CARBOHYDRATE AND PROTEIN DETERMINANTS ARE INVOLVED IN THYROGLOBULIN RECOGNITION BY FRTL-5 CELLS

To avoid premature lysosomal degradation, thyrocytes have a system abl e to recycle internalized immature thyroglobulin molecules (Tg) to the follicular lumen via the Golgi apparatus. It has been shown that this quality control system depends on recognition of exposed N-acetylgluc osamine (GlcNAc) determinants (Miquelis et al., J Cell Biol, 1993, 123 , 1695) present on immature Tg (Bastiani et al., 1995, Endocrinology, 1995, 136, 4204). However, the same in vitro kinetics studies also sho wed that GlcNAc residues alone induce only weak recycling. The latter finding led us to investigate the possibility that protein determinant s might also be involved in binding. For this purpose, we studied bind ing of Tg to FRTL 5 cells, a widely available TSH-dependent cell line and found that binding to plasma membranes occurred at acidic pH in th e presence of calcium, i.e. under conditions previously reported for b inding of GlcNAc-BSA to porcine thyroid cell membranes. As expected, b inding was GlcNAc- and oligosaccharide-dependent because Bandeiraea Si mplificiola II affinity column analysis indicated that GlcNAc-bearing Tg were preferentially bound and N-glycanase treatment of Tg inhibited interaction. Ovomucoid, GlcNAc-BSA, and porcine Tg oligosaccharides d id not inhibit binding, indicating that carbohydrates were not the sol e determinants for binding. The fact that pronase digestion of Tg tota lly abolished binding implied that peptide determinants were involved in the interaction. This involvement is supported by the observation t hat porcine, rat, bovine, and human Tg bound FRTL 5 cell membranes and that monoclonal antibodies raised against human Tg interfered with th e binding of both human and porcine Tg. Based on these findings we con clude that, besides the involvement of GlcNAc-bearing oligosaccharides , Tg receptors form a stable bond with peptide determinants.