Evolutionary and Functional Analysis of Monoamine Oxidase F: A Novel Member of the Monoamine Oxidase Gene Family

Figures

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  Fig. 1.. Maximum-likelihood tree showing relationships among monoamine oxidases of vertebrates. Numbers on the nodes correspond to support from the aBayes and ultrafast bootstrap values. The scale denotes substitutions per site, and shading represents MAO lineages. Lysine-specific demethylase 1A and 1B of the human (H. sapiens) and house mouse (M. musculus) were used as outgroups.

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  Fig. 2.. An evolutionary model regarding the evolution of the monoamine oxidases in vertebrates. Our model proposes that the vertebrate ancestor had a single copy gene (MAO A/B/F), a condition that cyclostomes maintain. This gene underwent a duplication event in the ancestor of jawed vertebrates, giving rise to MAO A/B and MAO F. These genes are found in cartilaginous fish, ray-finned fish, coelacanths, and West African lungfish. In the ancestor of tetrapods, the MAO F gene was lost. In contrast, the MAO A/B gene underwent a duplication event in the tetrapod ancestor, originating MAO A and MAO B. Silhouette images were downloaded from PhyloPic (http://phylopic.org/).

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  Fig. 3.. Expression of Indo-Pacific tarpon (Megalops cyprinoides) MAO A/B and MAO F in human cells. a and b) HeLa cells grown in glass coverslips were transfected to express full-length MAO A/B a) or MAO F b) tagged with three copies of the c-Myc epitope at the N-terminus (3myc-MAO A/B and 3myc-MAO F, respectively), followed by incubation with the mitochondrial probe MitoTracker Orange (red channel). Cells were fixed, permeabilized, and labeled with mouse monoclonal antibody against the c-Myc epitope, followed by incubation with Alexa-Fluor-488-conjugated donkey anti-mouse IgG (green channels), and nuclei were stained with the DNA probe DAPI. Stained cells were examined by fluorescence microscopy. In the merge channels, yellow indicates the colocalization of each MAO protein with MitoTracker at mitochondria, and red depicts mitochondria in nontransfected cells. Bar, 10 μm. c) The indicated cells were left untreated (Non-transfected; lanes 1 and 5), transfected with empty pCMV-3Tag-2A vector (lanes 2 and 6), or transfected with pCMV-3Tag-2A vector encoding either 3myc-MAO A/B (lanes 3 and 7) or 3myc-MAO F (lanes 4 and 8). Detergent-soluble extracts were prepared, and samples were processed…

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  Fig. 4.. UV-Vis absorbance spectra of IPT-MAO A/B and IPT-MAO F (10 μM) incubated with 10-fold molar excess of either clorgyline or deprenyl. Measurements were registered on the native enzyme (black) immediately after the addition of the inhibitor (red) and at 10 (blue) and 30 (green) min.

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  Fig. 5.. Zoomed view of the superposed 3D structures of MAO enzymes showing the active site. IPT-MAO A/B (green) and IPT-MAO F (light blue) are models predicted by AlphaFold3, whereas human MAO A (beige) and MAO B (gray) are crystal structures (PDB codes 2Z5X and 2V5Z, respectively; inhibitors bound to the structures were removed from the coordinate files). Structures are represented as semi-transparent ribbon diagram with FAD and the residues facing the active site represented as sticks (oxygen, nitrogen, and sulfur atoms are red, blue, and yellow, respectively). Labels are indicated according to the ribbon colors mentioned above; for residues that are fully conserved in all four proteins labels are reported only for the IPT homologues. In the crystal structures, the water molecule bridging the conserved lysine residue (Lys305 in human MAO A and Lys296 in human MAO B) and the N5 atom of the FAD cofactor is shown as red sphere. The figure was prepared using the software CCP4 mg (McNicholas et al. 2011).

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  Fig. 6.. Expression of MAO A and MAO B in the mouse brain, and MAO A/B/F in the lamprey brain. a) UMAP plot depicting the expression of lamprey MAO A/B/F in immune cells, habenular neurons (HB), monoaminergic neurons (MA), peptidergic neurons (PEP), excitatory neurons from the telencephalon, diencephalon and mesencephalon (Exc TE/DE/ME), inhibitory neurons from the telencephalon (Inh TE), astrocytes, and vascular cells. b) Violin plot depicting the expression levels of lamprey MAO A/B/F in specific cell clusters. Photoreceptors (Photo), spinal cord (SC), rhomboencephalon (RE), choroid plexus epithelium (CP Epi), ependymal cells (Epen), meningeal fibroblasts (Men Fibro), erythrocytes (Eryt). c) Left: UMAP plot depicting the expression of mouse MAO A in vascular cells, astrocytes, oligodendrocytes and oligodendrocyte precursor cells (OPC), cerebellum glutamatergic neurons (CB Glut), dopamine-β-hydroxylase expressing glutamatergic neurons from the nucleus of the solitary tract (NTS Dbh Glut), habenular neurons (HB Glut). Right: UMAP plot depicting the expression of mouse MAO B in histaminergic neurons (Hist), serotonergic neurons (Sero), and glutamatergic neurons from the thalamus (TH Glut).…