A functional alternative splicing mutation in human tryptophan hydroxylase-2

The brain serotonergic system has an essential role in the physiological functions of the central nervous system and dysregulation of serotonin (5-HT) homeostasis has been implicated in many neuropsychiatric disorders. The tryptophan hydroxylase-2 (TPH2) gene is the rate-limiting enzyme in brain 5-HT synthesis, and thus is an ideal candidate gene for understanding the role of dysregulation of brain serotonergic homeostasis. Here, we characterized a common, but functional single-nucleotide polymorphism (SNP rs1386493) in the TPH2 gene, which decreases efficiency of normal RNA splicing, resulting in a truncated TPH2 protein (TPH2-TR) by alternative splicing. TPH2-TR, which lacks TPH2 enzyme activity, dominant-negatively affects full-length TPH2 function, causing reduced 5-HT production. The predicted mRNA for TPH2-TR is present in postmortem brain of rs1386493 carriers. The rs13864923 variant does not appear to be overrepresented in either global or multiplex depression cohorts. However, in combination with other gene variants linked to 5-HT homeostasis, this variant may exhibit important epistatic influences

Functional domains of human tryptophan hydroxylase 2 (hTPH2)

Tryptophan hydroxylase (TPH) is the rate-limiting enzyme in serotonin biosynthesis. A novel gene - termed TPH2 - has recently been described. This gene is preferentially expressed in the central nervous system, while the original TPH1 is the peripheral gene. We have expressed human tryptophan hydroxylase 2 (hTPH2) and two deletion mutants (N150 and N150/C24) using IPTG-free auto-induction in E. coli. This expression system produced active wild type TPH2 with relatively low solubility. The solubility was increased for mutants lacking the N-terminal regulatory domain. The solubility of hTPH2, N150 and N150/C24 are 6.9%, 62% and 97.5%, respectively. Removal of the regulatory domain also produced a more than 6-fold increase in enzyme stability (t1/2 at 37oC). The wild type hTPH2, like other members of the aromatic amino acid hydroxylase superfamily, exists as a homotetramer (236 kDa on size exclusion chromatography). Similarly, N150 also migrates as a tetramer (168 kDa). In contrast, removal of the N-terminal domain and the C terminal, putative leucine zipper tetramerization domain produces monomeric enzyme (39 kDa). Interestingly, removal of the N-terminal regulatory domain did not affect the Michaelis constants for either substrate, but did increase Vmax values. These data identify the N-terminal regulatory domain as the source of hTPH2 instability and reduced solubility