Thyroid sialyltransferase mRNA level and activity are increased in Graves' disease

THYROID Volume 15, Number 7, 2005 © Mary Ann Liebert, Inc. Thyroid Sialyltransferase mRNA Level and Activity Are Increased in Graves’ Disease Jacek Kiljański,1 Michal Ambroziak,2 Janusz Pachucki,1 Krystian Jaz·dz·ewski,3 Wieslaw Wiechno,4 Elżbieta Stachlewska,5 Barbara Górnicka,6 Magdalena Bogdańska,6 Janusz Nauman1,7 and Zbigniew Bartoszewicz1,7 [ [ Sialylation of cell components is an important immunomodulating mechanism affecting cell response to hormones and adhesion molecules. To study alterations in sialic acid metabolism in Graves’ disease (GD) we measured the following parameters in various human thyroid tissues: lipid-bound sialic acid (LBSA) content, ganglioside profile, total sialyltransferase activity, and the two major sialyltransferase mRNAs for sialyltransferase-1 (ST6Gal I) and for sialyltransferase-4A (ST3Gal I). Fragments of toxic thyroid nodules (TN), nontoxic thyroid nodules (NN) and nontumorous tissue from patients with nodular goiter or thyroid cancer were used as a control (C). The LBSA content and sialyltransferase activity were the highest in the GD group (164 ⫾ 4.44 versus 120 ⫾ 2.00 nmoL/g, p ⬍ 0.005 and 1625 ⫾ 283.5 versus 324 ⫾ 54.2 cpm/mg of protein, p ⬍ 0.005 compared to control group C). Ganglioside profile in the GD group was similar to that in control tissues. Sialyltransferase1 mRNA and sialyltransferase-4A mRNA levels were significantly higher in the GD group than in the control group (12.52 ⫾ 6.90 versus 2.54 ⫾ 1.24 arbitrary units, p ⬍ 0.005 and 2,49 ⫾ 1.16 versus 1.23 ⫾ 0.46 arbitrary units, p ⬍ 0.05, respectively). There was a positive correlation between the increased sialyltransferase-1 mRNA level and the TSH-receptor antibody titer determined by the TRAK test. These results indicate that sialyltransferases expression and activity are increased in GD. Exact mechanism of this upregulation remains unknown, though one of possible explanations is the activation of the thyrotropin (TSH) receptor. Introduction I t IS WELL KNOWN that altered glycosylation of glycolipids and glycoproteins affects structure and function of the key pathogenic elements of many diseases. Similarly, changes in the amount, linkage, and acetylation of sialic acids were reported in various pathologic conditions (1,2). Sialic acid is usually the last glycosyl group transferred into glycoconjugates and therefore has a potential for changing protein-toprotein interactions (3). Sialylation of cell components is an important mechanism for the regulation of immune response and hormone-receptor interaction (3,4). Gangliosides, sialic acid containing glycosphingolipids, are structural elements of such a cell membrane components like receptors and ligands for adhesion molecules. Their impact on differentiation, signal transduction, cell-to-cell communication, im- munogenicity, neoplastic transformation, and ability to form metastases has been shown by numerous studies (5–8). However, there are only few studies focusing on sialic acid metabolism in thyroid tissue. Several groups studied thyroid ganglioside profiles and their role in health and disease. Most data came from studies on the bovine thyroid (9,10), yet the ganglioside content and profile of the human thyroid differs considerably from the bovine (11). Moreover, there is evidence for the existence of several cell populations of thyroid cells showing a distinct profile of glycosphingolipids (12). Early studies, based on thyrotropin (TSH) affinity chromatography, suggested that gangliosides were structurally and functionally an important element of thyrothropin receptor (TSHR) affecting TSH binding and thyrocyte function (13–18). One may speculate that potential changes of its immunogenicity might modify the course of Graves’ disease. 1Department of Internal Medicine and Endocrinology, Medical University of Warsaw, Warsaw, Poland. of Biochemistry, Medical Centre for Postgraduate Medical Education, Warsaw, Poland. 3Department of Hypertension and Diabetes, Medical University of Gdańsk, Gdańsk, Poland. 4Department of General and Thoracic Surgery, Medical University of Warsaw, Warsaw, Poland. 5Division of Endocrine Surgery, Institute of Oncology, Warsaw, Warsaw Poland. 6Department of Pathology, Medical University of Warsaw, Warsaw, Poland. 7Department of Endocrinology, Medical Research Centre, Polish Academy of Science, Warsaw, Poland. 2Department 645 646 On the other hand, sialic acid metabolism in the thyroid is regulated by thyrotropin (19). Sialylation is a late posttranslational modification of thyroglobulin that can affect synthesis and secretion of the thyroid hormone (19) and the thyroid hormone itself may control sialic acid content of various glycoproteins and glycolipids (20,21). The synthesis of glycolipids and glycoproteins depends on the activity of specific glycotransferases. Sialyltransferases belong to the family of glycotransferases catalysing the transfer of sialic acid from cytidine monophosphosialic acid (CMP)-sialic acid to terminal position of sugar chains of glycolipids and glycoproteins. The nomenclature of sialyltransferases similarly to other glycotransferases is quite confusing. It is becoming clearer that the rule of one enzyme one activity is not always obvious (22). Until now more than a dozen of distinct sialyltransferases have been characterized and their genes have been cloned in various species (23,24). Among them sialyltransferase-1 (␤-galactoside ␣-2,6-sialyltransferase, previously known as ST6Gal I or CD75) and sialyltransferase-4A (␤-galactoside ␣-2,3-sialyltransferase, previously known as ST3Gal I) are well defined in many species including humans. Their corresponding human genes were cloned and named SIAT1 and SIAT4A, respectively. Changes in their expression and cell distribution in organs, other than the thyroid, were found in some pathological conditions including: various kinds of cancer (25–28), liver cirrhosis (28), and von Willebrand disease (29). The presented results showed that the sialic acid content and metabolism is altered in Graves’ disease. To analyze sialic acid metabolism we measured lipid-bound sialic acid (LBSA), namely, ganglioside content, ganglioside profile, total sialyltransferase activity, and the two sialyltransferase mRNA levels for sialyltransferase-1 and for sialyltransferase4A. Further studies are needed to address potential effects of the changes in sialic acid metabolism on thyroid function and pathophysiology of Graves’ disease. Material and Methods Patients and tissue samples The material consisted of 103 tissue samples obtained from 80 patients (56 females, 24 males; ages 13 to 71, mean, 37 years). Twenty-five specimens came from patients with Graves’ disease (GD). Twenty-eight samples came from 19 patients with toxic nodular goiter (19 from nodules (TN) and 9 from the nontumorous part of the same thyroid [TC]). Thirty-six samples came from 22 patients with nontoxic nodular goiters (22 from benign nodules [NN] and 14 from the nontumorous surrounding tissue [NC]). Nontumorous tissues (9 from toxic goiters, 14 from nontoxic nodular goiters, and 14 from patients with thyroid papillary carcinoma) were used as a control group, C. In some assays this group was split into subgroups: normal thyroid control tissue coming from toxic nodular goiter (TC) and from nontoxic nodular goiter (NC). All patients were diagnosed on the basis of physical examination, hormone level measurements (TSH, free thyroxine [FT4] and free triiodothyronine [FT3]), ultrasonography, iodine scyntygraphy, and/or fine-needle aspiration biopsy of the thyroid nodule if needed. The thyrotropin binding inhibitory immunoglobulin (TBII) level was measured in some patients with GD using the LumiTest TRAKhuman kit (Brahms, Hennigsdorf bei Berlin, Germany). KILJA ŃSKI ET AL. FT3, FT4, and TSH were measured on the Axsym system automatic analyzer (Abbott Laboratory, Abbott Park, IL) using Abbott assays based on the microparticle enzyme immunoassay technology. The ultrasensitive hTSH II assay has analytical sensitivity of 0.03 uIU/mL. Normal value ranges for FT3 were 1.64 to 3.45 pg/mL, for FT4 were 0.71 to 1.85 ng/dL and for TSH were 0.49 to 4.67 ␮U/mL. The laboratory is controlled by The Randox International Quality Assessment Scheme (RIQAS) of Immunoassay Programme (Randox Laboratories Ltd, Crumlin, Co., Antrim, UK). Patients with GD and toxic nodular goiter were treated before operation with thiamazol (methimazol) and ␤-adrenolitic agent, propranolol in doses 10 to 30 mg or 30 to 160 mg per day, respectively. The duration of pharmacologic treatment ranged from 3 weeks to 2 months, according to the amelioration of hyperthyroidism. All but four patients with GD were treated with iodine solution. They received 170 mg of iodine daily, 5 to 7 days before thyroidectomy. FT4 level ranged from 0.76 to 1.87 ng/dL, mean 1.12 (normal range, 0.71–1.85). The TSH level ranged from the value below analytical sensitivity (0.03 uIU/mL) to 4.93 uIU/mL, mean 2.81 (normal range, 0.49–4.67). FT3 ranged from 0.68 to 3.28 pg/mL, mean, 2.84 (normal range, 1.64–3.45). Based on the clinical signs the patients were euthyroid at the time of surgery. The study was approved by the Ethical Committee of the Medical Research Centre of Polish Academy of Sciences. Tissue preparation for estimation of LBSA, ganglioside, and sialyltransferase activity After removal of a thyroid gland, samples were immediately put on ice and transported to a pathologist who selected proper fragments for scientific purposes and for a routine anatomopathologic examination. Tissue samples were homogenized with 3 volumes of water on ice bath. Homogenates were used for the estimation of the total sialyltransferase activity (ST) and after the addition of the complete protease inhibitors cocktail (Roche Diagnostics, Mannheim, Germany) for measurement of protein content and for extraction of gangliosides. Thyroid gangliosides were extracted using the following method published by Svennerholm (11) and Phillips et al. (30). Briefly, tissue lipids were extracted over 24 hours at 4°C with methanol and chloroform in a final chloroform: methanol: water ratio of 4:8:3. After centrifugation and reextraction of the pellet (12 hours at 4°C) combined extracts were evaporated to dryness, dissolved in a final volume of 20 mL of chloroform: methanol (4:1), sonicated, filtered, and applied to a 5 mL G 60 silica gel columns filled with the same solvent. Gangliosides were eluted with 11 mL of chloroform: methanol: water mixture (3:6:2 by volume). Eluent was subsequently purified on DEAE-Sephadex A-25 columns (BioRad, Hercules, CA) packed with a mixture chloroform:methanol:water 30:60:8. Acidic lipids were eluted with methanol/0.2 M NaOAc and solubilized at 37°C in 1 M methanolic NaOH. The samples were then lyophilized and reconstituted in water. The solution was acidified with 3 M AcOH to pH 4.5–4.6. Samples were then desalted on Sep-Pak C18 columns (Waters, Milford, MA) (31). Gangliosides were eluted with methanol and solution chloroform/methanol 2:1. Eluents were dissolved in a small volume of chlo- SIALYLTRANSFERASES IN GRAVES’ DISEASE roform:methanol 4:1 and applied to small-volume silica gel columns (0.5–1.0 mL). Gangliosides were eluted with chloroform:methanol:water 3:6:2. The samples were lyophilized again, dissolved in chloroform:methanol. 2:1 and used for thin-layer chromatography (TLC) or measurement of LBSA content. Measurement of LBSA content in ganglioside samples The estimation of LBSA content was based on the method published by Suzuki et al. (32) modified for small quantities of material studied. Briefly: ganglioside fractions were lyophilized and subsequently sonicated in 0.2 mL of distilled water. Then, 0.4 mL of resorcinol reagent was added and samples were incubated at 37°C for 15 minutes and cooled down. Mixture of butyl acetate and n-butanol at an 85:15 ratio was added. After vortexing and centrifugation absorption of the upper phase was measured at 620 nm. TLC of thyroid gangliosides TLC was performed along with standards on silica gel 60 glass-backed HPTLC (high-performance thin layer chromotography) plates (Merck, Darmstadt, Germany) suitable for small samples (nano-TLC) with chloroform:methanol: aqueous 0.25% CaCl2 60:25:4 or with chloroform:methanol: aqueous-0.2 % CaCl2 60:40:9. Gangliosides were visualized with resorcinol reagent and the bands were scanned with a Shimadzu Model CS-910 dual-wavelength scanning densitometer at 580 nm. Immunostaining of TLC thin-layer plates (33) was done to confirm identification of ganglioside FucGM1. Briefly, after pretreatment with 0.1% solution of polyizobutylmetakrylate in hexane and blocking with 1% bovine serum albumin (BSA) in phosphate-buffered saline (PBS) strips of plates were incubated overnight at 4°C with specific primary antibody dissolved 1:100 in 1% BSA in PBS. After incubation with peroxidase-linked secondary antibodies (1:500) staining was developed using 4-chloro-1-naphthol. Total sialyltransferase activity (ST) in tissue samples The total ST activity in homogenized thyroid tissues was measured according to Delannoy et al. (34) using [3H] CMPNANA and asialofetuin (Roche Diagnostics) or asialoglycophorin (Sigma, St. Louis, MO) as exogenous acceptors. Asialofetuin and asialoglycophorin are highly glycosylated glycoproteins, carrying diversity of N- and O-linked glycans and both are good high molecular weight acceptors for ␣2-3 (e.g., sialyltransferase-4A), ␣2-6 (e.g., sialyltransferase1) and ␣2-8 sialyltransferases. Incubations were performed for 1–5 hours at 37°C in a sodium cacodylate buffer. The reaction was stopped with phosphotunstic acid and centrifuged 14000g for 10 min at 4°C. The pellet was washed 3 times with 1 mL of ice–cold 5% trichloroacetic acid, water, and ethanol, centrifuged as above and processed for counting in 1 mL of OptiPhase SuperMix scintillation fluid (Fisons Chemicals, Loughborough Leics, UK) using MicroBeta Trilux counter (Wallac, Turku, Finland). 0.2 mU of 2.6 sialyltransferase from rat liver (Roche Diagnostics) was used as a positive control in assay instead of thyroid homogenate. 90% of radioactivity is incorporated to the exogenous acceptor in this condition. Homogenate of normal thyroid incubated 1 minute in 95°C was used as a negative control. 647 RNA isolation and Northern blots Total RNA was isolated from tissue samples by Chomczynski’s method (35) using TRIzol reagent (Invitrogen, Carlsbad, CA). The RNA concentrations in all samples were measured by light absorptymetry (Wallac). Northern blots were prepared from 0.9% agarose-formaldehyde gels (1⫻MOPS, 0.9% agarose, 3% formaldehyde with 10 ␮g total RNA per line) by overnight osmotic transfer onto Gene Screen Plus membranes (New England Nuclear, Boston, MA) using 10 ⫻ SSPE buffer (3 M NaCl, 0.2 M NaH2PO4-H2O, 0.02 M ethylenediaminetetraacetic acid [EDTA]; pH 7.4). Clones, used for preparation of specific probes, were obtained from IMAGE Consortium. Clone IMAGE 2371549 (corresponding to EST GeneBank sequence GI 5234676) was used for SIAT1 probe and clone IMAGE 360422 (corresponding to EST GeneBank sequence GI 1476738) was used for SIAT4A probe. The specific SIAT1 probe was made using a 480-bp fragment of IMAGE clone 2371549 (corresponding to fragment between nucleotides 52 and 530 of human SIAT1 cDNA, X17247) by PCR reaction using M13-forward and M13-reverse primers. Single labeled probe was made by linear PCR using 10 ng of human SIAT1 cDNA, 3 fmol of specific antisense primer ACTCCCTTTCTTCTTTTCCTTCC, 10 fmol of [␣-32P]-dCTP (3000 Ci/mmol; Gene Screen Plus; New England Nuclear) and 4 units of Taq polymerase (MBI Fermentas, Vilnius, Lithuania) in 20 ␮L standard Taq polymerase buffer supplemented with (-dCTP)dNTP (final concentration, 0.05 mM) and Mg2⫹ (final concentration, 1.5 mM) (36). The SIAT4A probe was made from the 420-bp fragment of IMAGE clone 360422 (corresponding to fragment between nucleotides 339 and 757 of human SIAT4A cDNA, NM_003033) by PCR reaction using M13-forward and M13reverse primers. SIAT4A probe labeling was made by the random primer method, using Prime-It Random Primer Labeling kit (Stratagene, La Jolla, CA) with random primers, Klenow fragment (MBI Fermentas, Vilnius, Lithuania) and [␣-32P]-dCTP (3000 Ci/mmol). All probes were purified after labeling on a Sephadex G50 column chromatography. The percentage of [␣-32P]-dCTP incorporation was checked in the Wallac Microbeta counter (Wallac). Membranes were hybridized for 16 to 24 hours at 42°C in hybridisation buffer (5⫻ SSPE, 50% deionized formamide, 5⫻ Denhardt’s solution, 1% sodium dodecyl sulfate [SDS], 10% dextran sulfate) supplemented with salmon sperm DNA (50 ␮g/mL) and with labeled, temperature-denaturated probe (1 million cpm/mL). Prehybridization was performed without probe for 2 hours at 42°C. Blots were washed after hybridization twice for 15–30 minutes in 2 ⫻ SSC, 0.5% SDS at 50°C and twice for 15–30 minutes in 0.1 ⫻ SSC, 0.1% SDS at 50°C. After 3 to 10 days of long exposure, autoradiographs were scanned using a Sharp JX-330 scanner and signals were quantified by densitometry. The results were corrected to the total amount of RNA transferred, which was determined by ethidium bromide staining. Statistical analysis All data are reported as mean ⫾ standard deviation (SE). Groups were compared using the Mann-Whitney test, Student’s t test, and the correlation between variables was assessed using the Spearman test. 648 KILJA ŃSKI ET AL. Sialyltransferase activity and mRNA levels in thyroid tissue FIG. 1. Lipid-bound sialic acid (LBSA) content in Graves’ goiter. LBSA content was measured in samples coming from control thyroid tissues (C), Graves’ goiters (GD), and benign thyroid nodules: nontoxic (NN) and toxic (TN). The LBSA content in the GD group was significantly higher from the LBSA content in other groups, C, NN, or TN (p ⬍ 0.005 by Student’s t test). LBSA content in all samples from the GD group, in contrast to control tissues, was higher from 145 nmol/g. The sample numbers (n) is shown below the graph. Results LBSA content and ganglioside profile in thyroid tissues The LBSA content was the highest in Graves’ thyroid tissues (mean value was 164 ⫾ 4.44 nmol/g, p ⬍ 0.005 when compared to all other groups studied Fig. 1). There were no significant differences in LBSA content between C, TN, and NN groups (mean values, 120 ⫾ 2.00, 126 ⫾ 6.95 and 116 ⫾ 3.12 nmol/g, respectively). There were no major differences in ganglioside profiles between various groups (Table 1). GM3 was found to be the predominate ganglioside in all groups of tissues (over 50% of total ganglioside content). Gangliosides GM3 and GD3 constituted 60%–80% of gangliosides present in all analyzed tissues (Table 1). To evaluate less abundant FucGM1 content we used TLC-immunooverlay. However we did not observe significant expression of this ganglioside in any tissue studied. Because of the relatively small weight of tissue samples we could estimate only total sialyltransferase activity without differentiating into specific isoenzymes. The highest sialyltransferase activity was found in GD (1625 ⫾ 283.5 cpm/mg of protein, Fig. 2). The activity in GD group was significantly higher from the control group, C (324 ⫾ 54.2 cpm/mg of protein, p ⬍ 0.005) or from the nontoxic thyroid nodules, NN (607 ⫾ 101.0 cpm/mg of protein, p ⬍ 0.05). The difference between the GD group and the toxic nodules (TN) was not statistically significant (mean value for TN was 1153 cpm/mg of protein, p ⫽ 0,09). Sialyltransferase activity in the TN group was also significantly higher than in the control group, C (p ⬍ 0.005). Northern blot for sialyltransferase-1 (SIAT1 gene product) and sialyltransferase 4A (SIAT4A gene product) showed single mRNA signal of the expected size in all tested groups (approximately 4 kb and 7 kb, respectively; Fig. 3) (37–39). There were no obvious additional bands that might have suggested the existence of exon splice variants or different transcription sites. The sialyltransferase-1 mRNA level was the highest in Graves’ thyroid tissues (12.52 ⫾ 6.90 arbitrary units[AU]) and statistically different from control tissues, C (2.54 ⫾ 1.24 AU, p ⬍ 0.005 in MannWhitney test), or nontoxic nodules NN (3.14 ⫾ 3.61 AU, p ⬍ 0.05, Fig. 4A). The sialyltransferase-1 mRNA level in the TN group was not statistically different from the other groups. Similar results were found for sialyltransferase-4A mRNA (Fig. 4B), where the mRNA level was the highest in Graves’ thyroids (means were for GD 2,49 ⫾ 1.16 AU, for C 1.23 ⫾ 0.46 AU, p ⬍ 0.05). The expression level found in the TN group was significantly higher than in their counterpart the TC group (1.37 ⫾ 0.09 versus 1.00 ⫾ 0.12 U, p ⬍ 0.05). Surprisingly, we found extreme variability in mRNA content for both mRNAs within the same groups (Fig. 3) reaching 85-fold in the case of sialyltransferase-4A mRNA level in Graves’ disease. We also found a significant, positive correlation between increased sialyltransferase-1 mRNA level, but not sialyltransferase-4A mRNA level, and TSH-receptor antibody titer determined by the TRAK test (p ⫽ 0,029; R ⫽ 0,6017). TABLE 1. GANGLIOSIDE PROFILES IN THYROID TISSUES Major ganglioside content in thyroid tissues (% of total ganglioside content ⫾ ranges) Ganglioside Control (C) (n ⫽ 14) GM3 GM1 3⬘LM1 GD3 GD1a GD1b GT1 ⫹ GQ1 55 ⫾ ⬍2 4.5 ⫾ 17.5 ⫾ 7.5 ⫾ 3⫾ 3.5 ⫾ 5 1.5 2.5 2.5 1 1.5 Benign thyroid nodules (NN ⫹ TN) (n ⫽ 13) 55 ⫾ 5 ⬍2 2.5 ⫾ 0.5 15 ⫾ 5 7.5 ⫾ 2.5 ⬍2 3.5 ⫾ 1.5 Graves’ goiter (GD) (n ⫽ 12) 60 ⫾ ⬍2 ⬍2 10 ⫾ 12.5 ⫾ ⬍2 2.5 ⫾ 5 5 2.5 0.5 Values represent the percent of the total ganglioside content (mean ⫾ ranges). Each sample was measured in duplicates. Number of samples tested (n) is shown above. All ganglioside symbols are derived from Svennerholm classification (49). SIALYLTRANSFERASES IN GRAVES’ DISEASE FIG. 2. Total sialyltransferase activity in Graves’ goiter. Total sialyltransferase activity was significantly higher in Graves’ disease (GD) than in control nontumorous thyroid tissues, C (1625.14 ⫾ 283.48 and 324.08 ⫾ 54.21, respectively). The difference between GD and nontoxic nodules group NN (631.93 ⫾ 101.04; p ⬍ 0.05) was also significant, in contrast to toxic thyroid nodules, TN (1153.00 ⫾ 190.88, p ⫽ NS). Discussion GD is one of the most common thyroid pathologies affecting up to 1% of general population in the United States and in many countries of the European Community (40). It is well accepted that anti-TSH-R autoantibodies are the key pathogenic factor responsible for the alteration in the thyroid function and morphology (40). However, little is known about the initiation and the modulation of the autoimmune process during the waxing and waning course of this disease. Prior studies done before TSHR cloning suggested that gangliosides are components of the TSHR (13–16). In cultured rat thyroid cell line, FRTL-5 or bovine and human primary thyroid cell culture gangliosides modulate TSHR signal transduction and cyclic adenosinemonophosphate (cAMP) synthesis (13–16). In FRTL-5 cells glycosphingolipid moiety associated with TSHR was shown to belong to the ganglioteratose family with both galactose molecules sialylated (18). Although these data need to be confirmed, one may speculate that changes in the ganglioside amount or profile might work as negative or positive feedback for the TSHR activation. Our study has shown that LBSA content in Graves’ thyroids was not only 30% higher than in any other group but the single separating line can be drawn between its level in individual tissue samples coming from Graves’ thyroids or control thyroids. This suggests that the observed changes are not only meaningless statistically significant differences between groups but are rather important changes affecting every individual Graves’ thyroid tissue. Measurements of Graves’ thyroid total lipid content can be potentially affected by morphological changes associated with TSHR activation (relatively smaller follicles, containing less colloid and more cellular components). As a result one can expect a higher total lipid content in Graves’ thyroid than in normal thyroid tissue. Therefore, further studies might be needed to verify our results in cellular compartment of Graves’ thyroid. Despite the slightly higher content of ganglioside GM3 in Graves’ thyroids, profiles of the major thyroid gangliosides seem to be unchanged. This suggests that the sialyltransferase activ- 649 ity is not restricted to a specific lipid product but rather affects all lipids equally. However, for an unknown reason ganglioside profiles varied significantly from patient to patient in all studied groups. It has been suggested that ganglioside FucGM1 is occasionally a target of autoimmune reaction in GD (41) and in thyroid cancer (42). Interestingly we were unable to detect significant expression of FucGM1 in thyroid tissue. The increased content of LBSA in Graves’ thyroid might reflect its increased synthesis or decreased degradation. However, the increased content of sialic acid correlated with the increased sialyltransferase activity and their mRNA level. This suggests that increased sialic acid content is mostly the result of its increased incorporation into lipids rather than decreased degradation of LBSA. Similar to LBSA content, morphologic changes in Graves’ thyroid might potentially alter sialyltransferase activity measurement because of contamination of thyroglobulin. However, because of scarcity of the thyroid tissue samples, to obtain uniform results from the same sample, we decided to homogenize the thyroid tissue samples first followed by dividing them into three parts for estimation of LBSA, ganglioside composition, and sialyltransferase activity using different procedures. We did not measure either specific sialoprotein level (like thyroglobulin) or total protein bound sialic acid. Nevertheless the same sialyltransferase activity is responsible for sialic acid transfer into lipids and proteins (3). The same sialyltransferase activity is also responsible for sialic acid transfer into various gangliosides (3). Lack of changes in ganglioside profiles also FIG. 3. Northern blots showing specific SIAT1 and SIAT4A gene transcripts in thyroid tissues. Total RNA was extracted from 10 different thyroid tissues coming from 10 different patients. Four samples came from patients with Graves’ disease (GD), 4 from patients with papillary thyroid cancer (PTC; from surrounding normal tissues, C and 2 from tumor, PTC) and 2 from nodules of toxic nodular goiters, TN. Upper panel represents the top part of Northern blot hybridized with specific human sialyltransferase-4A (SIAT4A) probe. Single signal approximately 7 kb in size was seen much above 28S ribosomal bound. Middle panel shows specific sialyltransferase-1 (SIAT1) signal approximately 4 kb in size (below 28S ribosomal bands) and bottom panel shows ribosomal RNA stained with ethidium bromide. 650 FIG. 4. Sialyltransferase-1 (SIAT1) and sialyltransferase-4A (SIAT4A) mRNA levels in Graves’ goiter. Specific SIAT1 (A) or SIAT4A (B) mRNA levels were measured by Northern blots using the same techniques as for Figure 3. Two control groups, either from macroscopically normal tissues of nontoxic nodular goiter (NC) or from toxic nodular goiter (TC) are parts of a larger control group C. In the Graves’ goiters (GD) SIAT1 and SIAT4A mRNA levels were significantly higher than in the C group. SIAT1 but not SIAT4A mRNA levels were significantly different between the GD group and tissues of nontoxic and toxic nodules (NN and TN, respectively). Sample number (n) is shown below the graphs. suggests that the increase in LBSA content is more likely to result from the increased sialyltransferase activity rather than from the changes in substrate profile. In one of the earliest studies on human thyroid ganglioside profile in cultured thyrocytes of patients with GD Lee et al. (43) observed qualitative differences between samples coming from Graves’ disease, toxic adenoma, and normal thyroid. However, conditions used for primary cell cultures are quite different from those seen in tissues. The increased sialyltranferase-1 mRNA and sialytransferase-4 mRNA suggest that the increased sialyltransferase activity in Graves’ thyroid is at least partially the result of pretranslational mechanisms. Because activation or deactivation of the TSHR by autoantibodies present in Graves’ thyroid disease switch transcriptional machinery of many hu- KILJA ŃSKI ET AL. man thyroid genes it is tentative to postulate that TSHR activation was responsible for transcriptional changes in sialyltransferase gene expression. However, we did not measure the transcription directly and mRNA level, similarly to protein level, could be affected by both synthesis and degradation. The results suggest that mechanisms for gene transcription activation of two sialyltransferases are not the same, since there was no correlation in sialyltransferase-1 and sialyltransferase-4A mRNA levels in some tissues (Fig. 3). Interestingly the sialytransferase-1 mRNA level correlated with the TSHR antibodies titer. It must be emphasized that patients with GD were treated with antithyroid medication and large doses of iodine before surgery. Both factors can potentially affect gene expression. Hormonal status at the time of the surgery was an unlikely factor affecting the observed changes, because all patients were clinically euthyroid at least few weeks before and only few had decreased TSH with normal thyroid hormone suggesting subclinical hyperthyroidism. However, it is well known that changes associated with long-standing severe thyrotoxicosis can persist for several weeks after normalization of thyroid hormone levels. Because of the missing data we were unable to correlate the length of euthyroid state prior to surgery with sialyltransferase activity or mRNA levels. We believe that it is more likely that TSHR activation by autoantibodies is responsible for observed changes in sialyltransferase activity and mRNA levels. It would be of value to evaluate if sialylation of TSHR can affect thyroid autoimmune process and/or TSHR responsiveness to autoantibodies. The differences in sialyltransferase activity and sialic acid content were not observed in toxic nodular goiter. It was recently postulated that, similar to toxic thyroid adenomas, activating mutation in TSHR is also present in toxic nodules of multinodular goiter (44). Indeed in 3 of 6 tissues coming from toxic nodule the sialyltransfease-1 mRNA was higher than in any control tissue tested, although the statistical analysis is hampered by small sample size. Studies on TSH-dependent regulation of thyroid glycosyltransferases used different experimental models, species, and led to contradictory conclusions. Most of them focused on thyroglobulin as one of major products of thyroid cells and a protein well characterized in terms of glycan structure. Unfortunately, most studies used cell culture as an experimental model. It has been demonstrated that glycosylation of proteins in cell culture differs from their native forms (45). Franc and colleagues (46) found that TSH stimulated several thyroid glycosyltransferases in cultured porcine cells, among them sialyltransferases. In cell culture TSH increased sialylation of thyroglobulin shifting terminal glycosylation from ␣-galactose to sialic acid (45). In rats the TSH-evoked increase in glycosylation was exemplified by results of an in vivo study demonstrating that propylthiouracyl treatment increased mannosyltransferase and galactosyltransferase levels (47). On the other hand, Grollman and colleagues (48) reported marked TSH-evoked decrease not only in the level of ␣2,6-bound sialic acid but also decreased other core monosaccharide constituents of thyroglobulin in FRTL-5 cells. There are no prior data concerning the TSH effect on glycosyltransferase expression and activity in human thyroid tissue. This study has shown that in GD increased sialic acid content correlated with increased sialyltransferase activity and SIALYLTRANSFERASES IN GRAVES’ DISEASE increased mRNA level. While more data and alternative methodological approaches are needed to draw the definite conclusion, it is possible that the changes in sialic acid metabolism may affect the thyroid function and the autoimmune process and may be an important part of pathophysiology of Graves’ thyroid disease. Further work is needed to elucidate the exact mechanism for observed changes of sialic acid metabolism in Graves’ thyroid. 651 17. 18. 19. Acknowledgments The study was supported by the State Committee for Scientific Research grant number 4 P05B 042 15. 20. References 1. Sillanaukee P, Ponnio M, Jaaskelainen IP 1999 Occurrence of sialic acids in healthy humans and different disorders. Eur J Clin Invest 29:413–425. 2. Kim YJ, Varki A 1997 Perspectives on the significance of altered glycosylation of glycoproteins in cancer. Glycoconjugate J 14:569–576. 3. Varki A 1999 Sialic acids. In: Varki A, Cunnings R, Esko J, Freeze H, Hart G, Marth J (eds) Essentials of Glycobiology: Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY pp. 195–209. 4. Lanoue A, Batista FD, Stewart M, Neuberger MS 2002 Interaction of CD22 with alpha2,6–linked sialoglycoconjugates: Innate recognition of self to dampen B cell autoreactivity? Eur J Immunol 32:348–355. 5. Lloyd KO, Furukawa K 1998 Biosynthesis and functions of gangliosides: Recent advances. Glycoconjugate J 15:627–636. 6. Malisan F, Testi R 2002 GD3 ganglioside and apoptosis. Biochim Biophys Acta 1585:179–187. 7. Allende ML, Proia RL 2002 Lubricating cell signaling pathways with gangliosides. Curr Opin Structural Biol 12: 587–592. 8. Hakomori Si SI 2002 Inaugural article: The glycosynapse. Proc Natl Acad Sci USA 99:225–232. 9. Van Dessel GA, Lagrou AR, Hilderson HJ, Dierick WS, Lauwers WF 1979 Structure of the major gangliosides from bovine thyroid. J Biol Chem 254:9305–9310. 10. Iwamori M, Sawada K, Hara Y, Nishio M, Fujisawa T, Imura H, Nagai Y 1982 Neutral glycosphingolipids and gangliosides of bovine thyroid. J Biochemi (Tokyo) 91:1875–1887. 11. Svennerholm L 1985 Gangliosides of human thyroid gland. Bioch Biophys Acta 835:231–235. 12. Bouchon B, Portoukalian J, Madec AM, Orgiazzi J 1990 Evidence for several cell populations in human thyroid with distinct glycosphingolipid patterns. Biochim Biophys Acta 1051:1–5. 13. Mullin BR, Fishman PH, Lee G, Aloj SM, Ledley FD, Winand RJ, Kohn LD, Brady RO 1976 Thyrotropin-ganglioside interactions and their relationship to the structure and function of thyrotropin receptors. Proc Natl Acad Sci USA 73:842–846. 14. Aloj SM, Kohn LD, Lee G, Meldolesi MF 1977 The binding of thyrotropin to liposomes containing gangliosides. Biochem Biophys Res Commun 74:1053–1059. 15. Gardas A, Nauman J 1981 Presence of gangliosides in the structure of membrane receptor for thyrotrophin. Acta Endocrinolo (Copenh) 98:549–555. 16. Gardas A, Adler G, Lewartowska A, Faryna M, Nauman J 1983 Influence of antiganglioside antibodies on thyrotrophin 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. binding and adenyl cyclase activity of thyroid plasma membranes. Acta Endocrinol (Copenh) 104:333–339. Kielczynski W, Harrison LC, Leedman PJ 1991 Direct evidence that ganglioside is an integral component of the thyrotropin receptor. Proc Natl Acad Sci USA 88:1991– 1995. Kielczynski W, Bartholomeusz RK, Harrison LC 1994 Characterization of ganglioside associated with the thyrotrophin receptor. Glycobiology 4:791–796. Grollman EF, Doi SQ, Weiss P, Ashwell G, Wajchenberg BL, Medeiros-Neto G 1992 Hyposialylated thyroglobulin in a patient with congenital goiter and hypothyroidism. J Clin Endocrinol Metab 74:43–48. Trojan J, Theodoropoulou M, Usadel KH, Stalla GK, Schaaf L 1998 Modulation of human thyrotropin oligosaccharide structures—Enhanced proportion of sialylated and terminally galactosylated serum thyrotropin isoforms in subclinical and overt primary hypothyroidism. J Endocrinol 158:359–365. Persani L, Borgato S, Romoli R, Asteria C, Pizzocaro A, BeckPeccoz P 1998 Changes in the degree of sialylation of carbohydrate chains modify the biological properties of circulating thyrotropin isoforms in various physiological and pathological states. J Clin Endocrinol Metabol 83:2486–2492. Martin LT, Marth JD, Varki A, Varki NM 2002 Genetically altered mice with different sialyltransferase deficiencies show tissue-specific alterations in sialylation and sialic acid 9–O-acetylation. J Biol Chem 277:32930–32938. Tsuji S, Datta AK, Paulson JC 1996 Systematic nomenclature for sialyltransferases. Glycobiology 6:v–vii. Harduin-Lepers A, Vallejo-Ruiz V, Krzewinski-Recchi MA, Samyn-Petit B, Julien S, Delannoy P 2001 The human sialyltransferase family. Biochimie 83:727–737. Petretti T, Schulze B, Schlag PM, Kemmner W 1999 Altered mRNA expression of glycosyltransferases in human gastric carcinomas. Biochim Biophys Acta 142:209–218. Lo NW, Dennis JW, Lau JT 1999 Overexpression of the alpha2,6–sialyltransferase, ST6Gal I, in a low metastatic variant of a murine lymphoblastoid cell line is associated with appearance of a unique ST6Gal I mRNA. Biochem Biophys Res Commun 264:619–621. Wang PH, Li YF, Juang CM, Lee YR, Chao HT, Ng HT, Tsai YC, Yuan CC 2001 Altered mRNA expression of sialyltransferase in squamous cell carcinomas of the cervix. Gynecol Oncol 83:121–127. Cao Y, Merling A, Crocker PR, Keller R, Schwartz-Albiez R 2002 Differential expression of beta-galactoside alpha2,6 sialyltransferase and sialoglycans in normal and cirrhotic liver and hepatocellular carcinoma. Lab Invest 82:1515–1524. Ellies LG, Ditto D, Levy GG, Wahrenbrock M, Ginsburg D, Varki A, Le DT, Warth JD 2002 Sialyltransferase ST3Gal-IV operates as a dominant modifier of hemostasis by concealing asialoglycoprotein receptor ligands. Proc Natl Acad Sci USA 99:10042–10047. Phillips J, Schulze-Specking A, Rump JA, Decker K 1985 Age- and hormone-dependent ganglioside patterns of rat hepatocytes in primary culture. Biol Chem Hoppe Seyler 366:1131–1140. Williams MA, McCluer RH. 1980 The use of Sep-Pak C18 cartridges during the isolation of gangliosides. J Neurochem 35:266–269. Suzuki K 1965 The pattern of mammalian brain gangliosides. II. Evaluation of the extraction procedures, postmortem changes and the effect of formalin preservation. J Neurochem 12:629–638. 652 33. Hirabayashi Y, Koketsu K, Higashi H, Suzuki Y, Matsumoto M, Sugimoto M, Ogawa T1986 Sensitive enzyme-immunostaining and densitometric determination of ganglio-series gangliosides on thin-layer plate: pmol detection of gangliosides in cerebrospinal fluid. Biochim Biophys Acta 876:178–182. 34. Delannoy P, Pelczar H, Vandamme V, Verbert A 1993 Sialyltransferase activity in FR3T3 cells transformed with ras oncogene: Decreased CMP-Neu5Ac:Gal beta 1–3GalNAc alpha-2,3– sialyltransferase. Glycoconj J 10:91–98. 35. Chomczynski P, Sacchi N 1987 Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162:156–159. 36. Pachucki J, Hopkins J, Peeters R, Tu H, Carvalho SD, Kaulbach H, Abel ED, Wondisford FE, Ingwall JS, Larsen PR 2001Type 2 iodothyronin deiodinase transgene expression in the mouse heart causes cardiac-specific thyrotoxicosis. Endocrinology 142:13–20. 37. Wen DX, Svensson EC, Paulson JC 1992 Tissue-specific alternative splicing of the beta-galactoside alpha 2,6-sialyltransferase gene. J Biol Chem 267:2512–2518. 38. Hanasaki K, Varki A, Stamenkovic I, Bevilacqua MP 1994 Cytokine-induced beta-galactoside alpha-2,6–sialyltransferase in human endothelial cells mediates alpha 2,6–sialylation of adhesion molecules and CD22 ligands. J Biol Chem 269:10637–10643. 39. Kitagawa H, Paulson JC 1994 Differential expression of five sialyltransferase genes in human tissues. J Biol Chem 269:17872–17878. 40. Weetman AP 2000 Graves’ disease. N Engl J Med 343: 1236–1248. 41. Adler G, Pacuszka T, Targonska I, Lewartowska A, Nauman J 1994 Incidental presence of antibodies against gangliosides in Graves’ disease. Autoimmunity 18:149–152. 42. Lewartowska A, Pacuszka T, Adler G, Panasiewicz M, Wojciechowska W 2002 Ganglioside reactive antibodies of IgG and IgM class in sera of patients with differentiated thyroid cancer. Immunol Lett 80:129–132. KILJA ŃSKI ET AL. 43. Lee G, Grollman EF, Aloj SM, Kohn LD, Winand RJ 1977 Abnormal adenylate cyclase activity and altered membrane gangliosides in thyroid cells from patients with Graves’ disease. Biochem Biophys Res Commun 77:139–146. 44. Tonacchera M, Agretti P, Chiovato L, Rosellini V, Ceccarini G, Perri AP, Naccarato AG, Miccoli P, Pinchera A, Vitti P 2000 Activating thyrotropin receptor mutations are present in nonadenomatous hyperfunctioning nodules of toxic or autonomous multinodular goiter. J Clin Endocrinol Metab 85:2270–2274. 45. Ronin C, Fenouillet E, Hovsepian S, Fayet G, Fournet B 1986 Regulation of thyroglobulin glycosylation. A comparative study of the thyroglobulins from porcine thyroid glands and follicles in serum-free culture. J Biol Chem 261:7287–7293. 46. Franc JL, Hovsepian S, Fayet G, Bouchilloux S 1984 Differential effects of thyrotropin on various glycosyltransferases in porcine thyroid cells. Biochem Biophys Res Commun 118:910–915. 47. Spiro MJ 1980 Preferential response of thyroid glycosyltransferases to changes in thyrotropin stimulation. Arch Biochem Biophys 202:35–42. 48. Grollman EF, Saji M, Shimura Y, Lau JT, Ashwell G 1993 Thyrotropin regulation of sialic acid expression in rat thyroid cells. J Biol Chem 268:3604–3609. 49. Svennerholm L 1963 Chromatographic separation of human brain gangliosides. J Neurochem 10:613–623. Address reprint requests to: Dr Jacek Kiljanski Department of Internal Medicine and Endocrinology Medical University of Warsaw ul. Banacha 1a 02–097 Warsaw Poland E-mail: kiljan@amwaw.edu.pl