Polyprenyl lipid synthesis in mammalian cells expressing human cis-prenyl transferase.
The level of cis-prenyl transferase activity has been implicated in controlling the level of biosynthesis of dolichol and dolichol intermediates. In this study, we isolated a cDNA encoding a human CPT (GenBank Accession No. ), which had substantial homology to other CPT isolated from human brain, bacteria, Arabidopsis, and Saccharomyces cerevisiae. Expression of this cDNA in two different insect cell lines confirmed the functionality of the protein in an in vitro assay. Western blot analysis revealed an expressed protein of approximately 38 kDa in HEK293 cells. Overexpression of the protein in HEK293 cells resulted in an increase in the level of total prenol in vivo. Furthermore, product characterization by thin layer chromatography (TLC) confirmed that the major product was a long-chain prenol with a chain length of 95 carbons. These results suggest a regulatory relationship between CPT activity and dolichol biosynthesis, and may implicate CPT in the levels of dolichol-oligosaccharide intermediate biosynthesis.
Jones
Jullian
J
Department of Chemical and Biomolecular Engineering, Baltimore, MD 21218, USA. beten@jhu.edu
Viswanathan
Karthik
K
Krag
Sharon S
SS
Betenbaugh
Michael J
MJ
D000818:Animals; D002460:Cell Line; D002855:Chromatography, Thin Layer; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004286:Dolichols / Q000096:biosynthesis / Q000378:metabolism; D045782:Hemiterpenes; D006801:Humans; D050356:Lipid Metabolism; D008055:Lipids / Q000096:biosynthesis* / Q000737:chemistry*; D008970:Molecular Weight; D000439:Pentanols / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018411:Spodoptera; D014166:Transferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D018076:DNA, Complementary; D004286:Dolichols; D045782:Hemiterpenes; D008055:Lipids; D000439:Pentanols; D011994:Recombinant Proteins; C009034:prenol; D014166:Transferases; C106821:cis-prenyl transferase