| Project/Area Number |
63044114
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| Research Category |
Grant-in-Aid for international Scientific Research
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| Allocation Type | Single-year Grants |
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| Section | Joint Research |
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| Research Institution | Oita Medical School, Faculty of Medicine |
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Principal Investigator |
KUWANO Michihiko Oita Medical School, Oita, Japan, 医学部, 教授 (80037431)
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| Co-Investigator(Kenkyū-buntansha) |
SCHLESSINGER David Washington University School of Medicine, St. Louis, USA, 医学部, 教授
KRIEGER Monty Massachusetts Institute of Technology, Boston, USA, 準教授
MERKLE Roberta University of Georgia Carbohydrate Research Center, Athens, USA, 炭水化物研究所, 主任研究員
CUMMINGS Richard University of Georgia, Athens, USA, 準教授
ONO Mayumi Oita Medical School, Oita, Japan, 医学部, 助手 (80128347)
KOHNO Kimitoshi Oita Medical School, Oita, Japan, 医学部, 助教授 (00153479)
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| Project Period (FY) |
1989 – 1990
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| Project Status |
Completed (Fiscal Year 1990)
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| Budget Amount *help |
¥4,900,000 (Direct Cost: ¥4,900,000)
Fiscal Year 1990: ¥1,500,000 (Direct Cost: ¥1,500,000)
Fiscal Year 1989: ¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 1988: ¥2,000,000 (Direct Cost: ¥2,000,000)
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| Keywords | low density lipoprotein (LDL) / LDL-receptor / familiar hypercholesterolemia / receptor mutation / somatic cell mutants / Golgi apparatus / O-linked sugar chains / cholesterol metabolism |
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| Research Abstract |
We have isolated, by independent selection, two different somatic cell mutants that show altered expression of LDL receptor. A compactin-resistant mutant, MF-2, was isolated from the Chinese hamster V79 cell line and a monensin-resistant (Mon^r-31) mutant from CHO. These two mutants showed a decreased response to LDL as well as aberrant cholesterol metabolism. The mature LDL receptors produced in these mutants were seemingly 5000 Mr smaller than that of the parental cells, and the mutation specifically alters O-linked sugar chains, not N-linked sugar chains. These two mutants fall into the same complementation int group. Further detailed characterization of the Ser/Thr-linked oligosaccharides in the LDL receptor of the int mutant was done in collaboration with R. Merkle and R. Cummings at the University of Georgia. The Ser/Thr-linked oligosaccharides in the receptors from both the parental and Mon^r-31 cells are mono- and di-sialylated species having the common core structure Gal-GalNAc. The receptor from Mon^r-31 cells, however, contains about one-third fewer Ser/Thr-linked oligosaccharides than the receptor from parental CHO cells. The mature form of the human mutant receptor lacking the clustered Ser/Thr-linked oligosaccharides in CHO has an apparent M_r of 120000 while that in Mon^r-31 has a M_r of 110000. The apparent relative molecular mass of the sialidase-treated human mutant receptor in Mor^r-31 cells is equivalent to that of the receptor in CHO cells that is treated with both sialidase and O-glycanase. These results demonstrate that the LDL receptor produced by the Mon^r-31 cells contains Ser/Thr-linked oligosaccharides in the clustered domain, but is missing the Ser/Thr-linked oligosaccharides in the unclustered LDL-binding domains of the receptor. The O-glycosylation at the LDL-binding domain may be critical for LDL receptor function.
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