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. 1997 Nov 25;94(24):12880-5.
doi: 10.1073/pnas.94.24.12880.

Disruption of the murine gene encoding phosphatidylethanolamine N-methyltransferase

C J Walkey  1 , L R Donohue, R Bronson, L B Agellon, D E Vance
Affiliations

Disruption of the murine gene encoding phosphatidylethanolamine N-methyltransferase

C J Walkey et al. Proc Natl Acad Sci U S A. .

Abstract

All nucleated cells make phosphatidylcholine via the CDP-choline pathway. Liver has an alternative pathway in which phosphatidylcholine is made by methylation of phosphatidylethanolamine catalyzed by phosphatidylethanolamine N-methyltransferase (PEMT). We investigated the function of PEMT and its role in animal physiology by targeted disruption of its gene, Pempt2. A targeting vector that interrupts exon 2 was constructed and introduced into mice yielding three genotypes: normal (+/+), heterozygotes (+/-), and homozygotes (-/-) for the disrupted PEMT gene. Only a trace of PE methylation activity remained in Pempt2(-/-) mice. Antibody to one form of the enzyme, PEMT2, indicated complete loss of this protein from Pempt2(-/-) mice and a decrease in Pempt2(+/-) mice, compared with Pempt2(+/+) mice. The levels of hepatic phosphatidylethanolamine and phosphatidylcholine were minimally affected. The active form of CTP:phosphocholine cytidylyltransferase, the regulated enzyme in the CDP-choline pathway, was increased 60% in the PEMT-deficient mice. Injection of [L-methyl-3H]methionine demonstrated that the in vivo PEMT activity was eliminated in the Pempt2(-/-) mice and markedly decreased in the Pempt2(+/-) mice. This experiment also demonstrated that the choline moiety derived from PEMT in the liver can be distributed via the plasma throughout the mouse where it is found as phosphatidylcholine, lysophosphatidylcholine, and sphingomyelin. Mice homozygous for the disrupted Pempt2 gene displayed no abnormal phenotype, normal hepatocyte morphology, normal plasma lipid levels and no differences in bile composition. This is the first application of the "knockout mouse" technique to a gene for phospholipid biosynthesis.

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Figures

Figure 1
Figure 1
(A) The normal Pempt2 gene, the gene targeting vector, and the disrupted Pempt2 allele. The disrupted allele was identified by Southern blot analysis of EcoRI-digested DNA, using the 5′ probe EX. (B) Genotyping of mice by Southern blot analysis. The normal Pempt2 allele yielded a 13-kb EcoRI fragment detected by probe EX, while the disrupted allele yielded a 5-kb EcoRI fragment. (C) Absence of PEMT2 protein in Pempt2 knockout mice. Immunoblot analysis of liver homogenates demonstrated that PEMT2 protein is present only in mice carrying at least one normal Pempt2 allele. Each lane contained 50 μg protein. PEMT2 has an apparent molecular mass of 19 kDa.
Figure 2
Figure 2
Elimination of methylation activity of PE, PMME, and PDME in Pempt2-disrupted mice. Total liver homogenates from mice of all three genotypes were assayed for PEMT activity. For all three genotypes, n = 4. Bars = SD.
Figure 3
Figure 3
Phospholipid composition of livers from mice of all three genotypes. Levels of PC and PE in liver homogenates were measured and expressed relative to total phospholipid. In all cases, n = 4. Bars = SD. *P < 0.05 compared with (+/+).
Figure 4
Figure 4
Membrane-bound CT activity was increased in Pempt2 knockout mice. CT activity was measured in soluble and membrane-bound fractions of liver homogenates. In all cases n = 4. Bars = SD. *P < 0.05 compared with (+/+).
Figure 5
Figure 5
Incorporation of radiolabeled methionine into choline-containing phospholipids. [l-methyl-3H]methionine was injected into the bloodstream of mice of each of the three genotypes. Plasma and organs were harvested after 1 h (A) or 24 h (B). Phospholipids were extracted and separated by thin-layer chromatography. Bands corresponding to choline-containing phospholipids (PC, lyso PC, and sphingomyelin) were scraped, and radiolabel incorporation measured, as well as total lipid phosphorous. Results are expressed as dpm per μmol choline-containing phospholipid. The mouse homozygous for the disrupted Pempt2 allele had only 4.4% the level of radiolabeled methionine incorporation into choline-containing phospholipid seen in the normal mouse after 1 h, and 4.9% the level of incorporation after 24 h.
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