The role of constant region carbohydrate in the assembly and secretion of human IgD and IgA1.
Immunoglobulins are glycoproteins, containing N- linked carbohydrates in the heavy chain constant regions of all isotypes and O-linked carbohydrates in the hinge regions of human IgA1 and IgD. A previous study showed that IgD synthesized in the presence of tunicamycin and lacking the three N-linked glycans on the heavy chain was not secreted (Shin, S. U., Wei, D. F., Amin, A. R., Thorbecke, G. J., and Morrison, S. L. (1992) Hum. Antibodies 3, 65-74). The contribution of each of the carbohydrates in the Fc of IgD to assembly and secretion was now analyzed by eliminating the carbohydrate addition sequence, Asn-X-Ser/Thr, through site-directed mutagenesis. Only the carbohydrate nearest the sole disulfide bond between heavy chains, which remained high mannose and appeared to be buried within the folded molecule, was found to be essential for secretion. When IgD lacked that glycan, assembly reached only the heavy/light chain half-molecule stage, and heavy chains were held inside the endoplasmic reticulum. Using benzyl 2-acetamido-2-deoxy-alpha-d-galactopyranoside (BADG) to inhibit complete O-linked glycosylation, we found that IgA1 and IgD with incomplete hinge carbohydrates were assembled and secreted from cells. Thus, one N-linked glycan plays a structural role in IgD and is required for proper assembly and secretion, but the O-linked carbohydrates in the hinge of IgD and IgA1 are not required for folding and export.
Gala
Francoise A
FA
Department of Microbiology, University of California, Los Angeles, California 90095-1489, USA.
Morrison
Sherie L
SL
D000117:Acetylglucosamine / Q000031:analogs & derivatives* / Q000737:chemistry; D000818:Animals; D015153:Blotting, Western; D016466:CHO Cells; D050260:Carbohydrate Metabolism; D002241:Carbohydrates / Q000737:chemistry*; D003001:Cloning, Molecular; D006224:Cricetinae; D004220:Disulfides; D004591:Electrophoresis, Polyacrylamide Gel; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005822:Genetic Vectors; D006026:Glycoside Hydrolases / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D006868:Hydrolysis; D007070:Immunoglobulin A / Q000378:metabolism*; D007072:Immunoglobulin D / Q000378:metabolism*; D008358:Mannose / Q000737:chemistry; D051379:Mice; D008856:Microscopy, Fluorescence; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D014162:Transfection
D002241:Carbohydrates; D004220:Disulfides; D007070:Immunoglobulin A; D007072:Immunoglobulin D; C044848:10-N-(bromoacetyl)amino-1-decylglucopyranoside; D006026:Glycoside Hydrolases; D008358:Mannose; D000117:Acetylglucosamine