Glycosylation of human truncated Fc epsilon RI alpha chain is necessary for efficient folding in the endoplasmic reticulum.
The high affinity immunoglobulin E (IgE) receptor is an alpha beta gamma 2 tetrameric complex. The truncated extracellular segment (alpha t) of the heavily glycosylated alpha chain is sufficient for high affinity binding of IgE. Here we have expressed various alpha t mutants in eukaryotic and prokaryotic cells to analyze the role of glycosylation in the folding, stability, and secretion of alpha t. All seven N-linked glycosylation sites in alpha t are glycosylated and their mutations have an additive effect on the folding and secretion of alpha t. Mutation of the seven N-glycosylation sites (delta 1-7 alpha t) induces misfolding and retention of alpha t in the endoplasmic reticulum. Similarly, tunicamycin treatment reduces substantially the folding efficiency of wild-type alpha t. In contrast, no difference in folding efficiency is detected between wild-type alpha t and delta 1-7 alpha t expressed in Escherichia coli. In addition, maturation of N-linked oligosaccharides and addition of O-linked carbohydrates are not required for either the transport or the IgE-binding function of alpha t. Furthermore, complete enzymatic deglycosylation does not affect the stability and the IgE-binding capacity of alpha t. Therefore, glycosylation is not intrinsically necessary for proper folding of alpha t but is required for folding in the endoplasmic reticulum. Our data are compatible with the concept that specific interactions between N-linked oligosaccharides and the folding machinery of the endoplasmic reticulum are necessary for efficient folding of alpha t in eukaryotic cells.
Letourneur
O
O
Molecular Allergy and Immunology Section, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, Maryland 20852, USA.
Sechi
S
S
Willette-Brown
J
J
Robertson
M W
MW
Kinet
J P
JP
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000276:immunology; D001692:Biological Transport; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D006031:Glycosylation; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010455:Peptides / Q000276:immunology; D017510:Protein Folding; D017455:Receptors, IgE / Q000235:genetics / Q000378:metabolism*; D014415:Tunicamycin / Q000494:pharmacology
D000906:Antibodies; D010455:Peptides; D017455:Receptors, IgE; D014415:Tunicamycin