Functional analysis of two isoforms of phosphatidylethanolamine N-methyltransferase.
The enzyme catalysing the conversion of PE (phosphatidylethanolamine) into PC (phosphatidylcholine), PEMT (PE N-methyltransferase), exists as two isoforms, PEMT-L (longer isoform of PEMT) and PEMT-S (shorter isoform of PEMT). In the present study, to compare the functions of the two isoforms of PEMT, we established HEK (human embryonic kidney)-293 cell lines stably expressing PEMT-L and PEMT-S. Both PEMT-L and PEMT-S were localized in the ER (endoplasmic reticulum). PEMT-L, but not PEMT-S, was N-glycosylated with high-mannose oligosaccharides. The enzymatic activity of PEMT-S was much higher than that of PEMT-L. By using novel enzymatic assays for measuring PC and PE, we showed that PEMT-L and PEMT-S expression remarkably increased the cellular PC content, whereas the PE content was decreased by PEMT-S expression, but was hardly affected by PEMT-L expression. The cellular content of phosphatidylserine was also reduced by the expression of PEMT-L or PEMT-S. MS analyses demonstrated that the expression of PEMT-S led to more increases in the molecular species of PC and PC-O (ether-linked PC) with longer polyunsaturated chains than that of PEMT-L, whereas the PC-O species with shorter chains were increased more by PEMT-L expression than by PEMT-S expression, suggesting a difference in the substrate specificity of PEMT-L and PEMT-S. On the other hand, various PE and PE-O species were decreased by PEMT-S expression. In addition, PEMT-L and PEMT-S expression promoted the proliferation of HEK-293 cells. Based upon these findings, we propose a model in which the enzymatic activity and substrate specificity are regulated by the glycosylated N-terminal region of PEMT-L localized in the ER lumen.
Morita
Shin-ya
SY
Kobe Pharmaceutical University, Higashinada-ku, Kobe 658-8558, Japan. smorita@kobepharma-u.ac.jp
Takeuchi
Atsuko
A
Kitagawa
Shuji
S
D000818:Animals; D017931:DNA Primers; D014644:Genetic Variation; D057809:HEK293 Cells; D006801:Humans; D007700:Kinetics; D013058:Mass Spectrometry; D050918:Phosphatidylethanolamine N-Methyltransferase / Q000235:genetics / Q000378:metabolism*; D020033:Protein Isoforms / Q000378:metabolism; D014158:Transcription, Genetic; D014162:Transfection
D017931:DNA Primers; D020033:Protein Isoforms; C494489:PEMT protein, human; D050918:Phosphatidylethanolamine N-Methyltransferase