1. Glycosylation of Sera Thyroglobulin Antibody in Patients with Thyroid Diseases Department of Endocrinology, Peking University First Hospital, Beijing 100034, China Ying Gao, Lanlan Zhao, Mingming Liu, Youyuan Huang, Guizhi Lu, Yanming Gao, Xiaohui Guo

2. Backgrounds Thyroglobulin antibody (TgAb) is one of the major autoantibodies in thyroid diseases. In papillary thyroid cancer (PTC) In autoimmune thyroid disease (AITD): Hashimoto' thyroiditis (HT) and Graves' disease (GD) Spencer CA, et al. J Clin Endocrinol Metab. 2011,96:3615-3627. Calder EA, et al. Clin Exp Immunol. 1973, 14: 153-8.

3. Backgrounds TgAb is predominantly of IgG class IgG is a glycoprotein with a sugar moiety attached to each of the asparagin 297 residues in the CH2-domains of the two Fc-fragments. Fuc ： fucose Gal ： galactose Neu5AC: sialic acid Torrigiani G, et al. Clin Exp Immunol. 1968,3:621-630. Fc fragments CH2 domains Fab fragments

4. Shields RL, et al. J Biol Chem. 2002,277:26733-26740. Scallon BJ,et al. Mol Immunol. 2007,44:1524-1534. Kodar K et al. Glycoconj J. 2012,29:57-66. Backgrounds The absence of core fucose residues in the Fc glycans substantially increases the ADCC activity of IgG. Increased sialylation of Fc glycans results in decreased ADCC activity. Alteration of IgG-Fc galactosylation have been described not only in autoimmune diseases, but also in some malignancy diseases. These glycoforms can differ in their efficacy of effector function activation as it influences binding of IgG molecules to Fc receptors and C1q.

5. Objective The aim of our study was to investigate the glycosylation of sera TgAb in patients with different thyroid diseases including HT, GD, and PTC.

6. Materials and Methods Sera samples were collected in Peking University First Hospital. Eu: euthyroidism; sH: subclinical hypothyroidism; H: hypothyroidism

7. Thyroid function: Chemiluminescence immunoassays TgAb IgG: antigen specific ELISAs carbohydrate residues on sera TgAb: Lectin-ELISAs The relative amount of fucose in each TgAb IgG was calculated as: Comparisons were carried out by the Mann–Whitney test, ANOVA, Chi-Square test and Kruskal-Wallis H test. x 100% the percentage of fucose positive control the percentage of TgAb IgG positive control Materials and Methods

8. a P < 0.05 vs. GD; b P < 0.05 vs. thyroid cancer group. Table 1. Demographic data, thyroid functional status and TgAb IgG levels of the patients in different groups. GroupsHT (n=81)GD (n=16)Thyroid cancer (n=12) Age (years)50 (33-64) a 29 (23-41)38 (29-61) Gender (M/F)5/760/162/10 TT3 (nmol/l)1.51 (1.22-1.69) a, 5.81 (3.79-9.19) b 1.55 (1.28-1.66) TT4 (nmol/l)84.70 (61.70-102.80) a, b 220.60 (137.70- 262.10) b 102.75 (88.00-117.05) TSH (mIU/l)6.51 (4.12-22.81) a, b 0.01 (0.002-0.02) b 2.98 (1.09-4.81) Positive percentage of TgAb IgG (%) 53.03±17.7653.66±17.8149.63±18.49 Results

9. Table2. Demographic data, thyroid functional status and TgAb IgG levels of the patients in HT subgroups. a P < 0.05 vs. sH. ; b P < 0.05 vs. H. Groups Eu (n=26)sH (n=22)H (n=33) Age (years) 43.35±16.3449.36±14.2048±13.56 Gender (M/F)0/261/214/29 TT3 (nmol/l)1.57 (1.44-1.63) a, b 1.75 (1.58-1.99) b 1.18 (0.99-1.41) TT4 (nmol/l)98.10 (83.43-107.35) b 93.50 (83.53-105.40) b 52.20 (30.80-68.60) TSH (mIU/l) 2.87 (1.78-4.26) a, b 7.20 (6.10-11.55) b 38.38 (10.19-101.09) Positive percentage of TgAb IgG (%) 44.93 (29.43-56.45) a, b 54.38 (44.67-68.77)61.52 (40.24-72.37) Results

10. Fig.1. Comparisons of the relative amount of carbohydrate residues on each sera TgAb from patients with different thyroid diseases. Results

11. Fig.2. Comparisons of the relative amount of carbohydrate residues on each serum TgAb from Hashimoto ’ s thyroiditis patients with different thyroid functional status. Results

12. Fig.3. The correlation between the relative amount of carbohydrate residues on each TgAb and TgAb IgG in all the patients (n = 109). Results

13. The levels of fucosylation and sialylation on TgAb varied in different thyroid diseases. We speculated that TgAb with lower content of core fucose and terminal sialic acid might have stronger ability to participate in ADCC in HT. Terminal galactose content of IgG does not affect ADCC but complement dependent cytotoxicity (CDC). Discussions

14. There were no significant differences in the levels of glycosylation on each TgAb among the three HT subgroups. The levels of glycosylation on TgAb might not represent thyrocyte hyperplasia but merely reflect the capacity of inducing thyroid destruction Discussions

15. The levels of glycosylation on each TgAb had a negative relationship with TgAb IgG levels a consequence of elevated Ig synthesis by B cells. the individuals with thyroid antibodies might be at high risk of developing thyroid failure. Discussions Vanderpump MP, et al. Clin Endocrinol (Oxf). 1995, 43:55-68. Parekh R, et al. J Autoimmun. 1989, 2:101-114.

16. 1. Glycosylation of sera TgAb varied in the patients with different thyroid diseases. 2. The levels of glycosylation of TgAb might decrease with increasing TgAb levels. Conclusions

17. Acknowledgements Supported by Beijing Natural Science Foundation Beijing Nova Program Program for New Century Excellent Talents in University Sector funds of ministry of health (no. 201002002). Thank you for your attention!!

18. Fig.1. The biotinylated lectins binding to Tg in different oxidation time with sodium periodate.

19. Fig.2. TgAb lgG binding to thyroglobulin with a serial dilution (diluted 1:12.5 - 1:800) in different oxidation time with sodium periodate.