The dysfunctional LDL receptor in a monensin-resistant mutant of Chinese hamster ovary cells lacks selected O-linked oligosaccharides.
The Chinese hamster ovary (CHO) cell line Monr31, which is resistant to the cytotoxic ionophore monensin, produces a receptor for the low density lipoprotein (LDL) that has a lowered binding affinity for LDL and is approximately 5 kDa smaller in size than the receptor from parental CHO cells. It has been proposed that the reduced size and affinity for LDL are associated with a reduced level of O-glycosylation of Ser/Thr residues in the receptor. To examine this possibility in more detail, both parental CHO and Monr31 cells were metabolically radiolabeled with [3H]glucosamine, and the labeled LDL receptors were purified by immunoprecipitation and identified by SDS-PAGE-fluorography. The Ser/Thr-linked oligosaccharides in the receptors from both parental CHO and Monr31 cells are mono- and desialylated species having the common core structure Gal beta 1-3GalNAc. The receptor from Monr31 cells, however, contains about one-third fewer Ser/Thr-linked oligosaccharides than the receptor from parental CHO cells. Analysis of the glycopeptides derived from the Monr31 cell LDL receptors indicates that they contain Ser/Thr-linked oligosaccharides only in the clustered domain and are missing Ser/Thr-linked oligosaccharides in the unclustered regions of the protein. Additionally, analysis of a human LDL receptor lacking the domain for attachment of the clustered Ser/Thr-linked oligosaccharides and expressed in both parental CHO and Monr31 cells indicated that the truncated human receptor from Monr31 cells is devoid of Ser/Thr-linked oligosaccharides. In contrast, the truncated human receptor produced by parental CHO cells contains Ser/Thr-linked oligosaccharides contributing approximately 5 kDa to its apparent size. Collectively, these results demonstrate that the LDL receptor produced by the Monr31 cells contains Ser/Thr-linked oligosaccharides in the clustered domain but is missing Ser/Thr-linked oligosaccharides in the unclustered, NH2-terminal domains of the receptor.
Seguchi
T
T
Department of Biochemistry, Oita Medical School, Japan.
Merkle
R K
RK
Ono
M
M
Kuwano
M
M
Cummings
R D
RD
D000116:Acetylgalactosamine / Q000737:chemistry; D000117:Acetylglucosamine / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D001216:Asparagine / Q000737:chemistry; D002236:Carbohydrate Conformation; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D004351:Drug Resistance / Q000235:genetics; D005260:Female; D006020:Glycopeptides / Q000737:chemistry; D006031:Glycosylation; D006801:Humans; D008985:Monensin / Q000494:pharmacology*; D009154:Mutation; D009844:Oligosaccharides / Q000737:chemistry; D011973:Receptors, LDL / Q000187:drug effects / Q000235:genetics*; D012694:Serine / Q000737:chemistry; D013912:Threonine / Q000737:chemistry
D006020:Glycopeptides; D009844:Oligosaccharides; D011973:Receptors, LDL; D013912:Threonine; D012694:Serine; D001216:Asparagine; D008985:Monensin; D000116:Acetylgalactosamine; D000117:Acetylglucosamine