Molecular cloning of Chinese hamster ceramide glucosyltransferase and its enhanced expression in peroxisome-defective mutant Z65 cells.
To clarify the metabolic bases of characteristic increases in the concentrations of glucosylceramide (CMH) and GM3 in peroxisome-defective mutant Chinese hamster ovary (CHO) cells (Z65), we measured the ceramide glucosyltransferase (CGT) and beta-glucosidase activities in Z65 and CHO-K1 cells, and found that the former enzyme was responsible for the accumulation of CMH in Z65 cells. Inhibition of CGT by D,L-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) caused a marked reduction in a incorporation of [3-14C]serine to CMH in both CHO-K1 and Z65 cells, but resulted in the accumulation of ceramide in Z65 cells in a concentration higher than that in CHO-K1 cells. Then, we cloned the cDNA encoding CGT from CHO-K1 cells, which exhibited sequence homology with the human gene product (98.7%). Northern blot analysis of CGT revealed increased expression of it in Z65 cells compared with that in CHO-K1 cells, which probably caused the simultaneous increase in GM3. With an immunohistochemical procedure, GM3 was found to be more strongly expressed in the cell membrane of Z65 cells than in CHO-K1 cells.
Saito
Makiko
M
Department of Pediatrics, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan. ssmiko@fa2.so-net.ne.jp
Fukushima
Yasushi
Y
Tatsumi
Ken
K
Bei
Lin
L
Fujiki
Yukio
Y
Iwamori
Masao
M
Igarashi
Takashi
T
Sakakihara
Yoichi
Y
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D016466:CHO Cells; D003001:Cloning, Molecular; D006224:Cricetinae; D003412:Cricetulus; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005679:G(M3) Ganglioside / Q000276:immunology / Q000378:metabolism; D005964:Glucosyltransferases / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D009025:Morpholines / Q000494:pharmacology; D009154:Mutation; D020675:Peroxisomes / Q000235:genetics* / Q000378:metabolism; D013107:Sphingolipids / Q000096:biosynthesis; D001617:beta-Glucosidase / Q000378:metabolism
D004791:Enzyme Inhibitors; D005679:G(M3) Ganglioside; D009025:Morpholines; D013107:Sphingolipids; C033110:RV 538; D005964:Glucosyltransferases; C023246:ceramide glucosyltransferase; D001617:beta-Glucosidase