Biogenesis of the endoplasmic reticulum in activated B lymphocytes: Temporal relationships between the induction of protein N-glycosylation activity and the biosynthesis of membrane protein and phospholipid

An earlier report from this laboratory documented a substantial increase in the rates of dolichol-linked oligosaccharide intermediate synthesis and protein N-glycosylation in purified murine splenic B lymphocytes (B cells) activated by treatment with bacterial lipopolysaccharide (LPS). In this study the developmental patterns for the induction of lipid-mediated protein N-glycosylation, membrane protein, and phosphatidylcholine (PC) biosynthesis were compared during the proliferative response of B cells to LPS. By electron microscopy it could be seen that a distinct endoplasmic reticulum (ER) network began to develop by 24-48 h after exposure of the purified B cells to LPS. The rate of synthesis of membrane protein increased markedly during the first 10 h after activation, reaching a maximum at 30-40 h. The induction of protein N-glycosylation was delayed slightly relative to membrane protein synthesis, with glycoprotein synthesis increasing sharply approximately 20 h after activation. When phospholipid synthesis was monitored by measuring [CH₃-³H]choline incorporation into PC, the rate of labeling increased slowly during the first 35 h, but more substantially between 35 and 90 h. The incorporation of labeled choline into PC was drastically reduced by 5′-deoxy-5′-isobutylthio-3-deazaadenosine, an inhibitor of CDP-choline synthesis, indicating that the incorporation of radiolabeled choline is primarily a measurement of the rate of de novo synthesis of PC. In vitro assays revealed that while choline kinase activity was virtually unchanged, CDP-choline synthetase activity increased gradually throughout the activation period. Diacylglycerol cholinephosphotransferase activity, an ER-associated enzyme, was present at low levels between 0 and 35 h, but increased fivefold between 35 and 90 h. On the basis of the developmental patterns for the rates of protein N-glycosylation, membrane protein insertion, and PC biosynthesis determined by metabolic labeling experiments, we tentatively conclude that all of the ER-associated membrane proteins involved in these biosynthetic processes are not induced concurrently during the activation of B Cells by LPS.