Carbohydrate and protein determinants are involved in thyroglobulin recognition by FRTL 5 cells.
To avoid premature lysosomal degradation, thyrocytes have a system able 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-acetylglucosamine (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 showed that GlcNAc residues alone induce only weak recycling. The latter finding led us to investigate the possibility that protein determinants might also be involved in binding. For this purpose, we studied binding 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 the presence of calcium, i.e. under conditions previously reported for binding of GlcNAc-BSA to porcine thyroid cell membranes. As expected, binding was GlcNAc- and oligosaccharide-dependent because Bandeiraea Simplificiola 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 did not inhibit binding, indicating that carbohydrates were not the sole determinants for binding. The fact that pronase digestion of Tg totally abolished binding implied that peptide determinants were involved in the interaction. This involvement is supported by the observation that porcine, rat, bovine, and human Tg bound FRTL 5 cell membranes and that monoclonal antibodies raised against human Tg interfered with the binding of both human and porcine Tg. Based on these findings we conclude that, besides the involvement of GlcNAc-bearing oligosaccharides, Tg receptors form a stable bond with peptide determinants.
Mziaut
H
H
Laboratoire de Biochimie, Ingénierie des Proteines, URA 1455, Marseille, France.
Bastiani
P
P
Balivet
T
T
Papandreou
M J
MJ
Fert
V
V
Erregragui
K
K
Blanck
O
O
Miquelis
R
R
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D002241:Carbohydrates / Q000502:physiology*; D002417:Cattle; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D017124:Conserved Sequence; D006801:Humans; D009844:Oligosaccharides / Q000378:metabolism; D011506:Proteins / Q000502:physiology*; D051381:Rats; D011956:Receptors, Cell Surface / Q000378:metabolism; D013552:Swine; D013954:Thyroglobulin / Q000235:genetics / Q000378:metabolism*; D013961:Thyroid Gland / Q000166:cytology / Q000378:metabolism*; D013972:Thyrotropin / Q000502:physiology*
D000911:Antibodies, Monoclonal; D002241:Carbohydrates; D009844:Oligosaccharides; D011506:Proteins; D011956:Receptors, Cell Surface; C032198:thyroglobulin receptor; D013972:Thyrotropin; D013954:Thyroglobulin