Interactions between the mannose receptor and thyroid autoantigens

Abstract

Thyroid autoantigens require internalization and processing by antigen-presenting cells to induce immune responses. Besides pinocytosis, antigen uptake can be receptor-mediated. The mannose receptor (ManR) has a cysteine rich domain (CR) and eight carbohydrate recognition domains (CRD) that bind glycosylated proteins. The TSH receptor (TSHR), thyroid peroxidase (TPO) and thyroglobulin (Tg) are glycoproteins. To investigate a role for the ManR in thyroid autoimmunity, we tested the interaction between these autoantigens and chimeric ManRs. Plasmids encoding the CR-domain linked to IgG-Fc (CR-Fc) and CDR domains 4-7 linked to IgG-Fc (CDR4-7-Fc) were expressed and purified with Protein A. Enzyme-linked immunosorbent assay (ELISA) plates were coated with human thyroid autoantigens and CR-Fc or CRD4-7-Fc binding detected with peroxidase-conjugated anti-IgG-Fc. CRD4-7-Fc binding was highest for the TSHR, followed by Tg and was minimal for TPO. CR-Fc bound to Tg but not to TSHR or TPO. The interaction between the TSHR and CRD-Fc was calcium-dependent; it was inhibited by mannose (not galactose), and required a glycosylated TSHR A-subunit. Moreover, precomplexing the TSHR A-subunit with CRD-Fc (but not CR-Fc), or adding mannose (but not galactose), decreased in vitro responses of splenocytes from TSHR-immunized mice. Our data indicate that the ManR may participate in autoimmune responses to Tg and the TSHR but not to TPO. Most important, ManR binding of heavily glycosylated TSHR A-subunits suggests a mechanism by which the minute amounts of A-subunit protein shed from the thyroid may be captured by antigen-presenting cells located in the gland or in draining lymph nodes.

Fig. 1

Structure of the mannose receptor (a) and the chimeric proteins CR-Fc (b) and CDR4-7-Fc (c). CRDs, carbohydrate recognition domains; CR, cysteine-rich domain; FNII, fibronectin type II domain; TM, transmembrane region; CT, cytoplasmic tail. Adapted with permission from Linehan et al. [12] and Martinez-Pomares et al. [13], with kind permission from the publishers: WILEY-VCH Verlag GmbH & Co. KGaA [12]; and reproduced from the Journal of Experimental Medicine 1996, 184: 1927–37, copyright permission of The Rockefeller University Press.

Fig. 2

Enzymatic deglycosylation of the three major thyroid autoantigens. TSHR A-subunit and TPO ectodomain (purified from transfected CHO cell cultures) and Tg (Calbiochem) were digested with endoglycosidase F (see Methods). Aliquots of each protein (∼ 5 µg) before and after digestion were subjected to SDS-PAGE (12% for TSHR A-subunit, 10% for Tg and 7·5% for TPO) under reducing conditions followed by staining with Coomassie blue. The solid arrows indicate the size of each protein before and after deglycosylation. In the TSHR A-subunit panel, the dotted arrow indicates endoglycosidase F.

Fig. 3

Interaction between CRD4-7-Fc or CR-Fc and the TSHR A-subunit, Tg and TPO. For both panels, ELISA wells were coated with two concentrations of each autoantigen and binding was tested using three concentrations of CRD4-7-Fc or CR-Fc. Data are reported as the OD 490 nm (mean + s.e.m., n = 3). (a) CR-Fc binds to ELISA wells coated with Tg but not to wells coated with the TSHR A-subunit, TPO or (as a control) BSA. (b) CRD4-7-Fc binds to ELISA wells coated with the TSHR A-subunit and Tg but not to TPO or BSA.

Fig. 4

Binding of CRD4-7-Fc to the TSHR requires calcium. ELISA wells were coated with recombinant TSHR A-subunit (a, b) or the porcine TSH holoreceptor (c, d). Binding of CRD4-7-Fc or CR-Fc was studied in the presence of 10 mm CaCl₂ (a, c) or the presence of 50 mm EDTA (b, d). Data are reported as OD 490 nm (mean + s.d., n = 2).

Fig. 5

Specificity of TSHR A-subunit binding to the mannose receptor CRD. ELISA wells were coated with native, glycosylated TSHR A-subunit protein (4 µg/ml). For both panels, data are shown as OD 490 nm (duplicates + s.d.); *P < 0·05; buffer versus denatured TSHR A-subunit (10 µg/ml) or mannose (10 mm), Kruskal–Wallis one-way analysis of variance. (a) CRD4-7-Fc (0·4 µg/ml) was preincubated (30 min at room temperature) with buffer alone (‘0’) or with the indicated concentrations of either denatured TSHR A-subunits or denatured, deglycosylated TSHR A-subunits. The mixtures were then added to the ELISA wells and CRD4-7-Fc binding detected with conjugated anti-IgG-Fc. (b) CRD4-7-Fc (0·4 µg/ml) was mixed with buffer alone (‘0’) or with the indicated concentrations of either mannose or galactose. The mixtures were then added to the ELISA wells and CRD4-7-Fc binding detected with conjugated anti-IgG-Fc.

Fig. 6

Interactions between the TSHR A-subunit and mannose receptors tested in memory responses of splenocytes from TSHR-immunized mice. Data for both (a) and (b) are shown as the mean (+ range) of duplicate cultures with or without TSHR A-subunit (4 µg/ml). (a) Inclusion of mannose (10 mm) decreases IFN-γ secretion following challenge with A-subunit protein. In contrast, inclusion of galactose (also 10 mm) had a minimal inhibitory effect. (b) Precomplexing TSHR A-subunit protein with CRD4-7-Fc (but not CF-Fc) decreases IFN-γ production by splenocytes from TSHR-adenovirus (TSHR-Ad) immunized BALB/c mice.