Induction of dolichyl-saccharide intermediate biosynthesis corresponds to increased long chain cis-isoprenyltransferase activity during the mitogenic response in mouse B cells.
There are large increases in the rates of Glc3-Man9GlcNAc2-P-P-Dol (Oligo-P-P-Dol) biosynthesis and protein N-glycosylation during the proliferative response of murine B lymphocytes (B cells) to bacterial lipopolysaccharide (LPS). To learn more about the regulation of dolichyl-saccharide biosynthesis, the possible relationships between developmental changes in specific steps in dolichyl phosphate (Dol-P) and N-acetyl-glucosaminylpyrophosphoryldolichol (GlcNAc-P-P-Dol) biosynthesis and the induction of Oligo-P-P-Dol biosynthesis were investigated. These studies describe an impressive induction of long chain cis-isoprenyltransferase (cis-IPTase) activity, an enzyme system required for the chain elongation stage in de novo Dol-P synthesis, which corresponded to the striking increase in the rate of Oligo-P-P-Dol biosynthesis in LPS-activated B cells. The cellular level and specific activity of cis-IPTase increase 15-fold in LPS-treated cells with relatively unaltered affinity for isopentenyl pyrophosphate. The rates of Dol-P and Oligo-P-P-Dol synthesis increased substantially when cis-IPTase activity was induced, suggesting a regulatory relationship between the level of cis-IPTase activity and lipid intermediate synthesis. Distinctly different developmental patterns were observed for cis-IPTase and HMG-CoA reductase activity, and when sterol biosynthesis was drastically inhibited by lovastatin, the rate of synthesis of Dol-P was slightly higher in the presence of the drug. Modest elevations in the cellular levels of dolichol kinase, Dol-P phosphatase, and UDP-GlcNAc:Dol-P N-acetylglucosaminylphosphoryltransferase (L-G1PT) activities were also observed, but these changes were relatively small compared with the increases in cis-IPTase activity and the rates of Dol-P, Gl-cNAc-P-P-Dol, and Oligo-P-P-Dol synthesis. The expression of the L-G1PT gene is an early event in the developmental program for Oligo-P-P-Dol synthesis, but GlcNAc-P-P-Dol formation is apparently not rate-limiting. In summary, large increases in cis-IPTase activity and the rate of Dol-P biosynthesis appear to play a key regulatory role in the induction of Oligo-P-P-Dol biosynthesis during the proliferative response of B cells to LPS, and the biosynthetic pathways for Dol-P and cholesterol are regulated independently in dividing B cells.
Crick
D C
DC
Department of Biochemistry, A. B. Chandler Medical Center, University of Kentucky College of Medicine, Lexington 40536.
Scocca
J R
JR
Rush
J S
JS
Frank
D W
DW
Krag
S S
SS
Waechter
C J
CJ
D019883:Alkyl and Aryl Transferases; D000818:Animals; D001402:B-Lymphocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D001483:Base Sequence; D002240:Carbohydrate Sequence; D002454:Cell Differentiation / Q000235:genetics; D002478:Cells, Cultured; D017931:DNA Primers; D004790:Enzyme Induction; D005260:Female; D006903:Hydroxymethylglutaryl CoA Reductases / Q000378:metabolism; D008070:Lipopolysaccharides / Q000494:pharmacology; D008213:Lymphocyte Activation; D051379:Mice; D008811:Mice, Inbred DBA; D008934:Mitogens / Q000494:pharmacology*; D008969:Molecular Sequence Data; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism; D011105:Polyisoprenyl Phosphate Sugars / Q000378:metabolism*; D014166:Transferases / Q000096:biosynthesis / Q000378:metabolism*
D017931:DNA Primers; C066723:Glc(3)Man(9)(GlcNAc)(2)-diphosphate-dolichol; D008070:Lipopolysaccharides; D008934:Mitogens; D011105:Polyisoprenyl Phosphate Sugars; D006903:Hydroxymethylglutaryl CoA Reductases; D014166:Transferases; D019883:Alkyl and Aryl Transferases; C084921:cis-isoprenyltransferase; D017853:Phosphotransferases (Alcohol Group Acceptor); C020087:dolichol kinase; D010744:Phosphoric Monoester Hydrolases; C020088:dolichyl-phosphatase