Abstract

Selenium-binding protein 1 (SELENBP1) has been associated with several cancers, although its exact role is unknown. We show that SELENBP1 is a methanethiol oxidase (MTO), related to the MTO in methylotrophic bacteria, that converts methanethiol to H2O2, formaldehyde, and H2S, an activity not previously known to exist in humans. We identified mutations in SELENBP1 in five patients with cabbage-like breath odor. The malodor was attributable to high levels of methanethiol and dimethylsulfide, the main odorous compounds in their breath. Elevated urinary excretion of dimethylsulfoxide was associated with MTO deficiency. Patient fibroblasts had low SELENBP1 protein levels and were deficient in MTO enzymatic activity; these effects were reversed by lentivirus-mediated expression of wild-type SELENBP1. Selenbp1-knockout mice showed biochemical characteristics similar to those in humans. Our data reveal a potentially frequent inborn error of metabolism that results from MTO deficiency and leads to a malodor syndrome.

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Acknowledgements

The authors gratefully thank the patients and families for their help in this study. The contributions of W. Lehnert, I. Goldschmidt, and C. von Schnakenburg to early investigations on family A are gratefully acknowledged. M. Antoine and T. van Alen are kindly acknowledged for their technical expertise, and Ö. Eyice-Broadbent is acknowledged for work on identifying the bacterial MTO. G. Linthorst, F. Wijburg, and H. Blom are acknowledged for help in obtaining good-quality patient samples. The authors thank P. Klaren for statistical consultation. The work was supported by the following grants: a UK Biotechnology and Biological Sciences Research Council grant to H.S. (reference BB/H003851/1), an ERC grant to H.J.M.O.d.C. (ERC 669371-Volcano), and a grant from the E.C. Noyons Foundation to R.A.W.

Author information

Author notes

  1. Arjan Pol and G. Herma Renkema contributed equally to this work. 

  2. Huub J. M. Op den Camp and Ron A. Wevers jointly directed this work.

Affiliations

  1. Department of Microbiology, IWWR, Faculty of Science, Radboud University, Nijmegen, The Netherlands

    • Arjan Pol
    •  & Huub J. M. Op den Camp
  2. Translational Metabolic Laboratory, Department of Laboratory Medicine, Radboud University Nijmegen Medical Centre (RUNMC), Nijmegen, The Netherlands

    • G. Herma Renkema
    • , Udo F. Engelke
    • , Lambert van den Heuvel
    • , Marijn Oude Elberink
    • , Richard J. Rodenburg
    •  & Ron A. Wevers
  3. Department of Internal Medicine, RUNMC, Nijmegen, The Netherlands

    • Albert Tangerman
  4. Center for Dentistry and Oral Hygiene, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands

    • Edwin G. Winkel
  5. Clinic for Periodontology, Amsterdam, The Netherlands

    • Edwin G. Winkel
  6. Department of Human Genetics, RUNMC, Nijmegen, The Netherlands

    • Arjan P. M. de Brouwer
    •  & Christian Gilissen
  7. Department of Surgery, School of Medicine, and Mouse Biology Program, University of California, Davis, Davis, CA, USA

    • Kent C. Lloyd
  8. Mouse Biology Program, University of California, Davis, Davis, CA, USA

    • Renee S. Araiza
  9. Department of Pediatrics, RUNMC, Nijmegen, The Netherlands

    • Lambert van den Heuvel
    •  & Richard J. Rodenburg
  10. Klinik für Kinder und Jugendmedizin, Universitätsklinikum Münster, Münster, Germany

    • Heymut Omran
    •  & Heike Olbrich
  11. Bioanalytics and Biochemistry, Department of Natural Sciences, Bonn-Rhein-Sieg University of Applied Sciences, Rheinbach, Germany

    • Jörn Oliver Sass
  12. Department of Pediatrics and Adolescent Medicine, University Hospital Freiburg, Freiburg, Germany

    • K. Otfried Schwab
  13. School of Life Sciences, University of Warwick, Coventry, UK

    • Hendrik Schäfer
  14. Centre for Molecular and Biomolecular Informatics, RUNMC, Nijmegen, The Netherlands

    • Hanka Venselaar
  15. Metabolic Unit–Pediatric Department, Hospital de Dona Estefânia, CHLC, Lisbon, Portugal

    • J. Silvia Sequeira

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Contributions

H.J.M.O.d.C. and R.A.W. conceived the study and coordinated and supervised the different teams; H.J.M.O.d.C., A.P., and G H.R. designed the assays of the sulfur metabolites and MTO enzyme activity; A.P., U.F.E., and A.T. measured sulfur metabolites and/or MTO enzyme activity; E.G.W. performed the Oral Chroma technique for halitosis detection; U.F.E. was responsible for the body-fluid NMR measurements; A.P.M.d.B. provided the tissue expression data of SELENBP1; K.C.L. and R.S.A. made the KO mouse line; L.v.d.H. and M.O.E. performed Sanger sequencing of SELENBP1; H. Omran and H. Olbrich performed linkage analysis and whole-exome sequencing in family A; C.G. performed the frequency calculations for the SELENBP1 defect; R.J.R. and G H.R. performed the cell culture and lentiviral complementation studies; J.O.S., K.O.S., J.S.S., and A.T. performed clinical and biochemical phenotyping of the patients and family members; H.S. and H.J.M.O.d.C. provided the gene sequence of methanethiol oxidase from Hyphomicrobium strain VS; H.V. made the SELENBP1 three-dimensional model and mapped the affected amino acid residues; A.T. collected initial measurements of sulfur-containing metabolites in the blood and breath and carried out several studies on individuals with halitosis; A.P., G.H.R. and R.A.W. prepared the manuscript, to which various coauthors contributed. All coauthors edited and reviewed the final manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Ron A. Wevers.

Integrated Supplementary Information

  1. Supplementary Figure 1 Breath analysis in the dental clinic

    Breath samples of the extra-oral halitosis patient CII-2 (dotted line), a control person (dashed line), and an intra-oral halitosis patient (solid line) analysed in a portable gas chromatograph (OralChromaTM). Vertical lines indicate the retention times of H2S, MT and DMS. The vertical line indicated with * is a non-sulfur volatile background peak as described in: Hanada, M. et al. Portable oral malodor analyzer using highly sensitive In2O3 gas sensor combined with a simple gas chromatography system. Analytica Chimica Acta 475, 27-35 (2003).

  2. Supplementary Figure 2 Alignment of the Hyphomicrobium strain VS MT-oxidase and the human SELENBP1

    The full-length alignment was performed using the Pairwise Alignment tool of the Protein Information Resource (http://pir.georgetown.edu/pirwww/search/). The Smith-Waterman score was 398. The proteins show 26.0% identity (highlighted in yellow and by *) and 54.2% similarity (highlighted in green) in 461 aa overlap. The signal peptide of the bacterial enzymes is underlined. In red putative TTQ residues (W) and copper ligands (H).  

  3. Supplementary Figure 3 Newly established assay for MTO activity

    a MT oxidation results in H2S production. The amounts of MT (circles) and H2S (squares) were followed in time. Two standard assay mixtures (in 250 ml serum bottles) containing MT with- (closed symbols) or without (open symbols) Zn (0.2 mM) were started by the addition of 2 µl human control erythrocyte extract as a MTO source at the time point indicated by the arrowhead. Without Zn in the medium the molar amount of MT that has been converted is seemingly higher that the amount of H2S formed. Zn-ions will capture formed H2S. After MT was depleted the reaction mixtures were acidified thus releasing the H2S again (indicated with an arrow). In the incubation mixture with Zn the amount of sulfide formed is equimolar to the amount of MT that has been converted. In the absence of Zn-ions the formed H2S can be further enzymatically metabolized towards thiosulfate (Ref) thus explaining the lower molar recovery of H2S in the absence of Zn-ions. b Kinetic analysis of the MTO assay. Two identical standard assays using 10 µl human control erythrocyte extract were started at t = 0 under anaerobic conditions. The closed arrowhead indicates the addition of 20 ml of oxygen to incubation indicated with . The open arrowhead indicates the addition of oxygen to the other incubation () twenty minutes later. c MTO activity in colon cancer cell lines HT29 (▲) and SW480 (■). Oxygen dependence of the reaction in 3 ml exetainers is shown with cell line HT29 under anoxic condition (Δ), arrow indicates the addition of 0.25 ml of oxygen. Background disappearance of MT (). Each data point is the average of duplicate incubations that showed less than 5% difference. d Michaelis-Menten plot of MTO activity measurements done with control erythrocyte extract using different substrate concentrations. The apparent Km is indicated. e-g Kinetic analysis of MTO activity in human erythrocyte extract e, HT29 colon cancer cell extracts f and wild type mouse liver extracts (g, duplicate measurements as open and closed symbols) under low starting concentrations of MT. In each panel the line is fitted on the data using Michaelis-Menten kinetics. This resulted in apparent Km values of 5, 4.7 and 6 nM, respectively. Ref: Szabo, C. et al. Regulation of mitochondrial bioenergetic function by hydrogen sulfide. Part I. Biochemical and physiological mechanisms. Br J Pharmacol 171, 2099-122 (2014).

  4. Supplementary Figure 4 Intracellular localization of SELENBP1

    Stably transduced patient cells were stained for indirect fluorescence analysis with anti-V5 antibodies that detect the C-terminal tag of SELENBP1 (red). Nuclei were stained with Hoechst dye (blue).

  5. Supplementary Figure 5 THAP4-mutation analysis in pedigree A

    Sanger sequencing of the THAP4 mutation in family A shows that the c.1400C>A mutation occurs in homozygous form in the male individual AII-3 with the malodour and the neurological features while in heterozygous form in the female individual AII-2 with the malodour syndrome without neurological features. None of the other 3 sibs in this family has this THAP4 loss of function mutation in homozygous form. Black symbols in the pedigree indicate the malodour in individuals AII-2 and AII-3.

Supplementary information

  1. Supplementary Text and Figures

    Supplementary Figures 1–5, Supplementary Table 2 and Supplementary Note.

  2. Life Sciences Reporting Summary

  3. Supplementary Table 1

    Variants from ExAc and in-house databases for CFTR, PAH and SELENBP1. CADD_PHRED scores and variant frequencies were used to calculate the presumed frequency for SELENBP1 mutations, as described in the Supplementary Note

  4. Supplementary Data 1

    Full length blots