Crick D C, Scocca J R, Rush J S, Frank D W, Krag S S, Waechter C J
Department of Biochemistry, A. B. Chandler Medical Center, University of Kentucky College of Medicine, Lexington 40536.
J Biol Chem. 1994 Apr 8;269(14):10559-65.
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.
在小鼠B淋巴细胞（B细胞）对细菌脂多糖（LPS）的增殖反应过程中，Glc3-Man9GlcNAc2-P-P-Dol（寡糖-P-P-Dol）生物合成和蛋白质N-糖基化速率大幅增加。为了更深入了解多萜醇糖生物合成的调控机制，研究了磷酸多萜醇（Dol-P）和N-乙酰葡糖胺焦磷酸多萜醇（GlcNAc-P-P-Dol）生物合成特定步骤中的发育变化与寡糖-P-P-Dol生物合成诱导之间的可能关系。这些研究描述了长链顺式异戊二烯基转移酶（cis-IPTase）活性的显著诱导，该酶系统是从头合成Dol-P链延长阶段所必需的，这与LPS激活的B细胞中寡糖-P-P-Dol生物合成速率的显著增加相对应。在LPS处理的细胞中，cis-IPTase的细胞水平和比活性增加了15倍，而异戊烯基焦磷酸的亲和力相对未改变。当cis-IPTase活性被诱导时，Dol-P和寡糖-P-P-Dol的合成速率大幅增加，表明cis-IPTase活性水平与脂质中间体合成之间存在调控关系。观察到cis-IPTase和HMG-CoA还原酶活性具有明显不同的发育模式，并且当洛伐他汀强烈抑制甾醇生物合成时，在药物存在下Dol-P的合成速率略高。还观察到多萜醇激酶、Dol-P磷酸酶和UDP-GlcNAc:Dol-P N-乙酰葡糖胺磷酸基转移酶（L-G1PT）活性的细胞水平适度升高，但与cis-IPTase活性以及Dol-P、Gl-cNAc-P-P-Dol和寡糖-P-P-Dol合成速率的增加相比，这些变化相对较小。L-G1PT基因的表达是寡糖-P-P-Dol合成发育程序中的早期事件，但GlcNAc-P-P-Dol的形成显然不是限速步骤。总之，cis-IPTase活性和Dol-P生物合成速率的大幅增加似乎在B细胞对LPS的增殖反应过程中寡糖-P-P-Dol生物合成的诱导中起关键调控作用，并且在分裂的B细胞中Dol-P和胆固醇的生物合成途径是独立调控的。
