obtained was separated into four acidic fractions by paper electrophoresis. The four acidic fractions were confirmed to be mixtures of mono-, di-, tri-, and tetrasialyloligosaccharides. Both N-acetyl- and N-glycolylneuraminic acids were found as sialic acids of interphotoreceptor retinol-binding protein. The monosialylated oligosaccharide fraction, which accounted for 40 molar per cent of the total oligosaccharides liberated, was a mixture of the following hybrid-type oligosaccharides: (Formula: see text) This is the first time that fucosylated hybrid-type oligosaccharides have been found in any glycoprotein. The di-, tri-, and tetrasialyloligosaccharide fractions were composed of biantennary complex-type oligosaccharides, the outer chains of which are either Sia alpha 2----(3- or 6-linked)Gal beta 1----3(Sia alpha 2----6)GlcNac or Sia alpha 2----(3- or 6-linked)Gal beta 1----4GlcNAc.     

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.     

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.     

The importance of trimming reactions on asparagine-linked oligosaccharides for protein quality control

Although the biochemistry of early trimming reactions by glucosidases and ER mannosidases occurring on asparagine-linked oligosaccharides has been known for a long time, their involvement in quality control of protein folding has become apparent only more recently. Here we review the evidence for the involvement of specific oligosaccharide trimming intermediates such as Glc(1)Man(9)GlcNAc(2) and Man(8)GlcNAc(2) B isomer in this fundamental cellular process and the subcellular distribution of components of the protein quality control machinery which indicates the involvement of both the ER and pre-Golgi intermediates in this process. In addition, recent studies on the subcellular distribution of endomannosidase in conjunction with previously obtained biochemical data will be reviewed which demonstrate that an alternative deglucosylation pathway exists in pre-Golgi intermediates and the Golgi apparatus.     

Initiation of poly-N-acetyllactosamine chain biosynthesis occurs preferentially on complex multiantennary asparagine-linked oligosaccharides

An N-acetyl-beta-D-glucosaminyltransferase activity involved in the initiation of poly-N-acetyllactosamine chain biosynthesis can be solubilized from Ehrlich ascites tumor cell microsomal membranes. The ability of this enzyme to act on linear and branched acceptor substrates has been studied. The results indicate that complex-type tri- and tetra-antennary oligosaccharides exhibiting the branching pattern beta-D-Galp-(1----4)-beta-D-GlcpNAc-(1----6)-[beta-D-Galp-(1----4)-beta- D- Glcp-NAc-(1----2)]-D-Man are the preferred substrates for the enzyme, and therefore, may represent the structures upon which the generation of poly-N-acetyllactosamine chains proceeds more efficiently.     

Novel methods to determine the epitopes on the asparagine-linked oligosaccharides of glycoproteins

Oligosaccharides obtained from glycoproteins by hydrazinolysis followed by N-acetylation were covalently coupled to an amino-bonded thin-layer plate or to a poly-L-lysine coating on the microtiter plate. Bound oligosaccharides can be directly detected by immunostaining or by enzyme-linked immunosorbent assay (ELISA) using a mouse monoclonal antibody. These methods are simple and require small amounts of oligosaccharides and antibodies. The methods were successfully applied to determine the epitope of F48-60, a monoclonal antibody which reacts with the N-linked sugar chains of nonspecific cross-reacting antigen 2, but not with those of carcinoembryonic antigen. The results revealed that the antibody recognizes the Gal beta 1----3GlcNAc beta 1----group.     

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.