Indolethylamine N-methyltransferase (INMT) is not essential for endogenous tryptamine-dependent methylation activity in rats

Abstract

Indolethylamine N-methyltransferase (INMT) is a transmethylation enzyme that utilizes the methyl donor S-adenosyl-L-methionine to transfer methyl groups to amino groups of small molecule acceptor compounds. INMT is best known for its role in the biosynthesis of N,N-Dimethyltryptamine (DMT), a psychedelic compound found in mammalian brain and other tissues. In mammals, biosynthesis of DMT is thought to occur via the double methylation of tryptamine, where INMT first catalyzes the biosynthesis of N-methyltryptamine (NMT) and then DMT. However, it is unknown whether INMT is necessary for the biosynthesis of endogenous DMT. To test this, we generated a novel INMT-knockout rat model and studied tryptamine methylation using radiometric enzyme assays, thin-layer chromatography, and ultra-high-performance liquid chromatography tandem mass spectrometry. We also studied tryptamine methylation in recombinant rat, rabbit, and human INMT. We report that brain and lung tissues from both wild type and INMT-knockout rats show equal levels of tryptamine-dependent activity, but that the enzymatic products are neither NMT nor DMT. In addition, rat INMT was not sufficient for NMT or DMT biosynthesis. These results suggest an alternative enzymatic pathway for DMT biosynthesis in rats. This work motivates the investigation of novel pathways for endogenous DMT biosynthesis in mammals.

Figure 1

The Mammalian DMT biosynthetic pathway. The methylation of tryptamine to form N,N-Dimethyltryptamine is thought to occur via a two-step reaction catalyzed by the enzyme indolethylamine-N-methyltransferase (INMT), with N-methyltryptamine (NMT) as an intermediate. The co-factor S-adenosyl-L-methionine (SAM) serves as the methyl donor, with S-adenosyl-L-homocysteine (SAH) as a byproduct of this methyl transfer.

Figure 2

Enzymatic methylation of tryptamine does not differ between WT and INMT-KO rats (a) The gene structure of rat INMT with exons represented in black boxes. The location and sequence of the INMT guide RNA (gRNA) is shown with a red triangle. (b) The 2-base pair deletion responsible for generating the KO is shown below the wild type (WT) sequence with the location of the gRNA underlined. (c) Western blot showing the presence of INMT (green bands) in WT, but not KO rat lung tissues. The original image including all lanes in the molecular weight marker is included as Supplementary Figure S1. (d) Radiometric enzyme assays with tissue extracts from rabbit lung, as well as rat brain and rat lung from both WT and INMT-KO rats. Each point represents the average of 3 triplicate experiments from an individual animal for rabbits (n = 3), and rats (n = 6) for each genotype. Plots show averages with error bars indicating standard deviations. CPM: counts per minute. Rabbit lung tissue extracts (n = 3) showed tryptamine-dependent activity > 20-fold higher than rat tissues (average specific activity [SA] = 322.2 ± 8.81, 95% confidence interval [CI] of mean = 300.3–344.0). Tryptamine-dependent activity did not differ between WT and KO rats in brain (t = 0.30, mean difference = − 0.36, 95% CI = − 3.05–2.33, p = 0.77) or lung tissues (t = 0.52, mean difference = − 0.25, 95% CI = − 1.33–0.84, p = 0.62). In brain, average SA was 14.68 ± 2.34, 95% CI of mean = 12.22–17.13 in WT, and 14.32 ± 1.76, 95% CI of mean = 12.47–16.16 in KO. In lung, average SA was 6.36 ± 0.66, 95% CI of mean = 5.66–7.05 in WT, and 6.11 ± 0.96, 95% CI of mean = 5.10–7.12 in KO. Tryptamine-dependent activity was significantly higher in brain tissues relative to lung (t = 8.38, mean difference = 8.32, 95% CI = 5.87–10.77, p = 0.0002 for WT and t = 10.04, mean difference = 8.21, 95% CI = 6.31–10.11, p < 0.0001 for KO).

Figure 3

Brain and lung tissues from WT and KO rats produce products that are not isographic with NMT or DMT. Phosphor imaging scan of radiometric enzyme assay products using extracts from brain and lung tissues of WT and KO rats following separation on silica gel plates using a mobile phase of N-butanol:acetic acid:water (12:3:5). A standard mixture (Std) containing tryptamine, NMT and DMT were clearly separated using these chromatography methods (left most lane). Uncropped and unprocessed phosphor imaging scan of the graph is included as Supplementary Figure S2.

Figure 4

Rat INMT is not sufficient for tryptamine methylation. (a) Western blot showing expression of GST-INMT fusion proteins of rat, rabbit and human INMTs in E. coli. The control lane is showing expression of an empty vector GST, where INMT is absent. Uncropped image of the western blot is included as Supplementary Figure S3. (b) Radiometric enzyme assays with extracts from E. coli expressing GST-fusion recombinant rat, rabbit, and human INMT proteins. Plots show the average of 6 individual experiments per condition with error bars indicating standard deviations. CPM = counts per minute. The assay revealed tryptamine-dependent activity of both rabbit INMT (average SA = 350.9 ± 23.08, 95% CI of mean = 326.7–375.1) and human INMT (average SA = 171.8 ± 12.38, 95% CI of mean = 158.8–184.8). Activity was significantly higher in rabbit INMT relative to human INMT (t = 16.75, mean difference = 179.1, 95% CI = 154.3–204, p < 0.0001). The activity of rat INMT (average SA = 0.64 ± 0.21, 95% CI of mean = 0.42–0.85) was significantly lower (t = 3.68, mean difference = − 0.48, 95% CI = − 0.77 to − 0.19, p = 0.004) than empty vector GST extracts (average SA = 1.12 ± 0.24, 95% CI of mean = 0.86–1.37).

Figure 5

Rabbit and human, but not rat INMT methylate tryptamine to produce NMT and DMT. Phosphor imaging scan of radiometric enzyme assay products using recombinant GST-INMT fusion proteins following separation on silica gel plates using a mobile phase of N-butanol:acetic acid:water (12:3:5). A standard (Std) mixture containing tryptamine, NMT and DMT were clearly separated using these chromatography methods (left most lane). The standards showed respective R_F values of 0.58, 0.49, and 0.39. Rabbit INMT, human INMT, and rabbit lung each showed two spots isographic with both NMT and DMT. For rabbit INMT, NMT R_F = 0.48 DMT R_F = 0.41. For human INMT, NMT R_F = 0 48 and DMT R_F = 0.41. For rabbit lung (positive control), NMT R_F = 0.50 and DMT R_F = 0.41. Neither empty vector GST preparations nor rat GST-INMT showed detectable products, which indicates a lack of methylation activity. Uncropped, unprocessed phosphor imaging scan of the same graph is included as Supplementary Figure S6.