No abstract

Molybdenum cofactor deficiency is an autosomal recessive inborn error of metabolism, which results from mutations in genes involved in Moco biosynthesis. Moco serves as a cofactor of several enzymes, including sulfite oxidase. MoCD is clinically characterized by intractable seizures and severe, rapidly progressing neurodegeneration leading to death in early childhood in the majority of known cases. Here we report a patient with an unusual late disease onset and mild phenotype, characterized by a lack of seizures, normal early development, a decline triggered by febrile illness and a subsequent dystonic movement disorder. Genetic analysis revealed a homozygous c.1338delG MOCS1 mutation causing a frameshift (p.S442fs) with a premature termination of the MOCS1AB translation product at position 477 lacking the entire MOCS1B domain. Surprisingly, urine analysis detected trace amounts (1% of control) of the Moco degradation product urothione, suggesting a residual Moco synthesis in the patient, which was consistent with the mild clinical presentation. Therefore, we performed bioinformatic analysis of the patient's mutated MOCS1 transcript and found a potential Kozak-sequence downstream of the mutation site providing the possibility of an independent expression of a MOCS1B protein. Following the expression of the patient's MOCS1 cDNA in HEK293 cells we detected two proteins: a truncated MOCS1AB protein and a 22.4 kDa protein representing MOCS1B. Functional studies of both proteins confirmed activity of MOCS1B, but not of the truncated MOCS1AB. This finding demonstrates an unusual mechanism of translation re-initiation in the MOCS1 transcript, which results in trace amounts of functional MOCS1B protein being sufficient to partially protect the patient from the most severe symptoms of MoCD.

Molybdenum cofactor (Moco) biosynthesis is a highly conserved multistep pathway. The first step, the conversion of GTP to cyclic pyranopterin monophosphate (cPMP), requires the bicistronic gene molybdenum cofactor synthesis 1 (MOCS1). Alternative splicing of MOCS1 within exons 1 and 9 produces four different N-terminal and three different C-terminal products (type I–III). Type I splicing results in bicistronic transcripts with two open reading frames, of which only the first, MOCS1A, is translated, whereas type II/III splicing produces MOCS1AB proteins. Here, we first report the cellular localization of alternatively spliced human MOCS1 proteins. Using fluorescence microscopy, fluorescence spectroscopy, and cell fractionation experiments, we found that depending on the alternative splicing of exon 1, type I splice variants (MOCS1A) either localize to the mitochondrial matrix (exon 1a) or remain cytosolic (exon 1b). MOCS1A proteins required exon 1a for mitochondrial translocation, but fluorescence microscopy of MOCS1AB variants (types II and III) revealed that they were targeted to mitochondria independently of exon 1 splicing. In the latter case, cell fractionation experiments displayed that mitochondrial matrix import was facilitated via an internal motif overriding the N-terminal targeting signal. Within mitochondria, MOCS1AB underwent proteolytic cleavage resulting in mitochondrial matrix localization of the MOCS1B domain. In conclusion, MOCS1 produces two functional proteins, MOCS1A and MOCS1B, which follow different translocation routes before mitochondrial matrix import for cPMP biosynthesis involving both proteins. MOCS1 protein maturation provides a novel alternative splicing mechanism that ensures the coordinated mitochondrial targeting of two functionally related proteins encoded by a single gene.

Therapy of molybdenum cofactor (Moco) deficiency has received US Food and Drug Administration (FDA) approval in 2021. Whereas urothione, the urinary excreted catabolite of Moco, is used as diagnostic biomarker for Mocodeficiency, its catabolic pathway remains unknown. Here, we identified the urothione-synthesizing methyltransferase using mouse liver tissue by anion exchange/size exclusion chromatography and peptide mass fingerprinting. We show that the catabolic Moco S-methylating enzyme corresponds to thiopurine S-methyltransferase (TPMT), a highly polymorphic drug-metabolizing enzyme associated with drug-related hematotoxicity but unknown physiological role. Urothione synthesis was investigated in vitro using recombinantly expressed human TPMT protein, liver lysates from Tpmt wild-type and knock-out (Tpmt −/− ) mice as well as human liver cytosol. Urothione levels were quantified by liquid-chromatography tandem mass spectrometry in the kidneys and urine of mice. TPMT-genotype/phenotype and excretion levels of urothione were investigated in human samples and validated in an independent population-based study. As Moco provides a physiological substrate (thiopterin) of TPMT, thiopterin-methylating activity was associated with TPMT activity determined with its drug substrate (6-thioguanin) in mice and humans. Urothione concentration was extremely low in the kidneys and urine of Tpmt −/− mice. Urinary urothione concentration in TPMT-deficient patients depends on common TPMT polymorphisms, with extremely low levels in homozygous variant carriers (TPMT*3A/*3A) but normal levels in compound heterozygous carriers (TPMT*3A/*3C) as validated in the populationbased study. Our work newly identified an endogenous substrate for TPMT and shows an unprecedented link between Moco catabolism and drug metabolism. Moreover, the TPMT example indicates that phenotypic consequences of genetic polymorphisms may differ between drug-and endogenous substrates.