Deletion of clustered O-linked carbohydrates does not impair function of low density lipoprotein receptor in transfected fibroblasts.
A single exon in the gene for the receptor for plasma low density lipoprotein (LDL) encodes a region of clustered serine and threonine residues that is immediately external to the membrane-spanning sequence. This region has been proposed as the site of clustered O-linked carbohydrate chains. In the current studies we have deleted the 144 base pairs (48 amino acids) that encode this serine- and threonine-rich region from the cDNA for the human LDL receptor. Upon transfection into receptor-deficient hamster fibroblasts, this mutated cDNA encoded a shortened receptor that no longer showed an anomalously high molecular weight on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Labeling with [3H]glucosamine confirmed the lack of clustered O-linked sugars and further revealed that the shortened receptor and the normal receptor both contained isolated O-linked carbohydrate chains attached to the NH2-terminal portion of the protein. The ratio of clustered to isolated O-linked sugar chains in the normal receptor was estimated to be approximately 4-6 to 1. Despite the loss of clustered O-linked carbohydrate, the LDL receptor encoded by the deletion-bearing cDNA bound and internalized LDL normally. It also recycled normally and exhibited a normal half-life. We conclude that: 1) the serine- and threonine-rich region of the LDL receptor is the site for addition of clustered O-linked carbohydrates; 2) the receptor contains a small number of isolated chains of O-linked carbohydrates in addition to the clustered chains; and 3) the clustered O-linked carbohydrates are not essential for LDL receptor function in cultured hamster fibroblasts.
D000595:Amino Acid Sequence; D000818:Animals; D002241:Carbohydrates / Q000032:analysis*; D002460:Cell Line; D002738:Chloroquine / Q000494:pharmacology; D002846:Chromatography, Affinity; D002854:Chromatography, Paper; D006224:Cricetinae; D003412:Cricetulus; D003513:Cycloheximide / Q000494:pharmacology; D004247:DNA / Q000032:analysis; D005260:Female; D005347:Fibroblasts / Q000378:metabolism*; D005944:Glucosamine / Q000378:metabolism; D006801:Humans; D008970:Molecular Weight; D008985:Monensin / Q000494:pharmacology; D010053:Ovary / Q000032:analysis; D011973:Receptors, LDL / Q000378:metabolism*; D013539:Simian virus 40 / Q000235:genetics; D014162:Transfection; D014415:Tunicamycin / Q000494:pharmacology
D002241:Carbohydrates; D011973:Receptors, LDL; D014415:Tunicamycin; D002738:Chloroquine; D004247:DNA; D008985:Monensin; D003513:Cycloheximide; D005944:Glucosamine