Role of oligosaccharides in the processing and function of human transferrin receptors. Effect of the loss of the three N-glycosyl oligosaccharides individually or together.
When the coding sequence for human transferrin receptors was expressed in a Chinese hamster ovary cell line lacking endogenous transferrin receptors, 86-kDa molecules containing three N-glycosidically linked oligosaccharides were synthesized. These rapidly dimerized to form 172-kDa molecules which increased in size to 190 kDa. After site-directed mutagenesis of all three N-glycosylation sites, 80-kDa receptors were synthesized and only a few dimers were formed. 84-kDa monomers were synthesized in the absence of the oligosaccharide attached to Asn727 or Asn317. Dimerization and maturation through the Golgi body of the Asn727 mutant receptors were much slower than the wild type whereas the Asn317 mutant receptors behaved more similarly to the wild type. Lack of the oligosaccharide at Asn251 gave rise to 73-kDa monomers because of proteolytic processing (Hoe, M. H., and Hunt, R. C. (1992) J. Biol. Chem. 267, 4916-4923), but a second mutation at a potential cleavage site allowed the formation of 84-kDa receptors. These also dimerized at a similar rate to wild type receptors. The three-site mutant receptors were degraded in the endoplasmic reticulum but all three 84-kDa single site mutant receptor species migrated to the cell surface. However, receptors lacking the oligosaccharide at Asn727 bound and internalized little transferrin as a result of reduced affinity.
Yang
B
B
Department of Microbiology and Immunology, University of South Carolina School of Medicine, Columbia 29208.
Hoe
M H
MH
Black
P
P
Hunt
R C
RC
D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D006224:Cricetinae; D004247:DNA; D005455:Fluorescent Antibody Technique; D006031:Glycosylation; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009844:Oligosaccharides / Q000378:metabolism*; D011233:Precipitin Tests; D011499:Protein Processing, Post-Translational; D011990:Receptors, Transferrin / Q000235:genetics / Q000378:metabolism*
D009844:Oligosaccharides; D011990:Receptors, Transferrin; D004247:DNA