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.     

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.     

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 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.     

The role of O-linked and N-linked oligosaccharides on the structure-function of glycoprotein hormones: development of agonists and antagonists

Thyrotropin (TSH) and the gonadotropins; follitropin (FSH), lutropin (LH) and human chorionic gonadotropin (hCG) are a family of heterodimeric glycoprotein hormones. These hormones composed of two noncovalently linked subunits; a common alpha and a hormone specific beta subunits. Assembly of the subunits is vital to the function of these hormones. However, genetic fusion of the alpha and beta subunits of hFSH, hCG and hTSH resulted in active polypeptides. The glycoprotein hormone subunits contain one (TSH and LH) or two (alpha, FSHbeta and hCGbeta) asparagine-linked (N-linked) oligosaccharides. CGbeta subunit is distinguished among the beta subunits because of the presence of a carboxyl-terminal peptide (CTP) bearing four O-linked oligosaccharide chains. To examine the role of the oligosaccharide chains on the structure-function of glycoprotein hormones, chemical, enzymatic and site-directed mutagenesis were used. The results indicated that O-linked oligosaccharides play a minor role in receptor binding and signal transduction of the glycoprotein hormones. In contrast, the O-linked oligosaccharides are critical for in vivo half-life and bioactivity. Ligation of the CTP bearing four O-linked oligosaccharide sites to different proteins, resulted in enhancing the in vivo bioactivity and half-life of the proteins. The N-linked oligosaccharide chains have a minor role in receptor binding of glycoprotein hormones, but they are critical for bioactivity. Moreover, glycoprotein hormones lacking N-linked oligosaccharides behave as antagonists. In conclusion, the O-linked oligosaccharides are not important for in vitro bioactivity or receptor binding, but they play an important role in the in vivo bioactivity and half-life of the glycoprotein hormones. Addition of the O-linked oligosaccharide chains to the backbone of glycoprotein hormones could be an interesting strategy for designing long acting agonists of glycoprotein hormones. On the other hand, the N-linked oligosaccharides are not important for receptor binding, but they are critical for bioactivity of glycoprotein hormones. Deletion of the N-linked oligosaccharides resulted in the development of glycoprotein hormone antagonists. In the case of hTSH, development of an antagonist may offer a novel therapeutic strategy in the treatment of thyrotoxicosis caused by Graves' disease and TSH secreting pituitary adenoma.     

Separation of anionic oligosaccharides by high-performance liquid chromatography

We have developed methods for rapid fractionation of anionic oligosaccharides containing sulfate and/or sialic acid moieties by high-performance liquid chromatography (HPLC). Ion-exchange HPLC on amine-bearing columns (Micropak AX-10 and AX-5) at pH 4.0 is utilized to separate anionic oligosaccharides bearing zero, one, two, three, or four charges, independent of the identity of the amnionic moieties (sulfate and/or sialic acid). Ion-exchange HPLC at pH 1.7 allows separation of neutral, mono-, di-, and tetrasialylated, monosulfated, and disulfated oligosaccharides. Oligosaccharides containing three sialic acid residues and those bearing one each of sulfate and sialic acid, however, coelute at pH 1.7. Since the latter two oligosaccharide species separate at pH 4.0, analysis at pH 4.0 followed by analysis at pH 1.7 can be utilized to completely fractionate complex mixtures of sulfated and sialylated oligosaccharides. Ion-suppression amine adsorption HPLC has previously been shown to separate anionic oligosaccharides on the basis of net carbohydrate content (size). In this study we demonstrate the utility of ion-suppression amine adsorption HPLC for resolving sialylated oligosaccharide isomers which differ only in the linkages of sialic acid residues (alpha 2.3 vs alpha 2.6) and/or location of alpha 2,3- and alpha 2,6-linked sialic acid moieties on the peripheral branches of oligosaccharides. These two methods can be used in tandem to separate oligosaccharides, both analytically and preparatively, based on their number, types, and linkages of anionic moieties.     

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.     

Evaluation of desialylation during 2-amino benzamide labeling of asparagine-linked oligosaccharides

Labeling of released asparagine-linked (N-linked) oligosaccharides from glycoproteins is commonly performed to aid in the separation and detection of the oligosaccharide. Of the many available oligosaccharide labels, 2-amino benzamide (2-AB) is a popular choice for providing a fluorescent product. The derivatization conditions can potentially lead to oligosaccharide desialylation. This work evaluated the extent of sialic acid loss during 2-AB labeling of N-linked oligosaccharides released from bovine fetuin, polyclonal human serum immunoglobulin G (IgG), and human α₁-acid glycoprotein (AGP) as well as of sialylated oligosaccharide reference standards and found that for more highly sialylated oligosaccharides the loss is greater than the <2% value commonly cited. Manufacturers of glycoprotein biotherapeutics need to produce products with a consistent state of sialylation and, therefore, require an accurate assessment of glycoprotein sialylation.     

Differential processing of Asn-linked oligosaccharides on pituitary glycoprotein hormones: implications for biologic function

Luteinizing hormone, follicle-stimulating hormone, and thyroid-stimulating hormone from pituitary and chorionic gonadotropin from placenta are a family of glycoproteins, each consisting of an and subunit. Within an animal species, the subunit of all four hormones contains the identical amino acid sequence, while each subunit is distinct and confers biologic specificity to the hormone dimer. Despite sharing common subunits, these hormones bear Asn-linked oligosaccharides which differ in structure. Whereas chorionic gonadotropin contains exclusively neutral and sialylated oligosaccharides, the pituitary hormones bear neutral, sialylated, sulfated, and sialylated/sulfated structures. The sulfated oligosaccharides are unique in structure and are more prevalent on certain pituitary hormones, indicating that the synthesis of these unusual oligosaccharides is tightly regulated. The differences in oligosaccharide structures in conjunction with the highly specific endocrine responses elicited by these hormones, suggest an important functional role for the oligosaccharides, such as metabolic clearance, control of hormone response, modulation of hormone potency, and/or intracellular sorting of hormones into separate secretory granules.     

Beta-subunits of equine chorionic gonadotropin and lutenizing hormone with an identical amino acid sequence have different asparagine-linked oligosaccharide chains.

The glycoprotein hormones, equine chorionic gonadotropin (eCG) and lutenizing hormone (eLH), possess a beta-subunit with an identical amino acid sequence. The Asn-linked oligosaccharide chains of eCG beta and eLH beta were quantitatively liberated as tritium-labeled oligosaccharides by hydrazinolysis followed by N-acetylation and NaB3H4-reduction. Paper electrophoresis in combination with sialidase digestion and solvolytic desulfation indicated that eCG beta contained neutral and sialylated oligosaccharides, while eLH beta contained neutral, sialylated, sulfated, and both sialylated and sulfated oligosaccharides. In addition, elution profiles on a Bio-Gel P-4 column of the neutralized oligosaccharide mixtures of eCG beta and eLH beta were different, indicating that the molecular masses of oligosaccharides of the two glycoproteins are different. Therefore, this suggests that the structures of the Asn-linked oligosaccharide chains of eCG beta and eLH beta are different although they have an identical amino acid sequence.     

Pro-opiomelanocortin synthesized by corticotrophs bears asparagine-linked oligosaccharides terminating with SO4-4GalNAc beta 1,4GlcNAc beta 1,2Man alpha.

We have determined that greater than or equal to 80% of the Asn-linked oligosaccharides on the glycosylated form of mouse adrenocorticotropin (15-kDa adrenocorticotropin (ACTH)) bear one or more branches terminating with the sequence SO4-4GalNAc beta 1,4GlcNAc beta 1,2Man alpha (S4GGnM). Proopiomelanocortin (POMC), the precursor of ACTH, is the first example of a glycoprotein that is not a member of the glycoprotein hormone family to bear such sulfated structures. Like lutropin and thyrotropin, 15-kDa ACTH bears dibranched oligosaccharides terminating with SO4-4-GalNAc; however, at least half of the oligosaccharides on 15-kDa ACTH terminating with SO4-4-GalNAc consist of more highly branched structures that have not previously been described. Both the GalNAc beta 1,4GlcNAc beta 1,2Man-4-sulfotransferase and the glycoprotein hormone-specific GalNAc-transferase are expressed in the corticotroph-derived AtT-20 cell line. A tripeptide recognition sequence, Pro-Val-Lys, similar to the Pro-Leu-Arg sequence required for recognition of glycoprotein hormone alpha- and beta-subunits by the glycoprotein hormone-specific GalNAc-transferase, is present 8 residues amino-terminal to the glycosylated Asn of 15-kDa ACTH. Thus, POMC has the features expected for specific addition of the S4GGnM sequence to its oligosaccharides. The recent discovery of a receptor in hepatic endothelial cells that recognizes oligosaccharides terminating with S4GGnM suggests these sulfated oligosaccharides will regulate the circulatory half-life of glycosylated POMC cleavage products.     

The role of O-linked and N-linked oligosaccharides on the structure–function of glycoprotein hormones: Development of agonists and antagonists

Thyrotropin (TSH) and the gonadotropins; follitropin (FSH), lutropin (LH) and human chorionic gonadotropin (hCG) are a family of heterodimeric glycoprotein hormones. These hormones composed of two noncovalently linked subunits; a common α and a hormone specific β subunits. Assembly of the subunits is vital to the function of these hormones. However, genetic fusion of the α and β subunits of hFSH, hCG and hTSH resulted in active polypeptides. The glycoprotein hormone subunits contain one (TSH and LH) or two (α, FSHβ and hCGβ) asparagine-linked (N-linked) oligosaccharides. CGβ subunit is distinguished among the β subunits because of the presence of a carboxyl-terminal peptide (CTP) bearing four O-linked oligosaccharide chains. To examine the role of the oligosaccharide chains on the structure–function of glycoprotein hormones, chemical, enzymatic and site-directed mutagenesis were used. The results indicated that O-linked oligosaccharides play a minor role in receptor binding and signal transduction of the glycoprotein hormones. In contrast, the O-linked oligosaccharides are critical for in vivo half-life and bioactivity. Ligation of the CTP bearing four O-linked oligosaccharide sites to different proteins, resulted in enhancing the in vivo bioactivity and half-life of the proteins. The N-linked oligosaccharide chains have a minor role in receptor binding of glycoprotein hormones, but they are critical for bioactivity. Moreover, glycoprotein hormones lacking N-linked oligosaccharides behave as antagonists. In conclusion, the O-linked oligosaccharides are not important for in vitro bioactivity or receptor binding, but they play an important role in the in vivo bioactivity and half-life of the glycoprotein hormones. Addition of the O-linked oligosaccharide chains to the backbone of glycoprotein hormones could be an interesting strategy for designing long acting agonists of glycoprotein hormones. On the other hand, the N-linked oligosaccharides are not important for receptor binding, but they are critical for bioactivity of glycoprotein hormones. Deletion of the N-linked oligosaccharides resulted in the development of glycoprotein hormone antagonists. In the case of hTSH, development of an antagonist may offer a novel therapeutic strategy in the treatment of thyrotoxicosis caused by Graves' disease and TSH secreting pituitary adenoma.     

The asparagine-linked oligosaccharides on tissue factor pathway inhibitor terminate with SO4-4GalNAc beta 1, 4GlcNAc beta 1,2 Mana alpha.

Tissue factor pathway inhibitor (TFPI) produced by endothelial cells contains sulfated Asn-linked oligosaccharides. We have determined that greater than 70% of the oligosaccharides on recombinant TFPI expressed in 293 cells terminate with the sequence SO4-4GalNAc beta 1, 4GlcNAc beta 1, 2Man alpha. Oligosaccharides terminating with this sequence have previously been described on lutropin, thyrotropin, and pro-opiomelanocortin: glycoproteins synthesized in the anterior pituitary. A GalNAc-transferase that recognizes the tripeptide motif Pro-Xaa-Arg/Lys 6-9 residues N-terminal to Asn glycosylation sites accounts for the specific addition of GalNAc to the oligosaccharide acceptor on these glycoproteins, whereas a GalNAc beta 1,4GlcNAc beta 1, 2Man alpha-4-sulfotransferase accounts for the addition of sulfate. The sulfated oligosaccharides present on these hormones are responsible for their rapid clearance from plasma by a receptor in hepatic reticuloendothelial cells. GalNAc- and sulfotransferase activities with the same properties as those expressed in the pituitary are detected at high levels in 293 cells and at lower levels in endothelial cells. Chinese hamster ovary (CHO) cells do not contain detectable levels of either transferase and rTFPI expressed in CHO cells does not contain sulfated Asn-linked oligosaccharides. TFPI contains the sequence Pro-Phe-Lys, 9 residues N-terminal to the glycosylation site at position 228; this tripeptide may act as the recognition sequence for the GalNAc-transferase. rTFPI produced by 293 cells, but not that produced by CHO cells, is bound by the receptor on hepatic reticuloendothelial cells suggesting the sulfated structures play a role in the biologic behavior of TFPI.     

Differential sorting of lutropin and the free alpha-subunit in cultured bovine pituitary cells.

The glycoprotein hormones lutropin (LH) and follitropin (FSH) are both synthesized by gonadotrophs in the anterior pituitary but are stored in separate secretory granules prior to secretion. Despite having highly homologous beta-subunits and alpha-subunits with the identical amino acid sequence, the Asn-linked oligosaccharides on LH terminate with SO4-GalNAc while those on FSH terminate with sialic acid-Gal. In addition to LH and FSH, gonadotrophs secrete uncombined (free) alpha-subunit which bears the same sulfated oligosaccharides as LH. We have examined the synthesis and secretion of LH and free alpha-subunit in primary cultures of bovine pituitary cells in order to determine if the sulfated oligosaccharides have any impact on sorting. Our results show that newly synthesized free alpha-subunit is secreted exclusively via the constitutive pathway with a t1/2 of 1.8 h and is never found in dense-core secretory granules. In contrast, LH dimer is secreted by both the constitutive and the regulated pathways. Constitutive secretion and arrival in a dense secretory granule both occur with t1/2 values of 1-1.5 h for newly synthesized LH. Sulfation occurs immediately prior to arrival of LH in the secretory granule and is followed by a period of 1-1.5 h before the LH-containing granules become sensitive to release by gonadotropin releasing hormone. As a result the t1/2 for LH secretion in the presence of gonadotropin releasing hormone is 3.5 h. Sulfation of the free alpha-subunit oligosaccharides is not, therefore, sufficient to direct the alpha-subunit to secretory granules, and the information required for directing LH to granules must reside either in the beta-subunit or the alpha beta-complex.     

Human choriogonadotropin

Human choriogonadotropin, a hormone derived from the syncytiotrophoblast cells of the placenta, is a member of the glycoprotein hormone family which also contains the pituitary hormones lutropin, follitropin and thyrotropin. These four hormones are comprised of two dissimilar subunits, one (α) being common to all four and the other (β) conferring hormonal specificity. Information is rapidly accumulating on the nature and regulation of the genes for these subunits, as well as the structural aspects, mechanism-of-action and physiological roles of these complex hormones. This mini review considers some of the recent advances in our understanding of human choriogonadotropin.     

Free alpha-like material from bovine pituitaries. Removal of its O-linked oligosaccharide permits combination with lutropin-beta

Further characterization of the free alpha subunit immunoreactive material, not combined with beta subunit in extracts of bovine pituitaries, shows that the only significant modifications, relative to alpha subunits themselves, are the oligosaccharide O-linked to threonine-43, and heterogeneity of the carboxyl terminus. Removal of the O-linked carbohydrate with a mixture of glycosidases from Streptococcus pneumoniae results in an alpha-like material capable of combining with lutropin beta subunit and, thus, the presence of the oligosaccharide is responsible for the inability of the free alpha-like material to combine with beta subunits. Amino acid compositions of tryptic peptides spanning the entire sequence indicate no change in amino acid sequence of the free alpha-like material as compared to lutropin alpha. Further, based on the similar behavior reverse phase high performance liquid chromatography of the tryptic peptides as compared to their lutropin alpha counterparts, it is concluded that no additional post-translational modifications are present. The N-linked oligosaccharides of the free alpha-like material most likely contain terminal O-sulfated N-acetylhexosamines (as do the asparagine-linked carbohydrates from the pituitary hormones) as indicated by the presence of 3 mol of sulfate/mol of free alpha-like material and the resistance of these oligosaccharides to enzymatic deglycosylation. The O-linked oligosaccharide does not contain sulfated residues.     

Incompletely processed LH molecules synthesized by rat gonadotrophs treated with inhibitors of oligosaccharide processing

Inhibitors of N-linked oligosaccharide processing are useful tools for studies on the biological function of the oligosaccharide structures in glycoprotein hormones. We have synthesized molecules of lutropin (LH) containing high-mannose- and hybrid-type oligosaccharides using rat gonadotroph-enriched primary cultures in the presence of castanospermine (a glucosidase I inhibitor) or swainsonine (a mannosidase II inhibitor), in order to compare the actions of these molecules with that of the hormone containing complex-type oligosaccharides in the activation of the receptor-adenylate cyclase system. Treatment of gonadotrophs with the above inhibitors caused an increase in the synthesis of highly basic LH molecules (pI 9.6-10.0), because addition of charged carbohydrate moieties to these molecules was prevented. Characterization of the oligosaccharide structure performed by enzymatic treatment (endoglycosidase H and neuraminidase) and the use of immobilized lectins (wheat germ agglutinin and Ricinus communis agglutinin-120) showed that these inhibitor-synthesized LH molecules contained high-mannose- and hybrid-type (asialo and sialylated) oligosaccharides. Their immunological properties were similar to that of complex-type oligosaccharide LH, but they had significantly higher receptor-binding ability in comparison with a sialylated complex-type oligosaccharide LH (about 12-fold) and an asialo complex-type oligosaccharide LH (about 3-fold). It was noted that the incompletely processed molecules were less potent than complex-type oligosaccharide LH in the activation of adenylate cyclase of Leydig cells, showing about 40-60% of the activity induced by the sialylated complex-type oligosaccharide molecule. The present data indicate that the inhibition of terminal processing of N-linked oligosaccharides by castanospermine and swainsonine impairs the full hormonal function of rat LH.     

Sulfated N-linked oligosaccharides in mammalian cells. I. Complex-type chains with sialic acids and O-sulfate esters

The structures of sulfated N-linked oligosaccharides have been reported for a few specific proteins. We recently demonstrated that such oligosaccharides occur in many different types of tissue culture cell lines (Freeze, H. H., and Varki, A. (1986) Biochem. Biophys. Res. Commun. 140, 967-973). Here we report improved methods to metabolically label cell lines with 35SO4 and to release sulfated N-linked oligosaccharides with peptide:N-glycosidase F as well as the partial structure of some of these novel oligosaccharides. The released 35SO4-labeled chains from Chinese hamster ovary (CHO) cells and bovine pulmonary artery endothelial cells (CPAE) were characterized by gel filtration, anion exchange and lectin affinity chromatography, and various enzymatic and chemical treatments. Each cell line contains a class of sulfated oligosaccharide chains bearing from two to six negative charges in varying combinations of O-sulfate esters and sialic acids. These molecules represent a significant proportion of both the total 35SO4 label and the total anionic N-linked oligosaccharides. They are also relatively enriched in a CHO mutant that is deficient in glycosaminoglycan chain synthesis. Lectin affinity chromatography of such molecules from CPAE cells indicates that the majority are sialylated multiantennary complex-type chains. The sulfate esters are exclusively of the primary type. Sequential exoglycosidase digestions, including beta-hexosaminidase A treatment at low pH, demonstrate that at least one-third of these sulfate esters are found in the following structure, (formula; see text) where R is the remainder of the underlying oligosaccharide, and SA is sialic acid. In addition to these molecules, a more highly charged group of sulfated N-linked oligosaccharides sharing structural features with glycosaminoglycans was found in CPAE cells, but not in CHO cells. These are described in the following paper (Sundblad, G., Holojda, S., Roux, L., Varki, A., and Freeze, H. H. (1988) J. Biol. Chem. 263, 8890-8896).     

Sepharose-linked concanavalin A in the purification and characterization of glycoprotein hormones of the bovine pituitary

Affinity chromatography on concanavalin A-Sepharose is a time saving step in both large and small scale isolations of the bovine pituitary glycoprotein hormones. After ion-exchange chromatography, the final yield of purified lutropin is 40-50% of material in starting concentrates and of purified thyrotropin is approximately 20%. The final products have the same electrophoretic and immunological properties and amino acid compositions as previous preparations. Less than 3% of the immunoreactive lutropin, follitropin and thyrotropin are present as non-glycosylated forms in either crude pituitary extracts or concentrates. Thyrotropin and follitropin elute from the immobilized lectin as a single fraction, whereas lutropin separates into two glycosylated fractions. Gel filtration of both crude extracts and the glycoprotein fractions shows that less than 5% of the immunoreactivity of the hormones is present as material of apparently high molecular weight. Substantial alpha subunit immunoreactivity, however, is in three fractions (as found by others in human pituitary extracts) corresponding to "high molecular weight material" (7%), intact hormones (46%) and free subunit (47%).     

Detailed structural analysis of asparagine-linked oligosaccharides of the nicotinic acetylcholine receptor from Torpedo californica.

The structures of the major oligosaccharide moieties of the nicotinic acetylcholine receptor (AcChoR) protein from Torpedo californica have been reported [Nomoto, H., Takahashi, N., Nagaki, Y., Endo, S., Arata, Y. and Hayashi, K. (1986) Eur. J. Biochem. 157, 233-242] to be high-mannose types. Here we report detailed analyses of the structures of the remaining oligosaccharides in this receptor. The sialylated oligosaccharides released by glycopeptidase (almond) digestion were separated according to the number of sialic acid residues using high-performance anion-exchange chromatography with pulsed amperometric detection. After removal of sialic acid from each fraction, the resulting neutral oligosaccharides were separately pyridylaminated and were analyzed by a combination of sequential exoglycosidase digestion and HPLC, then identified on a two-dimensional sugar map. The structures of two desialylated pyridylamino-oligosaccharides were further analyzed by high-resolution proton NMR. Each oligosaccharide was composed of species containing varying numbers of sialic acids. The desialylated complex-type oligosaccharides of AcChoR consisted of ten, eight and one different biantennary, triantennary and tetraantennary oligosaccharide, respectively. The biantennary oligosaccharides were divided into two groups; oligosaccharides with fucose at the proximal N-acetylglucosamine (six varieties) and oligosaccharides without fucose (four varieties). Each group consisted of species differing in the number of terminal galactose residues. The major component of the biantennary oligosaccharides had two galactose residues at the non-reducing termini. The terminal alpha-galactose residue(s) linked to C3 of beta-galactose were found in the fucose-containing biantennary oligosaccharides (two varieties). The triantennary oligosaccharides were also divided into two groups; oligosaccharides with (four varieties) and without (four varieties) besecting N-acetylglucosamine. These groups were composed of species differing in the number of terminal galactose residues. The major component of the triantennary oligosaccharides was fully galactosylated with three galactose residues. An unusual group, Gal beta 1-3GlcNAc, was present in low levels in the triantennary oligosaccharides. In contrast, the tetraantennary oligosaccharide was composed of only one species, which is fully galactosylated with four galactose residues.