Phosphocholine Transferase Research Articles

Legionella effector AnkX displaces the switch II region for Rab1b phosphocholination.

The causative agent of Legionnaires disease, Legionella pneumophila, translocates the phosphocholine transferase AnkX during infection and thereby posttranslationally modifies the small guanosine triphosphatase (GTPase) Rab1 with a phosphocholine moiety at S76 using cytidine diphosphate (CDP)-choline as a cosubstrate. The molecular basis for Rab1 binding and enzymatic modification have remained elusive because of lack of structural information of the low-affinity complex with AnkX. We combined thiol-reactive CDP-choline derivatives with recombinantly introduced cysteines in the AnkX active site to covalently capture the heterocomplex. The resulting crystal structure revealed that AnkX induces displacement of important regulatory elements of Rab1 by placing a β sheet into a conserved hydrophobic pocket, thereby permitting phosphocholine transfer to the active and inactive states of the GTPase. Together, the combination of chemical biology and structural analysis reveals the enzymatic mechanism of AnkX and the family of filamentation induced by cyclic adenosine monophosphate (FIC) proteins.

Exploring the Substrate Scope of the Bacterial Phosphocholine Transferase AnkX for Versatile Protein Functionalization.

Site-specific protein functionalization has become an indispensable tool in modern life sciences. Here, tag-based enzymatic protein functionalization techniques are among the most versatilely applicable approaches. However, many chemo-enzymatic functionalization strategies suffer from low substrate scopes of the enzymes utilized for functional labeling probes. We report on the wide substrate scope of the bacterial enzyme AnkX towards derivatized CDP-choline analogues and demonstrate that AnkX-catalyzed phosphocholination can be used for site-specific one- and two-step protein labeling with a broad array of different functionalities, displaying fast second-order transfer rates of 5×102 to 1.8×104 m-1 s-1 . Furthermore, we also present a strategy for the site-specific dual labeling of proteins of interest, based on the exploitation of AnkX and the delabeling function of the enzyme Lem3. Our results contribute to the wide field of protein functionalization, offering an attractive chemo-enzymatic tag-based modification strategy for in vitro labeling.

Legionella Effector AnkX Disrupts Host Cell Endocytic Recycling in a Phosphocholination-Dependent Manner.

The facultative intracellular bacterium Legionella pneumophila proliferates within amoebae and human alveolar macrophages, and it is the causative agent of Legionnaires' disease, a life-threatening pneumonia. Within host cells, L. pneumophila establishes a replicative haven by delivering numerous effector proteins into the host cytosol, many of which target membrane trafficking by manipulating the function of Rab GTPases. The Legionella effector AnkX is a phosphocholine transferase that covalently modifies host Rab1 and Rab35. However, a detailed understanding of the biological consequence of Rab GTPase phosphocholination remains elusive. Here, we broaden the understanding of AnkX function by presenting three lines of evidence that it interferes with host endocytic recycling. First, using immunogold transmission electron microscopy, we determined that GFP-tagged AnkX ectopically produced in mammalian cells localizes at the plasma membrane and tubular membrane compartments, sites consistent with targeting the endocytic recycling pathway. Furthermore, the C-terminal region of AnkX was responsible for association with the plasma membrane, and we determined that this region was also able to bind the phosphoinositide lipids PI(3)P and PI(4)P in vitro. Second, we observed that mCherry-AnkX co-localized with Rab35, a regulator of recycling endocytosis and with major histocompatibility class I protein (MHC-I), a key immunoregulatory protein whose recycling from and back to the plasma membrane is Rab35-dependent. Third, we report that during infection of macrophages, AnkX is responsible for the disruption of endocytic recycling of transferrin, and AnkX's phosphocholination activity is critical for this function. These results support the hypothesis that AnkX targets endocytic recycling during host cell infection. Finally, we have demonstrated that the phosphocholination activity of AnkX is also critical for inhibiting fusion of the Legionella-containing vacuole (LCV) with lysosomes.

Neurokinin 3 receptor and phosphocholine transferase: missing factors for pathogenesis of C-reactive protein in preeclampsia.

C-reactive protein (CRP), an innate immune mediator, is elevated in the circulation before symptoms in patients with preeclampsia, a severe hypertensive pregnancy disorder with high mortality and morbidity. However, the specific sources underlying increased CRP and the role of elevated CRP in preeclampsia are undefined. Here, we report that circulating CRP levels are significantly increased in a large cohort of normotensive pregnant individuals when compared with nulligravid women and is further increased in patients with preeclampsia. These findings led us to discover further that placental syncytiotrophoblasts are previously unrecognized cellular sources of CRP and underlie elevated CRP in normotensive pregnant women and the additional increase in patients with preeclampsia. Next, we demonstrated that injection of CRP induces preeclampsia features, including hypertension (157 mm Hg CRP treated versus 119 mm Hg control), proteinuria (35.0 mg/μg CRP treated versus 14.1 mg/μg control), kidney, and placental damage and increased levels of sFlt-1 in pregnant mice but not in nonpregnant mice. Our study implicates that phosphocholine transferase, a placental-specific enzyme post-translationally modifying neurokinin B, is essential for the pathogenic role of CRP in preeclampsia through activation of the neurokinin 3 receptor. Overall, our studies have provided significant new insight on the pathogenic role of CRP in preeclampsia and highlighted innovative therapeutic strategies.

Abstract 207: C-Reactive Protein Signaling via Neurokinin B Receptor Contributes to the Pathophysiology of Preeclampsia

Introduction: C-Reactive Protein (CRP) is an important immunomodulatory molecule. Early studies have indicated that CRP is elevated in preeclamptic patient sera, but its function remains unclear. CRP is known to bind with phosphocholine (PC) of bacteria membrane to kill bacteria by triggering innate immune response. Phosphocholination is a post-translational modification mediated by PC transferase, an enzyme reported expressed only in two tissues, including placenta and testis. Recent studies identified that Neurokinin B (NKB), a known, placenta-enriched pathogenic molecule for preeclampsia (PE), is phosphocholinated. This modification increases its stability, preferentially activates NK3 receptor (NK3R) and promotes PE features. Given CRP can bind to PC on bacterial membranes, the role that phosphocholination plays is a mystery. In light of NKB, being phosphocholinated in the endoplasmic reticulum and signals via the NK3R in the pathogenesis of PE, we feel that elevated CRP may be associated with NKB and contributes to the pathophysiology of PE via NK3R activation. Results: ELISA results show that CRP is significantly elevated in PE vs. control sera_65 ug/mL vs. 10 ug/ml; p<0.0001. Western blot analysis reveals that CRP, NKB, and NK3R are significantly increased in PE vs. control placental tissue. Immunohistochemistry studies indicate that all of these three molecules are abundant in syncytiotrophoblast cells. Co-immunoprecipitation demonstrates an interaction between CRP and NKB. In vitro , we provide the first evidence that CRP decreased the invasion of trophoblast cells from 0.7 ± 0.06 to 0.06 ± 0.001 invasion index; p<0.005, while treatment with SB222200 ameliorates shallow invasion from 0.28 ± 0.09 to 0.55 ± 0.07 invasion index; p<0.05. Significantly, infusion of CRP into pregnant mice induces hypertension (169.152 ± 14.7 mmHg vs. 141.916 ± 6.93 mmHg; p<0.05) and proteinuria (28.78 ± 13.56 mg albumin/μg Cr vs. 13.87 ± 3.277 mg albumin/μg Cr; p<0.05). Conclusion: Our findings demonstrate elevated CRP contributes to PE and NKB/NK3R is a new mechanism underlying CRP-mediated shallow invasion and disease development. These studies suggest novel pathogenic biomarkers and innovative therapies for PE.

Modulation of Rab GTPase function by a protein phosphocholine transferase

The intracellular pathogen Legionella pneumophila modulates the activity of host GTPases to direct the transport and assembly of the membrane-bound compartment in which it resides1–6. In vitro studies have suggested that the Legionella protein DrrA post-translationally modifies the GTPase Rab1 by a process called AMPylation7. Here, mass spectrometry was used to investigate post-translational modifications to Rab1 that occur during infection of host cells by Legionella. Consistent with in vitro studies, DrrA-mediated AMPylation of a conserved tyrosine residue in the switch II region of Rab1 was detected during infection. In addition, a modification to an adjacent serine residue in Rab1 was discovered, which was independent of DrrA. The Legionella effector protein AnkX was required for this modification. Biochemical studies determined that AnkX directly mediates the covalent attachment of a phosphocholine moiety to Rab1. This phosphocholine transferase activity used CDP-choline as a substrate and required a conserved histidine residue located in the FIC domain of the AnkX protein. During infection, AnkX modified both Rab1 and Rab35, which explains how this protein modulates membrane transport through both the endocytic and exocytic pathways of the host cell. Thus, phosphocholination of Rab GTPases represents a mechanism by which bacterial FIC domain-containing proteins can alter host cell functions.

Covalent Coercion by Legionella pneumophila

Adenylylation of Rab proteins appears to be an intriguing mechanism that Legionella pneumophila uses to modulate their activity during infection. Now the reverse reaction (deadenylylation) (Neunuebel etal., 2011; Tan and Luo, 2011) and a new posttranslational modification (phosphocholination) of Rab1 (Mukherjee etal., 2011) have been reported.

Sphingolipid Metabolism Inhibitors and Cell Function

It has been widely shown that ceramide, ceramide phosphate, sphingosine and sphingosine-1-phosphate are molecules involved in cell proliferation, differentiation and apoptosis. The regulation of the enzymes that control their metabolism has an important role for cellular fate. Many studies in the field have shed light on sphingolipid metabolism inhibition and on its role in cell processes such as gene expression, DNA duplication and RNA transcription. The aim of this review is to collect, in a systematic way, the recent advances in the field of sphingolipid metabolism inhibitors, with special emphasis on their effects on cell function. The name sphingolipid was born at the end of the XIX century on the basis of the molecule structure similar to the riddle of the sphinx. These molecules are complex lipids, localized in animal and plant membranes and in some forms of lower life, which contain sphingoid bases and have a very complex metabolism. The sphingolipid biosynthetic pathway initiates with the condensation of the serine with palmitoyl-CoA to form 3- ketodihydrosphingosine or 3-ketosphinganine, a serine pal- mitoyl transferase (SPT) catalyzed reaction. 3-ketodihy- drosphingosine is reduced to dehydrosphingosine or sphin- ganine by a NADPH dependent 3-ketosphinganine reduc- tase. An acylation in the presence of acyl-CoA produces di- hydroceramide by ceramide synthase (CerS). Dihydrocera- mide is then converted to ceramide through the introduction of the double bond trans-4,-5 by dihydroceramide desatu- rase. The ceramide can be transformed to sphingomyelin (SM) by two different ways: the first one involves the reaction with CDP-choline catalyzed by ceramide choline phos- photransferase and the second, described subsequently, con- sists in phosphocholine transfer from lecithin to ceramide by phosphatidylcholine: ceramide phosphocholine transferase or sphingomyelin synthase (SM-synthase). The synthesized SM can be used as source of phosphocholine to form phosphati- dylcholine in the presence of diacylglycerol by reverse- sphingomyelin-synthase (RSM-synthase) freeing ceramide, or can be degraded to ceramide and phosphocholine by acid or neutral sphingomyelinase (aSMase or N-SMase). The new-produced ceramide can be deacylated by acid or neutral ceramidase (AC or NC) to form sphingosine, which can be phosphorylated by a sphingosine kinase 1 (SphK1) or by sphingosine kinase 2 (SphK2) to sphingosine phosphate, can be phosphorylated by ceramide kinase (CerK) or can be

Effect of tricyclodecan-9-yl potassium xanthate (D609) on phospholipid metabolism and cell death during oxygen–glucose deprivation in PC12 cells

Alterations in lipid metabolism play an integral role in neuronal death in cerebral ischemia. Here we used an in vitro model, oxygen–glucose deprivation (OGD) of rat pheochromocytoma (PC12) cells, and analyzed changes in phosphatidylcholine (PC) and sphingomyelin (SM) metabolism. OGD (4–8 h) of PC12 cells triggered a dramatic reduction in PC and SM levels, and a significant increase in ceramide. OGD also caused increases in phosphatidylcholine-phospholipase C (PC-PLC) and phospholipase D (PLD) activities and PLD2 protein expression, and reduction in cytidine triphosphate:phosphocholine cytidylyltransferase-α (CCTα, the rate-limiting enzyme in PC synthesis) protein expression and activity. Phospholipase A 2 activity and expression were unaltered during OGD. Increased neutral sphingomyelinase activity during OGD could account for SM loss and increased ceramide. Surprisingly, treatment with PC-PLC inhibitor tricyclodecan-9-yl potassium xanthate (D609) aggravated cell death in PC12 cells during OGD. D609 was cytotoxic only during OGD; cell death could be prevented by inclusion of sera, glucose or oxygen. During OGD, D609 caused further loss of PC and SM, depletion of 1,2-diacylglycerol (DAG), increase in ceramide and free fatty acids (FFA), cytochrome c release from mitochondria, increases in intracellular Ca 2+ ([Ca 2+] i), poly-ADP ribose polymerase (PARP) cleavage and phosphatidylserine externalization, indicative of apoptotic cell death. Exogenous PC during OGD in PC12 cells with D609 attenuated PC, SM loss, restored DAG, attenuated ceramide levels, decreased cytochrome c release, PARP cleavage, annexin V binding, attenuated the increase in [Ca 2+] i, FFA release, and significantly increased cell viability. Exogenous PC may have elicited these effects by restoring membrane PC levels. A tentative scheme depicting the mechanism of action of D609 (inhibiting PC-PLC, SM synthase, PC synthesis at the CDP-choline-1,2-diacylglycerol phosphocholine transferase (CPT) step and causing mitochondrial dysfunction) has been proposed based on our observations and literature.

Sphingomyelin metabolites inhibit sphingomyelin synthase and CTP:phosphocholine cytidylyltransferase.

Tissue injury in inflammation involves the release of several cytokines that activate sphingomyelinases and generate ceramide. In the lung, the impaired metabolism of surfactant phosphatidylcholine (PC) accompanies this acute and chronic injury. These effects are long-lived and extend beyond the time frame over which tumor necrosis factor (TNF)-alpha and interleukin-1beta are elevated. In this paper, we demonstrate that in H441 lung cells these two processes, cytokine-induced metabolism of sphingomyelin and the inhibition of PC metabolism, are directly interrelated. First, metabolites of sphingomyelin hydrolysis themselves inhibit key enzymes necessary for restoring homeostasis between sphingomyelin and its metabolites. Ceramide stimulates sphingomyelinases as effectively as TNF-alpha, thereby amplifying the sphingomyelinase activation, and TNF-alpha, ceramide, and sphingosine all inhibit PC:ceramide phosphocholine transferase (sphingomyelin synthase), the enzyme that restores homeostasis between sphingomyelin and ceramide pools. Second, ceramide inhibits PC synthesis, probably because of its effects on CTP:phosphocholine cytidylyltransferase, the rate-limiting enzymatic step in de novo PC synthesis. The data presented here suggest that TNF-alpha may be an inhibitor of phospholipid metabolism in inflammatory tissue injury. These actions may be amplified because of the ability of metabolites of sphingomyelin to inhibit the pathways that should restore the normal ceramide-sphingomyelin homeostasis.

Phosphatidylcholine metabolism in nuclei of phorbol ester-activated LA-N-1 neuroblastoma cells.

The agonist stimulation of a variety of cells results in the induction of specific lipid metabolism in nuclear membranes, supporting the hypothesis of an important role of the lipids in nuclear signal transduction. While the existence of a phosphatidylinositol cycle has been reported in cellular nuclei, little attention has been given to the metabolism of phosphatidylcholine in nuclear signaling. In the present study the metabolism of phosphatidylcholine in the nuclei of neuroblastoma cells LA-N-1 was investigated. The incubation of LA-N-1 nuclei with radioactive choline, phosphocholine or CDP-choline led to the production of labelled phosphatidylcholine. The incorporation of choline and phosphocholine but not CDP-choline was enhanced in nuclei of TPA treated cells. Moreover the presence of choline kinase, phosphocholine cytidylyltransferase and phosphocholine transferase activities were detected in the nuclei and the TPA treatment of the cells stimulated the activity of the phosphocholine cytidylyltransferase. When cells prelabelled with [3H]palmitic acid were stimulated with TPA in the presence of ethanol, an increase of labelled diacylglycerol and phosphatidylethanol in the nuclei was observed. Similarly, an increase of labelled diacylglycerol and phosphatidic acid but not of phosphatidylethanol occurred in [3H]palmitic acid prelabelled nuclei stimulated with TPA in the presence of ethanol. However the production of phosphatidylethanol was observed when the nuclei were treated with TPA in the presence of ATP and GTPgammaS. The stimulation of [3H]choline prelabelled nuclei with TPA also generated the release of free choline and phosphocholine. The results indicate the presence of PLD and probably PLC activities in LA-N-1 nuclei and the involvement of phosphatidylcholine in the production of nuclear lipid second messengers upon TPA stimulation of LA-N-1 cells. The correlation of the disappearance of phosphatidylcholine, the production of diacylglycerol and phosphatidic acid with the stimulation of phosphatidylcholine synthesis in nuclei of TPA treated LA-N-1 suggests the existence of a phosphatidylcholine cycle in these nuclei.

Sphingomyelin synthase in rat liver nuclear membrane and chromatin

The presence of phospholipids in chromatin has been demonstrated, as well as the difference in composition and turnover compared to those present in the nuclear membrane. Recently, some enzymes were also evidenced in chromatin: the base exchange protein complex and neutral sphingomyelinase. The latter has a particular relevance, since sphingomyelin is one of the phospholipids more represented in chromatin. We therefore decided to study the synthesis of sphingomyelin in chromatin and in nuclear membrane isolated from liver nuclei. The evaluation of the enzyme was made (i) using [ 3H]phosphatidylcholine as donor of radioactive phosphorylcholine and (ii) by identifying the product isolated by thin layer chromatography. In both fractions the enzyme phosphatidylcholine:ceramide phosphocholine transferase or sphingomyelin synthase was present, although with higher activity in nuclear membrane. The enzyme present in the chromatin differs in pH optimum and K m, showing a higher affinity for the substrates than that of nuclear membrane. The results presented show that sphingomyelin synthase is present not only in the cytoplasm at the level of the Golgi apparatus, but also in the nuclei, at the level of either the nuclear membrane or the chromatin.

Dietary Pectin Lowers Sphingomyelin Concentration in VLDL and Raises Hepatic Sphingomyelinase Activity in Rats

There is evidence that cholesterol and sphingomyelin metabolism are interrelated, and thus the hypothesis tested was that dietary pectin, because it can alter hepatic cholesterol metabolism, would also alter hepatic sphingomyelin metabolism. For that purpose, 4-wk-old female Wistar rats were fed a diet without or with pectin (20 g/100 g) up to 21 d. In accordance with previous work, pectin consumption caused a significant (P < 0.001) reduction in hepatic (65%), whole plasma (37%), and VLDL (80%) cholesterol levels. Pectin also significantly reduced VLDL sphingomyelin concentrations (57%), but raised the amount of sphingomyelin in the high density lipoproteins (HDL)-2 fractions (58%), so that the level of sphingomyelin in whole plasma remained unaffected. Pectin did not affect the sphingomyelin concentration in the liver. Pectin consumption did not affect the hepatic sphingomyelin synthesizing enzymes, serine palmitoyltransferase, phosphatidylcholine:ceramide phosphocholine transferase, or phosphatidylethanolamine:ceramide phosphoethanolamine transferase. In contrast, dietary pectin activated both lysosomal (28%) and plasma membrane (26%) sphingomyelinase and thus may have enhanced sphingomyelin degradation. An attempt was made to describe the effects of dietary pectin on sphingomyelin metabolism in terms of altered fluxes through liver and plasma, with whole liver and whole plasma concentrations of sphingomyelin remaining unaffected.

Preparation of Golgi subfractions with free-solution isotachophoresis: analysis of sphingomyelin synthesis in Golgi subfractions from rat liver.

A new displacement electrophoresis technique, termed free-solution isotachophoresis (FS-ITP) was used for the analysis of sphingolipid metabolism in Golgi subfractions. The discontinuous electrolyte system enables tissue-derived membrane vesicles to be separated and purified due to their polarity patterns in a mobility gradient. In this study total Golgi apparatus obtained from rat liver by discontinuous density gradient centrifugation was subfractionated by preparative FS-ITP, yielding enzymatically active cis-, medial-, and trans-Golgi subfractions. These membrane vesicles were assayed by the following established enzyme marker activities: NADH cytochrome c reductase (cis-Golgi), NADP phosphatase (medial-Golgi), and thiamine pyrophosphatase (trans-Golgi). The activity of phosphatidylcholine:ceramide phosphocholine transferase, a sphingomyelin synthesizing enzyme, is attributed to the cis- and medial-Golgi-derived subfractions. Analysis of Golgi lipids revealed a decline in membranous ceramide along the cis- to trans-Golgi polarity axis. Furthermore, significant amounts of newly synthesized sphingomyelin and diacylglycerol are transferred from the medial/cis- to the trans-Golgi compartment. The FS-ITP system is well suited for micropreparative experimental applications, as demonstrated by studies on phosphatidylcholine:ceramide phosphocholine transferase activity in Golgi membrane vesicles of rat liver obtained by FS-ITP.

Inhibition of PAF synthesis by stimulated human polymorphonuclear leucocytes with cloricromene, an inhibitor of phospholipase A2 activation.

1. A phospholipase A2 (PLA2) represents the key enzyme in the remodelling pathway of platelet-activating factor (PAF) synthesis in human polymorphonuclear (PMN) leucocytes. 2. PLA2 activation is also the rate-limiting step for the release of the arachidonic acid utilized for the synthesis of leukotrienes in stimulated leucocytes; however, it is unknown whether the PLA2s involved in the two biosynthetic pathways are identical. 3. Cloricromene (8-monochloro-3-beta-diethylaminoethyl-4-methyl-7-ethoxy- carbonylmethoxy coumarin) is an antithrombotic coumarin derivative which inhibits platelet and leucocyte function and suppresses arachidonic acid liberation by interfering with PLA2 activation. 4. The aim of the present study was to assess whether chloricromene inhibits PAF synthesis by stimulated human polymorphonuclear leucocytes (PMNs). 5. Cloricromene (50-500 microM) inhibited in a concentration-dependent manner the release of PAF, as measured by h.p.l.c. bioassay, from A23187-stimulated PMNs. Significant inhibition (45%) of PAF-release was obtained with 50 microM cloricromene and the IC50 was 85 microM. Mepacrine (500 microM), a non-specific PLA2 inhibitor, strikingly reduced PAF release. 6. The incorporation of [3H]-acetate into [3H]-PAF induced by serum-treated zymosan in human PMNs was also inhibited concentration-dependently by cloricromene, with an IC50 of 105 microM. Mepacrine also suppressed [3H]-acetate incorporation into [3H]-PAF. 7. Cloricromene did not affect the activities of the enzymes involved in PAF-synthesis acetyltransferase or phosphocholine transferase. 8. Our data demonstrate that cloricromene, an inhibitor of PLA2-activation in human leucocytes, reduces the synthesis of PAF by stimulated PMNs. This finding has a twofold implication: the PLA2s (or the mechanisms that regulate their activation) involved in PAF synthesis and arachidonate release in human leucocytes are either identical or else indistinguishable by their sensitivity to cloricromene; the inhibition of PAF release by activated leucocytes may contribute to the antithrombotic and anti-ischaemic activities exerted by cloricromene.

Relationship between fatty acid accretion, membrane composition, and biologic functions

Dietary fat affects metabolic pathways for phospholipid biosynthesis in tissues in a coordinated fashion. This may be important to aspects of development that concern phosphatidylcholine metabolism or regulatory processes that depend on signals from a changing milieu in the microenvironment of the membrane. Dietary fat influences the phosphatidylethanolamine (PE) composition in many membranes of the brain and retina and may by altered by small changes in the content of 20:4(6) and 22:6(3). Membrane PE fatty acids that contain one, four, or six double bonds and the ratio of 22:5(6) to 22:6(3) in PE that contains four to six double bonds are also affected. An increase in the omega 6 fatty acid content of membranes is associated with increased PE methyltransferase activity and decreased phosphocholine transferase activity, thus indicating a mechanism by which change in an exogenous factor (e.g., dietary fat intake) may alter neural phospholipid biosynthesis. Small changes in the composition of dietary fat intake change the composition of brain membranes during development. It is provocative to ponder whether diet could be used to induce formation of membrane structures that are more resistant to specific insults that cause degeneration of brain structural material, to ensure optimal functional compositions, or to reverse degenerative changes that occur in neural membrane structure and function.

Conversion of diacylglycerol to phosphatidylcholine on the basolateral surface of epithelial (Madin-Darby canine kidney) cells. Evidence for the reverse action of a sphingomyelin synthase.

When N-6[7-nitro-2,1,3-benzoxadiazol-4-yl]aminohexanoyl-phosphatidic acid (C6-NBD-PA) is inserted into the plasma membrane of fibroblasts, it is metabolized by the cells to C6-NBD-diacylglycerol (DG), -triacylglycerol, -phosphatidylcholine (PC), and -phosphatidylethanolamine (PE) (Pagano, R. E., Longmuir, K. J., and Martin, O. C. (1983) J. Biol. Chem. 258, 2034-2040). In Madin-Darby canine kidney (MDCK) cells incubated at 10 degrees C with C6-NBD-PA, up to 70% of the newly synthesized C6-NBD-PC but no C6-NBD-PE could be depleted from the basolateral cell surface by the addition of bovine serum albumin to the medium. Preincubation of the cells with [3H]choline for 2 h at 37 degrees C prior to C6-NBD-PA addition at 10 degrees C labeled non-depletable C6-NBD-PC with a specific activity of > 10 times that of the depletable C6-NBD-PC on the basolateral cell surface, indicating that the latter had not been synthesized by the CDP-choline pathway. C6-NBD-DG could substitute for C6-NBD-PA as substrate for both intracellular and surface C6-NBD-PC synthesis. In addition, C6-NBD-PC synthesis on the cell surface was independent of the location of the C6-NBD-chain on the 1- or 2-position, indicating that the reaction occurred by transfer of phosphorylcholine. Using C6-NBD-ceramide, C6-NBD-sphingomyelin (SM) synthesis also was discovered on the basolateral but not on the apical cell surface. The conversion of PC plus ceramide to DG and SM on the basolateral MDCK cell surface suggests that the synthesis of C6-NBD-PC on this surface occurred via the reverse reaction of a SM synthase. Indeed, the surface C6-NBD-PC synthesis was reduced to 40-50% by addition of C6-NBD-ceramide or hydrolysis of cell surface SM by exogenous neutral sphingomyelinase. Since DG activates protein kinase C and ceramide indirectly inhibits this kinase but activates other kinase(s) and phosphatase(s), the phosphocholine transferase at the cell surface may have a regulatory role in signal transduction.

Cholecystokinin inhibits phosphatidylcholine synthesis via a Ca(2+)-calmodulin-dependent pathway in isolated rat pancreatic acini. A possible mechanism for diacylglycerol accumulation.

The effects of cholecystokinin (CCK) and other pancreatic secretagogues on phosphatidylcholine (PC) synthesis were studied in isolated rat pancreatic acini. When acini were incubated with [3H]choline in the presence of 1 nM CCK-octapeptide (CCK8) for 60 min, the incorporations of [3H]choline into both water-soluble choline metabolites and PC in acini were reduced by CCK8 to 74 and 41% of control, respectively. Pulse-chase study revealed that CCK8 reduced both the disappearance of phosphocholine and the synthesis of PC. Other Ca(2+)-mobilizing secretagogues such as carbamylcholine, bombesin, and Ca2+ ionophore A23187 also reduced PC synthesis to the same extent as did CCK8. When combined with 1 nM CCK8, A23187 or carbamylcholine did not further inhibit PC synthesis. Furthermore, W-7 or W-5, a calmodulin antagonist, reversed the inhibition by CCK8 of PC synthesis, suggesting that a Ca(2+)-calmodulin-dependent pathway may be involved in CCK-induced inhibition of PC synthesis in acini. By contrast, neither cAMP-dependent secretagogues such as secretin and dibutyryl cAMP nor a phorbol ester had any effect on PC synthesis in acini. Staurosporine or H-7, a protein kinase C inhibitor, did not affect the inhibition by CCK of PC synthesis. The analysis of enzyme activity involved in PC synthesis via CDP-choline pathway showed that CCK treatment of acini reduced CTP:phosphocholine cytidylyltransferase activity in both cytosolic and particulate fraction, a finding consistent with the delayed disappearance of phosphocholine induced by CCK in pulse-chase study. By contrast, CCK treatment of acini did not alter the activities of choline kinase and phosphocholine transferase in acini. The extent of inhibition by CCK of cytidylyltransferase activity became much larger when subcellular fractions of acini were prepared in the presence of phosphatase inhibitors. In addition, W-7 reversed the inhibitory effect of CCK treatment on cytidylyltransferase activity in acini. When acini were labeled with [3H]myristic acid and chased, CCK8 (1 nM) reduced the synthesis of [3H]myristic acid-labeled PC to 27% of control after a 60-min chase period. This inhibition of PC synthesis induced by CCK was accompanied by a delayed disappearance of [3H]diacylglycerol, the radioactivity of which was 225% of control at 60 min. These results indicate that CCK inhibits PC synthesis by inducing both the reduction of choline uptake into acini and the inhibition of CTP:phosphocholine cytidylyltransferase activity. Furthermore, the results suggest the possibility that the activation of Ca(2+)-calmodulin-dependent kinase in response to CCK may phosphorylate cytidylyltransferase thereby decreasing this enzyme activity in pancreatic acinar cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Changes in Phospholipid and Cholesterol Concentrations of the Rat Microvillus Membrane During Maturation

Summary: We studied the cholesterol and phospholipid content and the cholesterol-to-phospholipid molar ratio of the small intestinal microvillus membrane in fetal, weanling, and adult rats. We also investigated the effect of glucocorticoid administration on these lipids and the ontogeny of 1,2-diglycerol-CDP choline phosphocholine transferase (PCT). Cholesterol and phospholipid concentrations decreased with maturation, phospholipids declining more than cholesterol. Thus, the cholesterol-to-phospholipid molar ratio rose with maturation. A similar decline was seen with PCT activity. Glucocorticoid treatment had no effect on cholesterol, phospholipids, or PCT. However, the maturational increase in the cholesterol-to-phospholipid molar ratio was blunted by glucocorticoid administration in the weanling and adult rats.

Changes in Phospholipid and Cholesterol Concentrations of the Rat Microvillus Membrane During Maturation

We studied the cholesterol and phospholipid content and the cholesterol-to-phospholipid molar ratio of the small intestinal microvillus membrane in fetal, weanling, and adult rats. We also investigated the effect of glucocorticoid administration on these lipids and the ontogeny of 1,2-diglycerol-CDP choline phosphocholine transferase (PCT). Cholesterol and phospholipid concentrations decreased with maturation, phospholipids declining more than cholesterol. Thus, the cholesterol-to-phospholipid molar ratio rose with maturation. A similar decline was seen with PCT activity. Glucocorticoid treatment had no effect on cholesterol, phospholipids, or PCT. However, the maturational increase in the cholesterol-to-phospholipid molar ratio was blunted by glucocorticoid administration in the weanling and adult rats.