Role of asparagine-linked oligosaccharides in protein folding, membrane targeting, and thyrotropin and autoantibody binding of the human thyrotropin receptor

J Biol Chem. 1998 Dec 11;273(50):33423-8. doi: 10.1074/jbc.273.50.33423.

Abstract

The amino-terminal ectodomain of thyrotropin (TSH) receptor (TSHR) is heavily glycosylated with asparagine-linked (N-linked) oligosaccharides. The present studies were designed to evaluate how acquisition and processing of N-linked oligosaccharides play a role in the functional maturation of human TSHR. A glycosylation inhibitor tunicamycin, which inhibits the first step of N-linked glycosylation (acquisition of N-linked oligosaccharides), and a series of mutant Chinese hamster ovary (CHO)-Lec cells defective in the different steps of glycosylation processing were used. Inhibition of acquisition of N-linked oligosaccharides by tunicamycin treatment in CHO cells stably expressing TSHR produced nonglycosylated TSHR, which was totally nonfunctional. In contrast, all of the TSHRs synthesized in mutant CHO-Lec1, 2, and 8 cells (mannose-rich, sialic acid-deficient, and galactose-deficient oligosaccharides, respectively) bound TSH and produced cAMP in response to TSH with an affinity and an EC50 similar to those in TSHR expressed in parental CHO cells (CHO-TSHR; sialylated oligosaccharides). However, Lec1-TSHR and Lec2-TSHR were not efficiently expressed on the cell surface, whereas the expression levels of Lec8-TSHR and CHO-TSHR were essentially identical. All of the TSHRs expressed in CHO-Lec cells cleaved into two subunits. Finally, anti-TSHR autoantibodies from Graves' patients interacted with all of the TSHRs harboring different oligosaccharides to a similar extent. These data demonstrate that acquisition and processing of N-linked oligosaccharides of TSHR appear to be essential for correct folding in the endoplasmic reticulum and for cell surface targeting in the Golgi apparatus. We also show that complex type carbohydrates are not crucially involved in the interaction of TSHR with TSH and anti-TSHR autoantibodies.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Asparagine / chemistry
  • Asparagine / metabolism*
  • Autoantibodies / metabolism*
  • Blotting, Western
  • CHO Cells
  • Carbohydrate Sequence
  • Cell Membrane / metabolism
  • Clone Cells
  • Cricetinae
  • Cyclic AMP / biosynthesis
  • Humans
  • Molecular Sequence Data
  • Oligosaccharides / chemistry
  • Oligosaccharides / metabolism*
  • Precipitin Tests
  • Protein Binding
  • Protein Folding*
  • Receptors, Thyrotropin / metabolism*
  • Thyrotropin / metabolism*
  • Tunicamycin / pharmacology

Substances

  • Autoantibodies
  • Oligosaccharides
  • Receptors, Thyrotropin
  • Tunicamycin
  • Asparagine
  • Thyrotropin
  • Cyclic AMP