The TMEM189 gene encodes plasmanylehtanolamine desaturase which introduces the characterisitic vinyl ether double bond into plasmalogens

Werner ER, Keller MA, Sailer S, Lackner K, Koch J, Hermann M, Coassin S, Golderer G, Werner-Felmayer G, Zoeller RA, Hulo N, Berger J, and Watschinger K. (2020) The TMEM189 gene encodes plasmanylehtanolamine desaturase which introduces the characterisitic vinyl ether double bond into plasmalogens. PNAS, 117 (14): 7792-7798.

Plasmalogens are a special class of phospholipids containing a glycerol backbone with a vinyl-ether bond connecting a fatty alcohol at sn-1, an acyl linked fatty acid at sn-2, and either an ethanolamine or choline head group at sn-3. The vinyl-ether bond, which is unique to this class, is responsible for the biological functionality of plasmalogens. The double bond causes the side chains at sn-1 and sn-2 to have a parallel alignment, resulting in a more compact structure. It is because of this structure that plasmalogens play a critical role in membrane structure and vesicular fusion. As well, the vinyl bond can scavenge two radical oxygen species (ROS), giving plasmalogens antioxidant properties. A reduction in plasmalogens is associated with Alzheimer’s disease, Parkinson’s disease, multiple sclerosis, and Autism spectrum disorders. In addition, a complete deficiency in plasmalogens due to genetic mutations in the enzymes involved in their synthesis causes a severe form of dwarfism with cognitive deficits called rhizomelic chondrodysplasia punctata (RCDP). Most of the genes encoding the enzymes involved in the biosynthesis of plasmalogens have been known for decades and are well-studied in both in vivo and in vitro models. However, the gene encoding plasmanylethanolamine desaturase (PEDS), the enzyme responsible for introducing the vinyl-ether bond at sn-1 had not been previously identified. Werner et al found that the transmembrane protein 189 gene (TMEM189) encodes the PEDS enzyme and that Tmem189-deficient homozygous mice have significantly reduced PEDS activity and plasmalogen levels.

To identify the TMEM189 gene, Werner et al used four criteria when selecting the candidate genes from seven mouse tissues and eleven human cell lines, totaling 7,382 common genes between the two models. The criteria included:

1)    The messenger RNA (mRNA) amount correlating to the enzymatic activity in the tissue

2)    Features that align with it being a membrane protein

3)    Contains a characteristic histidine pattern similar to that seen in other lipid desaturases

4)    Only occur in species that can synthesize plasmalogens.

Of the 7,382 genes being analyzed, TMEM189 fit all four requirements best. To confirm the phenotype that a knockdown TMEM189 would reduce PEDS activity, plasmalogen levels, and vinyl-ether bond formation were studied using human cell culture.

Transfection of wild-type human cells with a plasmid containing myc-tagged TMEM189 (to allow endogenously expressed protein to be differentiated from the overexpressed protein) showed a 2.37 ± 0.18-fold increase in PEDS activity compared to cells that received a control transfection containing a green fluorescent protein (Figure 1, panel G). In addition, when TMEM189 was knocked-down in cells using small interfering RNA (siRNA), a 3-fold reduction in PEDS activity was seen (Figure 1, penel H). These findings indicate that the expression of TMEM189 is related to PEDS expression. In cells that were TMEM189-deficient, a 17-fold reduction in plasmalogen levels was detected. This finding would be expected as PEDS is responsible for the vinyl-ether bond and its reduced activity would inhibit the ability to synthesize these phospholipids. The ability to form the vinyl-ether bond was also determined by supplementing TMEM189-deficient cells with a modified alkyl glycerol, 1-O- pyrenedecyl-sn-glycerol, for 24 hours which can be taken up by the cells to produce plasmalogens. When tested against other desaturase proteins, only transfection with TMEM189-6x myc protein was shown to produce the vinyl-ether bond in the TMEM189-deficient cell line to ~30% of that seen in the wild-type cells, further supporting that TMEM189 is the gene encoding the PEDS enzyme.

Finally, a Tmem189-deficient mouse model was compared against a control line with intact PEDS activity to analyze plasmalogen levels in an in vivo system. Kidney tissue was chosen as it was found to have the highest PEDS activity. In the homozygous Tmem189-deficient mice, PEDS activity was reduced to below the level of detection at 0.2 pmol/mg/min compared to the wild-type at 1.5 pmol/mg/min in males and ~1.8 pmol/mg/min in females. As well, plasmalogen levels were nearly undetectable while the wild-type mice displayed levels of ~12 nmol/mg, further confirming the phenotype of this gene in this in vivo model.

Through many assays, Werner et al were able to provide support that the gene encoding the PEDS enzyme is TMEM189. This study has clearly shown that mutations in TMEM189 prevent plasmalogens from being formed, which would suggest that this mutation may be an additional cause of RCDP. All previous genes found to result in RCDP encode enzymes involved in the initial steps of plasmalogen synthesis and are found within the peroxisome; therefore, TMEM189 could be the first extra-peroxisomal cause of RCDP to date. Theoretically, a person with this mutation should be able to synthesize alkyl glycerol but be hindered at the final step when the vinyl-ether bond would be introduced, forming the plasmalogen. As the vinyl-ether bond sets this class of phospholipids apart from others, having the capacity to specifically study its function and determine how the effects of this mutation differ from typical RCDP peroxisomal mutations will allow further research avenues for learning about plasmalogen deficiencies.

Fig. 1. PEDS gene identification. (G) PEDS activities in WT HAP1 cells compared with TMEM189-deficient HAP1 cells (ΔTMEM189) at 48 h posttransfection with expression plasmids for GFP, for selected desaturase and hydroxylase proteins, and for TMEM189 protein. Data are mean ± SEM for three independent experiments. (H) Plasmalogen content of WT HAP1 cells compared with TMEM189-deficient HAP1 cells. Data are mean ± SEM for three independent experiments.