Loss of one asparagine-linked oligosaccharide from human transferrin receptors results in specific cleavage and association with the endoplasmic reticulum.
The coding sequence for the human transferrin receptor gene has been mutated in order to abolish the attachment of the oligosaccharide closest to the transmembrane sequence. Expression of the mutant receptor in Chinese hamster ovary cells resulted, after analysis under nonreducing conditions, in an 85- and a 73-kDa receptor species. After reduction, the 85-kDa receptors were mostly converted to 73 kDa, although with short labeling periods some 85-kDa receptor remained suggesting that the mutated gene was capable of coding for the entire polypeptide which was then proteolytically processed. This was supported by in vitro translation of mRNA from either wild type or mutant cells which in both cases yielded an 80-kDa protein, the full size of the nonglycosylated protein. The solubility characteristics of the mutant receptor suggest that it contains the COOH-terminal extracellular domain but not the transmembrane sequence. This is supported by the endoglycosidase H sensitivity of the 73-kDa protein which is compatible with the retention of two of the original three high mannose oligosaccharides. The receptor that lacks one oligosaccharide is unable to form intermonomer disulfide bridges or to migrate to the cell surface and is located in the endoplasmic reticulum where it is further degraded after a lag period of about 30 min.
Hoe
M H
MH
Department of Microbiology and Immunology, University of South Carolina Medical School, Columbia 29208.
Hunt
R C
RC
D000818:Animals; D001216:Asparagine / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D004591:Electrophoresis, Polyacrylamide Gel; D005455:Fluorescent Antibody Technique; D015870:Gene Expression; D006596:Hexosaminidases / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D009844:Oligosaccharides / Q000235:genetics* / Q000378:metabolism; D011233:Precipitin Tests; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics; D011990:Receptors, Transferrin / Q000235:genetics*; D014162:Transfection; D014168:Transferrin / Q000378:metabolism; D014415:Tunicamycin / Q000494:pharmacology
D009844:Oligosaccharides; D012333:RNA, Messenger; D011990:Receptors, Transferrin; D014168:Transferrin; D014415:Tunicamycin; D001216:Asparagine; D006596:Hexosaminidases