Effect of thyrotropin-releasing hormone on the carbohydrate structure of secreted mouse thyrotropin. Analysis by lectin affinity chromatography

Thyrotropin (TSH) is a glycoprotein hormone whose secretion from the anterior pituitary is regulated, in part, by the hypothalamic tripeptide thyrotropin-releasing hormone (TRH). We have used serial lectin affinity analysis to explore whether TRH, in addition to promoting TSH secretion, alters the carbohydrate structure of secreted TSH. Hypothyroid mouse hemipituitaries were incubated in medium containing [3H] mannose, [3H]glucosamine, or [3H]fucose either with or without 10(-7) M TRH. TSH was immunoprecipitated, proteolytically digested into glycopeptides, and chromatographed on serial lectin-Sepharose columns. Under basal conditions, 37% of secreted [3H]mannose-labeled TSH glycopeptides failed to bind to concanavalin A (ConA)-Sepharose, 55% bound and eluted with 10 mM alpha-methylglucoside, and 8% bound and eluted with 500 mM alpha-methylmannoside. Approximately 35% of glycopeptides not binding to ConA-Sepharose were bound by pea lectin-Sepharose, suggesting the presence of certain core fucosylated triantennary complex oligosaccharides. TRH caused a 2-fold increase in secretion of [3H]mannose-labeled TSH glycopeptides due almost exclusively to a specific increase in structures that bound to ConA-Sepharose and eluted with 10mM alpha-methylglucoside, corresponding to biantennary complex or unusual hybrid species. There was no change in the distribution of intrapituitary TSH glycopeptides with TRH. Acid hydrolysis of secreted proteins showed little metabolism of the tritiated sugar precursors, except for a 20% conversion of [3H]mannose to [3H]fucose. Moreover, ConA-Sepharose chromatography of secreted [3H]glucosamine- and [3H]fucose-labeled TSH glycopeptides showed similar increases in ConA-Sepharose binding with TRH as noted with [3H]mannose labeling. Subsequent lectin analysis of secreted [3H] mannose-labeled TSH glycopeptides on erythroagglutinating phytohemagglutinin-Sepharose and leukoagglutinating phytohemagglutinin-Sepharose disclosed no significant differences in TRH-treated versus control samples. These data suggest that secreted mouse TSH has greater carbohydrate heterogeneity than has been recognized previously. In addition, TRH in vitro promotes the secretion of specific TSH molecules apparently enriched in biantennary complex or unusual hybrid oligosaccharides.     

Human alpha-galactosidase A: characterization of the N-linked oligosaccharides on the intracellular and secreted glycoforms overexpressed by Chinese hamster ovary cells

Human alpha-galactosidase A (alpha-Gal A) is the lysosomal glycohydrolase that cleaves the terminal alpha-galactosyl moieties of various glycoconjugates. Overexpression of the enzyme in Chinese hamster ovary (CHO) cells results in high intracellular enzyme accumulation and the selective secretion of active enzyme. Structural analysis of the N -linked oligosaccharides of the intracellular and secreted glycoforms revealed that the secreted enzyme's oligosaccharides were remarkably heterogeneous, having high mannose (63%), complex (30%), and hybrid (5%) structures. The major high mannose oligosaccharides were Man5-7GlcNAc2 species. Approximately 40% of the high mannose and 30% of the hybrid oligosaccharides had phosphate monoester groups. The complex oligosaccharides were mono-, bi- , 2,4-tri-, 2,6-tri- and tetraantennary with or without core-region fucose, many of which had incomplete outer chains. Approximately 30% of the complex oligosaccharides were mono- or disialylated. Sialic acids were mostly N -acetylneuraminic acid and occurred exclusively in alpha2, 3-linkage. In contrast, the intracellular enzyme had only small amounts of complex chains (7.7%) and had predominantly high mannose oligosaccharides (92%), mostly Man5GlcNAc2 and smaller species, of which only 3% were phosphorylated. The complex oligosaccharides were fucosylated and had the same antennary structures as the secreted enzyme. Although most had mature outer chains, none were sialylated. Thus, the overexpression of human alpha-Gal A in CHO cells resulted in different oligosaccharide structures on the secreted and intracellular glycoforms, the highly heterogeneous secreted forms presumably due to the high level expression and impaired glycosylation in the trans- Golgi network, and the predominately Man5-7GlcNAc2 cellular glycoforms resulting from carbohydrate trimming in the lysosome.      

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.     

Regulation of thyroglobulin glycosylation. A comparative study of the thyroglobulins from porcine thyroid glands and follicles in serum-free culture

Porcine thyroid cells were cultured in serum-free medium and thyrotropin was or was not added at day 4 and [3H]glucosamine at day 6 for 24 h. The major glycoprotein secreted outside the follicles proved to be thyroglobulin by immunoprecipitation, polyacrylamide gel electrophoresis, and amino acid composition. Thyroglobulin glycopeptides were analyzed by sequential affinity chromatography on immobilized lectins and compared to chemically labeled carbohydrate chains released from thyroid-derived thyroglobulin by hydrazinolysis. 82% and 85% of the glucosamine-labeled oligosaccharides of thyroglobulin from control and stimulated cells, respectively, were unretained on concanavalin A (ConA)-Sepharose compared to 46% only for in vivo thyroglobulin. 35-42% and 33-35% of the ConA-unbound glycopeptides were retarded on erythrophytohemagglutinin and leukophytohemagglutinin under basal or stimulatory conditions, respectively, while none of the triantennary structures of in vivo thyroglobulin was. Moreover, binding to Bandieraea-agarose showed that 20% of these complex structures contained alpha-linked galactose in thyroglobulin secreted by control cells, but only 10% in the molecules derived from thyroid. When analyzed on ricin-agarose after neuraminidase treatment, the ConA-unbound glycopeptides were retained to an extent of 65% for those from control cells and 98% for those from stimulated cells. Furthermore, 15% of desialylated ConA-unbound glycopeptides from cellular origin were also found to bind to wheat germ agglutinin. Carbohydrate composition, gel chromatography, and exoglycosidase treatment further demonstrated that thyroglobulin carbohydrate chains synthesized under serum-free cell culture were essentially composed of heterogeneous multiantennary structures instead of usual biantennary and high mannose type species. Under thyrotropin stimulation, 85% of the carbohydrate chains of thyroglobulin was shown to be sialylated by high performance liquid chromatography analysis instead of 65% under basal conditions, suggesting that thyrotropin may shift terminal glycosylation of thyroglobulin from alpha-galactose to sialic acid.     

Sulfated N-linked oligosaccharides affect secretion but are not essential for the transport, proteolytic processing, and sorting of lysosomal enzymes in Dictyostelium discoideum

Although previous studies have indicated that N-linked oligosaccharides on lysosomal enzymes in Dictyostelium discoideum are extensively phosphorylated and sulfated, the role of these modifications in the sorting and function of these enzymes remains to be determined. We have used radiolabel pulse-chase, subcellular fractionation, and immunofluorescence microscopy to analyze the transport, processing, secretion, and sorting of two lysosomal enzymes in a mutant, HL244, which is almost completely defective in sulfation. [3H]Mannose-labeled N-linked oligosaccharides were released from immunoprecipitated alpha-mannosidase and beta-glucosidase of HL244 by digestion with peptide: N-glycosidase. The size, Man9-10GlcNAc2, and processing of the neutral species were similar to that found in the wild type, but the anionic oligosaccharides were less charged than those from the wild-type enzymes. All of the negative charges on the oligosaccharides for HL244 were due to the presence of 1, 2, or 3 phosphodiesters and not to sulfate esters. The rate of proteolytic processing of precursor forms of alpha-mannosidase and beta-glucosidase to mature forms in HL244 was identical to wild type. The precursor polypeptides in the mutant and the wild type were membrane associated until being processed to mature forms; therefore, sulfated sugars are not essential for this association. Furthermore, the rate of transport of alpha-mannosidase and beta-glucosidase from the endoplasmic reticulum to the Golgi complex was normal in the mutant as determined by the rate at which the newly synthesized proteins became resistant to the enzyme, endo-beta-N-acetylglucosaminidase H. There was no increase in the percentage of newly synthesized mutant precursors which escaped sorting and were secreted, and the intracellularly retained lysosomal enzymes were properly localized to lysosomes as determined by fractionation of cell organelles on Percoll gradients and immunofluorescence microscopy. However, the mutant secreted lysosomally localized mature forms of the enzymes at 2-fold lower rates than wild-type cells during both growth and during starvation conditions that stimulate secretion. Furthermore, the mutant was more resistant to the effects of chloroquine treatment which results in the missorting and oversecretion of lysosomal enzymes. Together, these results suggest that sulfation of N-linked oligosaccharides is not essential for the transport, processing, or sorting of lysosomal enzymes in D. discoideum, but these modified oligosaccharides may function in the secretion of mature forms of the enzymes from lysosomes.     

Processing and secretion of insulin-related peptides in an insulinoma cell line

Certain classes of prohormones and other neuroendocrine or endocrine-derived secretory proteins are post-translationally modified in the secretory storage granules. If such molecules were to be biosynthesized to acceptable quantity and yield using endocrine-derived cell lines, it would be important to understand the relationship between the secretory dynamics and the conversion and release of the immature and mature forms of the molecule. We studied aspects of such a relationship using the endocrine-derived cell line betaTC-3, which synthesizes murine proinsulin, sequesters it into secretory granules, and converts it into mature insulin. In T-flask experiments with confluent cultures of betaTC-3 cells, intracellular and secreted (pro)insulin was sampled before and after episodes of stimulated exocytosis and recharging and quantified by radioimmunoassay and reversed-phase high-performance liquid chromatography (HPLC). Under conditions of steady-state secretion in glucose-rich growth medium the cells turned over their (pro)insulin inventory (90 +/- 5% mature insulin) at 2-3% per hour through secretion of (pro)insulin which was less than 70% mature. During an episode of hyperstimulated exocytosis induced by the combined secretagogues carbachol (1 muM) and isobutylmethylxanthine (1 mM), approximately 80% of the intracellular (pro)insulin stores were depleted within 2 h and 84 +/- 4% of the secreted (pro)insulin was in the mature form. Following the discharging episode, exocytosis was suppressed to 10% of its steady-state rate with a treatment which attenuated calcium influx (20 muM verapamil with reduced levels of calcium in the medium). Under this condition the secreted protein was only approximately 50% converted to mature insulin, but 85 +/- 10% of the net (pro)insulin accumulating within the intracellular stores was converted to the mature form. The inverse relationship between rate of secretion and degree of conversion of secreted (pro)insulin is consistent with a previously observed phenomenon of preferential basal secretion from immature secretory granules. This tends to enrich the secreted peptides in immature forms relative to the total intracellular pool. Preferential early secretion can best be overcome by rapid discharging of the long-term and predominantly mature stores. Thus, a cyclic controlled secretion process wherein product is collected during intermittent discharging episodes would provide a better yield of mature product than would steady-state secretion. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 283-289, 1997.     

荧光标记电泳对透明质酸寡糖片段鉴定的方法建立

近年来,透明质酸寡糖片段（hyaluronan oligosaccharides, o-HA）的生物学活性引起国外学者的重视,因为o-HA具有一定的生物学活性,如参与免疫调节、刺激新生血管形成等.本研究建立一种经济、简便的ANTS（8-氨基奈-1,3,6-三磺酸）荧光标记电泳对透明质酸寡糖片段大小鉴定的新实验方法.实验原理为,ANTS能与糖分子发生还原反应,在反应时提供3个电子和1个荧光基团,通过高浓度PAGE分离,在特定波长下呈现颜色反应.采用酶消化法得到不同分子量大小的o-HA片段,测得不同片段大小的o-HA聚合度,分别与高效液相色谱（high-performance liquid chromatography, HPLC）和静电喷雾电离质谱（electrospray ionization mass spectrometry, ESI-MS）进行比较,结果吻合.研究提示,用荧光标记电泳法分析寡糖分子量,操作简单、设备低廉、灵敏度较高且检测速度快,是一种检测鉴定寡糖分子的较好方法.     

Structural analysis of the asparagine-linked oligosaccharides of rat haptoglobin metabolically labeled in a hepatocyte culture system

We analyzed the asparagine-linked oligosaccharide chains of rat haptoglobin which were synthesized and secreted by hepatocytes in primary culture. When the cells were incubated with either [3H]mannose, [3H]galactose, or [3H]fucose, all the radioactive precursors were incorporated into the beta subunit of haptoglobin. [3H]Mannose-labeled haptoglobin was purified from the culture medium by immunoaffinity chromatography, and [3H]oligosaccharides were prepared by strong alkali-borohydride treatment. The oligosaccharides obtained were analyzed by anion-exchange high-performance liquid chromatography, concanavalin-A--Sepharose chromatography and Bio-Gel P-4 chromatography before and after sequential exoglycosidase digestions. The oligosaccharides labeled with [3H]fucose or [3H]galactose were also characterized by the above methods. The results indicate that rat haptoglobin contains two complex-type oligosaccharide chains in each beta subunit; one with a possible structure of ( +/- NeuAc----Gal beta----GlcNAc beta----)3(Man alpha----)2 Man beta----GlcNAc----( +/- Fuc alpha----)GlcNAc and the other with ( +/- NeuAc----Gal beta----GlcNAc beta----Man alpha----)2 Man beta----GlcNAc----( +/- Fuc alpha----)GlcNAc.     

Characteristics of the functional properties of the spinal cord motoneurons of old rats]

The mean membrane potential (MP) of old rats did not differ significantly from that in young mature rats ((58.4 +/- +/-1,4 mV and 56.6 +/- 1.26 mV, respectively). At the same time the frequency of detection of motor neurons with the MP OF 70 mV and more fell by 18.6%, and with the MP of 50-59 mV -increased by 14.2% in the old, in comparison with the young animals. The direct excitability threshold in old rats decreased (3.0 +/- 3-10(-9) in young mature and 2.0 +/- 0.2-10(-9) a in old rats; P less than 0.02). The number of discharges per 50 msec of the neuron poliarization reached 4-5, constituting 80-100 pulse/min. When determined by the first two intervals the action potential frequency reached 125 pulse/sec, and in the young mature rats--over 300 pulse/sec. The duration of antidromic spikes was increased (1.02 +/- 0.09 msec in young mature animals and 1.65 +/- 0.14 msec in the old animals; P less than 0.001). The antidromic spikes of the neurons in old mature rats, as a rule, had no delayed depolarization.     

Structural analysis of N-linked oligosaccharides from glycoproteins secreted by Dictyostelium discoideum. Identification of mannose 6-sulfate

The N-linked oligosaccharides found on the lysosomal enzymes from Dictyostelium discoideum are highly sulfated and contain methylphosphomannosyl residues (Gabel, C. A., Costello, C. E., Reinhold, V. N., Kurtz, L., and Kornfeld, S. (1984) J. Biol. Chem. 259, 13762-13769). Here we report studies done on the structure of N-linked oligosaccharides found on proteins secreted during growth, a major portion of which are lysosomal enzymes. Cells were metabolically labeled with [2-3H]Man and 35SO4 and a portion of the oligosaccharides were released by a sequential digestion with endoglycosidase H followed by endoglycosidase/peptide N-glycosidase F preparations. The oligosaccharides were separated by anion exchange high performance liquid chromatography into fractions containing from one up to six negative charges. Some of the oligosaccharides contained only sulfate esters or phosphodiesters, but most contained both. Less than 2% of the oligosaccharides contained a phosphomonoester or an acid-sensitive phosphodiester typical of the mammalian lysosomal enzymes. A combination of acid and base hydrolysis suggested that most of the sulfate esters were linked to primary hydroxyl groups. The presence of Man-6-SO4 was demonstrated by the appearance of 3,6-anhydromannose in acid hydrolysates of base-treated, reduced oligosaccharides. These residues were not detected in acid hydrolysates without prior base treatment or in oligosaccharides first treated by solvolysis to remove sulfate esters. Based on high performance liquid chromatography quantitation of percentage of 3H label found in 3,6-anhydromannose, it is likely that Man-6-SO4 accounts for the majority of the sulfated sugars in the oligosaccharides released from the secreted glycoproteins.     

Milligram-scale preparation and purification of oligosaccharides of defined length possessing the structure of chondroitin from defructosylated capsular polysaccharide K4

Escherichia coli K4 bacterium synthesizes a nonsulfated capsule polysaccharide (K4) composed of a repeating disaccharide subunit of D-glucuronic acid (beta1-->3) and N-acetyl-D-galactosamine (beta1-->4) to which beta-fructofuranose units are linked to C-3 of D-glucuronic acid residues. The K4 polyanion is easily defructosylated under acid conditions with no fragmentation of the polymer to produce a polysaccharide having a repeated disaccharide unit of chondroitin consisting of D-glucuronic acid (beta1-->3) and N-acetyl-D-galactosamine (beta1-->4) (K4d). K4 and K4d were depolymerized by partial digestion with testicular hyaluronidase and separated into uniform-size oligosaccharides from 4-mers to 16-mers by preparative anion-exchange chromatography after removal of the hyaluronidase. The purity and size of each oligosaccharide was confirmed by using anion-exchange HPLC, HPSEC analysis, and FACE. Mg-scale K4d oligosaccharides were obtained from 50 mg K4d starting material. Under the conditions used to degrade the K4 polysaccharide by testicular hyaluronidase, fructose is slowly liberated forming the defructosylated K4. As a consequence, a mixture of uniform- size K4 and K4d oligosaccharide species, from approximately 4- to 20-mers, are generated and size-separated by anion-exchange chromatography. These pure, uniform-size, and large ranges of K4d oligosaccharides having the structure of a chondroitin, -->4)-GlcUA-beta(1-->3)GalNAc-beta(1-->, will be available for investigating important biological functions of this polymer.     

The nude rat is established as a model for endocrine studies: regulation of thyroid function and xenotransplantation of a thyroid tumor cell line.

The thyroid physiology of athymic nude rats, rnu/rnu, is characterized and established here as an animal model to study transplanted thyroid tumors. Male rats were catheterized 5 days before experiments were started. The mean thyroid-stimulating-hormone (TSH) plasma concentrations were 2.9 +/- 0.6 ng/ml during infusion of 0.25 ml/h of 0.9% NaCl (n = 12). T3 plasma concentrations were 2.6 +/- 0.4 ng/ml. T4 plasma levels were 22.0 +/- 5.6 micrograms/dl. A bolus of 0.1 mg thyrotropin-releasing hormone (TRH) significantly increased TSH plasma concentrations (P less than or equal to 0.001; from 2.9 +/- 0.6 to 7.8 +/- 1.1 ng/ml, n = 12). No pulsatile TSH secretion was observed in a 2-hour period with blood samples taken every 10 minutes (n = 12) and hourly sampling disclosed no circadian variation of TSH during a 24-hour period (n = 4). Successful xenografting was possible in 12 of 15 cases using a follicular thyroid carcinoma cell line (FTC 133). Measurement of human thyroglobulin (hTg) by a hTg IRMA revealed high levels in rats with functional FTC tumors, whereas no hTg was detected in untransplanted rats or animals with nonfunctional transplants.     

A study of immunoglobulin G glycosylation in monoclonal and polyclonal species by electrospray and matrix-assisted laser desorption/ionization mass spectrometry

N-linked oligosaccharides were released from human and bovine polyclonal immunoglobulin G (IgG) obtained from commercial sources and also from a monoclonal IgG(1) secreted by murine B-lymphocyte hybridoma cells (CC9C10) grown under different serum-free conditions. These conditions differed according to their steady-state dissolved oxygen concentrations. This work is based on a previous quantitative study where released glycans were characterized by fluorophore-assisted carbohydrate electrophoresis (FACE) and high-pH anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) (J. P. Kunkel, D. C. H. Jan, J. C. Jamieson, and M. Butler, 1998, J. Biotechnol. 62, 55-71). In the present article, peptide-N-glycosidase F-released glycans from different species of polyclonal IgG and murine monoclonal IgG were characterized qualitatively by high-performance liquid chromatography (HPLC) coupled to electrospray ionization mass spectrometry (ESI-MS). The glycans were also analyzed by matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS). The MALDI mass spectrometer used allowed acquisition of MS and tandem MS data, which were useful in structural investigations at a more detailed level than allowed by FACE and HPAEC-PAD. Predominant N-linked structures, as determined by all techniques, were core-fucosyl asialyl biantennary chains with varying galactosylation. Minor amounts of afucosyl, bisected, and monosialyl oligosaccharides were also detected. In contrast to FACE and HPAEC-PAD, MALDI-double quadrupole/time-of-flight MS and HPLC/ESI-MS also detected low-abundance high-mannose and hybrid structures in some of the species under investigation.     

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.     

Differential carbohydrate processing and secretion of thyrotropin and free alpha subunit. Effects of 1-deoxynojirimycin

In pulse-chase experiments we compared the kinetics of early carbohydrate processing and subsequent secretion of thyroid-stimulating hormone (TSH) and free alpha subunit under control conditions and after treatment with 1-deoxynojirimycin, an inhibitor of glucosidases I and II. Under control conditions TSH achieved resistance to endo-beta-N-acetylglucosaminidase H (endo H) more rapidly than free alpha (t1/2 0.3 h versus 0.9 h); however, free alpha was secreted more rapidly than TSH (t1/2 2.2 h versus 3.4 h). With 1-deoxynojirimycin, oligosaccharides co-migrating with G3Man9GlcNAc and G2Man9GlcNAc were demonstrated on TSH for the first time, suggesting that previous pulse-chase studies did not disclose these intermediates due to rapid removal of glucose residues from the common G3Man9GlcNAc2 precursor. 1-Deoxynojirimycin delayed the rate of attainment of endo H resistance for both TSH and free alpha, but there was no effect on subunit combination. With 5 mM 1-deoxynojirimycin the amount of secreted free alpha was reduced to 65% of control; secreted TSH was reduced markedly to 17% of control without intracellular accumulation, suggesting increased intracellular degradation. There was no significant toxicity from these doses of 1-deoxynojirimycin on the production or secretion of the two major nonglycosylated pituitary proteins, growth hormone and prolactin, or on at least 10 other secretory proteins. Basal differences in the relative rates of TSH and free alpha processing and secretion as well as differential sensitivity to 1-deoxynojirimycin suggest separate secretory pathways for these two closely related proteins.     

Quantitative mapping of the N-linked sialyloligosaccharides of recombinant erythropoietin: combination of direct high-performance anion-exchange chromatography and 2-aminopyridine derivatization.

A rapid quantitative analysis of the sialylated N-linked oligosaccharides of recombinant erythropoietin (EPO) expressed in Chinese hamster ovary (CHO) cells has been developed. The procedure utilizes a glycoamidase (glycopeptidase F) to release all of the N-linked oligosaccharides from the native glycoprotein, followed by direct chromatographic analysis using high-performance anion-exchange chromatography (HPAEC) with pulsed amperometric detection. The eight sialyloligosaccharides isolated from HPAEC were characterized by derivatizing with 2-aminopyridine followed by two-dimensional HPLC mapping of the pyridylaminated asialooligosaccharides (Tomiya et al., 1988, Anal. Biochem. 171, 73-90). Seven kinds of complex-type asialooligosaccharides were identified ranging from a biantennary structure to N-acetyllactosamine-extended tetraantennary structure. Approximately 3% of the terminal galactose residues of the oligosaccharides released from EPO were not sialylated whereas 97% contained an alpha(2-->3)-linked sialic acid. Quantitative oligosaccharide mapping of four different lots of EPO from CHO cells was performed to quantify the molar balance and distribution of the N-linked oligosaccharides. The sialyloligosaccharides were distributed with approximately 5% disialylated (single type), 20% trisialylated (six types), and 75% tetrasialylated (four types) oligosaccharides with an average molar recovery of 85% starting from 750 pmol of EPO.     

Sertoli cell glycosylation patterns as affected by culture age and extracellular matrix

This study evaluated the responsiveness of Sertoli cell glycosylation in vitro to changes in culture age and to the presence of a reconstituted basement membrane (Matrigel) or collagen IV/laminin substrata. Primary Sertoli cell cultures were prepared from 20-day-old rats and incubated with [3H]mannose, a monosaccharide specific for asparagine-linked oligosaccharides. The cells were harvested on Days 4, 6, or 10 of culture life. A supernatant enriched in cell-surface glycopeptides (the trypsinate) and a cell pellet stripped of surface glycoconjugates were evaluated separately. Glycopeptides derived from a Pronase digest of the two samples were fractionated using concanavalin-A lectin affinity chromatography into three major classes: multiantennary complex-type, biantennary complex-type, and high-mannose-type oligosaccharide structures. The proportion of radiolabeled glycopeptides appearing in each of the three classes did not differ between Days 4 and 6 of culture. In contrast, a significant increase in the percentage of radiolabeled glycopeptides containing multiantennary complex-type oligosaccharides was observed in cells harvested from the 10-day-old cultures. In other experiments, Sertoli cells were grown on various substrata: plastic; collagen IV/laminin; or Matrigel, a reconstituted basement membrane (RBM) composed of laminin, collagen IV, proteoglycan sulfate, entactin, and nidogen. Growth on RBM significantly increased multiantennary complex-type oligosaccharide formation compared to plastic, whereas the high-mannose-type glycopeptides increased in cells grown on collagen IV/laminin. These studies suggest that environmental and physiological conditions such as culture age and the presence of extracellular matrix significantly affect glycosylation patterns in Sertoli cell cultures.     

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.     

Abnormalities of the thyroid hormone negative feedback regulation of TSH secretion in spontaneously hypertensive rats.

Spontaneously hypertensive rats (SHR) are characterized by several neuroendocrine abnormalities including a chronic hypersecretion of thyrotropin (TSH) of unknown etiology. We hypothesized that the inappropriately high TSH secretion in SHR may be the result of an impaired thyroid hormone negative feedback regulation of hypothalamic thyrotropin-releasing hormone (TRH) and/or pituitary TSH production. To test this hypothesis, SHR or their normotensive Wistar-Kyoto (WKY) controls were treated with either methimazole (0.02% in drinking water) to induce hypothyroidism or administered L-thyroxine (T4) at a dose of 0.8 or 2.0 micrograms/100 g body weight/day to induce hyperthyroidism. All treatments were continued for 14 days after which animals were killed under low stress conditions. TSH concentrations in plasma and anterior pituitary tissue were 2-fold higher (P less than 0.01) in euthyroid SHR compared to WKY control rats while thyroid hormone (T3 and T4) levels were in the normal range. Hypothyroidism induced by either methimazole or thyroidectomy caused a significant (P less than 0.01) rise of plasma TSH levels in both WKY and SHR rats. However, relative to the TSH concentrations in control animals, the increase of plasma TSH in SHR was significantly blunted (P less than 0.01) in comparison to the WKY group. Hypothyroidism caused a significant depletion of TRH in stalk-median eminence (SME) tissue in both groups of rats. However, no differences between SHR and WKY rats were observed. The administration of thyroid hormone caused a dose dependent suppression of plasma TSH levels in both strains of rats. However, at both doses tested plasma TSH concentrations in SHR rats were significantly less suppressed (P less than 0.05) than those in WKY animals. Under in vitro conditions basal and potassium induced TRH release from SMEs derived from SHR was significantly (P less than 0.05) higher than that from WKY rats, whether expressed in absolute terms or as percent of content. These findings suggest that the thyroid hormone negative feedback regulation of TSH secretion may be impaired in SHR rats. Our data do not allow conclusions as to whether defects in the regulation of TSH production are located exclusively at the hypothalamic level. Since the overproduction of hypothalamic TRH and hypophysial TSH should lead to an increased thyroid hormone biosynthesis other defects in the hypothalamus-pituitary-thyroid-axis may contribute to the abnormal regulation of TSH secretion in SHR rats.     

Carbohydrates of human immunodeficiency virus. Structures of oligosaccharides linked to the envelope glycoprotein 120

Human T-cells (H9), persistently infected with the HTLV-III strain of human immunodeficiency virus, were metabolically labeled with D-[2-3H]mannose or D-[6-3H]glucosamine. The viral envelope glycoprotein, gp120, was isolated either from cell lysates or from cell-free culture supernatant. After proteolytic digestion, the radiolabeled oligosaccharides were sequentially liberated from glycopeptides by treatment with endo-beta-N-acetylhexosaminidase H and peptide:N-glycosidase F. Oligosaccharides released were separated from residual (glyco)peptides and fractionated according to size, charge, and fucose content. The individual oligosaccharide species obtained were characterized by digestion with exoglycosidases and by chromatographic comparison with standard oligosaccharides. Our results demonstrate that the intracellular gp120 carries predominantly oligomannosidic glycans comprising nine or eight mannose residues. The secreted glycoprotein is equally substituted by oligomannosidic species, containing seven to nine mannose residues, and by fucosylated, partially sialylated bi- and triantennary complex-type oligosaccharides.