Molecular analysis of lipid synthesis and its role in vesicle trafficking, de novo cloning, expression, and characterization of two human cholinephosphotransferases
Bibliographic record
Abstract
Phosphatidylcholine (PC) and phosphatidyletlianolamine (PE) are the most abundant phospholipids in eukaryotic cells, comprising 50% and 25% of membrane phospholipid mass, respectively. Cholinephosphotransferase catalyzes the final step in the de novo biosynthesis of PC through the Kennedy pathway via the transfer of phosphocholine from CDP-choline to diacylglycerol, with the release of CMP. Ethanolaminephosphotransferaser catalyzes an analogous reaction with CDP-ethanolamine as the phosphobase donor for the de novo biosynthesis of PE. Together, these two enzyme activities determine both the site of synthesis and the fatty acyl composition of PC and PE synthesized de novo. Attempts to purify these enzymes have been largely unsuccessful due to their integral membrane bound nature, and thus, most of the studies to date have used microsomal membrane preparations to characterize these enzymes. The recent cloning of the Sacccharomyces cerevisiae choline- and ethanolaminephosphotransferases (CPT1 and EPT1) has facilitated the search for mammalian cDNAs encoding these enzyme activities. This work reports the cloning expression, and characterization of the first mammalian choline- and ethanolaminephosphotransferase cDNAs. Human choline/ethanolaminephosphotransferase 1 (hCEPT1) codes for a 416 amino acid protein that can catalyze the de novo biosynthesis of both PC and PE in vitro and in vivo. Human cholinephosphotransferase 1 (hCPT1) encodes a 406 amino acid protein specific for the enzymatic synthesis of PC in vitro and in vivo. In vitro assessment of hCEPT1 and hCPT1-derived cholinephosphotransferase activity revealed differences in diradylglycerol specificities including their capacity to synthesize platelet-activating factor and platelet-activating factor precursor. While hCEPT1 mRNA was found in similar abundance in all tissues tested, expression of hCPT1 mRNA varied greater than 100-fold between tissues, and was most abundant in testis, followed by colon, small intestine, heart, prostate, and spleen. Immunofluorescence studies revealed that hCEPT1p was localized to the ER and the nuclear membrane, while hCPT1p was localized to the Golgi apparatus. Site-directed mutagenesis of hCEPT1 demonstrated that glycine156 plays a role in CDP-ethanolamine binding, while threonine214, valine 216, and isoleucine221 appear to play a role in determining the specificity of DAG utilized by hCEPT1p. Mutation of glutamate215 suggested that it might play a role in both DAG and CDP-aminoalcohol binding, although the exact roles remain to be elucidated.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.001 | 0.001 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".