Mutations in SELENBP1, encoding a novel human methanethiol oxidase, cause extraoral halitosis

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.

Figure 1. Sulfur metabolism

Diet, bacterial metabolism and endogenous metabolism contribute to the levels of MT, DMS, DMSO and DMSO₂ and catabolites in the body. The main conversion of MT to H₂O₂, formaldehyde and H₂S by the enzyme MTO (indicated by the cross) is deficient in the patients. The lower part shows the MTO reaction. All underlined metabolites have been confirmed in our assay.

Figure 2. Extra-oral halitosis families

Panel A, Schematic representation of the family trees of the affected families. Double horizontal line in families A and B indicate consanguineous marriages. Dark symbols represent affected individuals, crossed out symbols are deceased individuals. Panels B and C, One-dimensional ¹H-NMR spectroscopy of human plasma measured at pH 2.50. Plasma from patient CII-2 (B) and a control sample (C). The spectra illustrate the increased concentration of DMSO₂ in the patient. For quantitative data see Table 1B.

Figure 3. Analysis of missense mutations in SELENBP1

Panel A, Neighbor-Joining phylogenetic tree of methanethiol oxidases and putative selenium-binding proteins. The evolutionary distances were computed using the Dayhoff matrix based method and are in the units of the number of amino acid substitutions per site. Bootstrap values (500 replicates) are shown next to the branches for values > 60. Evolutionary analyses were conducted in MEGA6 . Panel B, The SELENBP1 sequence was modeled on the X-ray structure of the hypothetical selenium-binding protein from Sulfolobus tokodaii (PDB ID: 2ECE). The protein forms a typical WD40 fold. The positions of the mutations Gly225Trp (on the right) and His329Tyr (on the left) are indicated in red. Panel C, Detail of the amino acid changes that are found in family C. Green residue is the original, red the replacement in the patients. Panel D, Alignments of the regions of SELENBP1 of multiple species indicating the conserved residues found mutated in family C. The amino acid numbering is according to the human sequence. Asterisk indicates a fully conserved residue, colon indicates conservation between amino acids of strongly similar properties, period indicates conservation between amino acids of weakly similar properties.

Figure 4. Analysis of SELENBP1 expression and MTO activity in human cell lines

Panel A, MTO activity in human erythrocytes in control (n=30) versus patients AII-3 and CII-2. Box indicates the median and the 25 - 75 percentile, whiskers indicate the minimal and maximal value. Panel B, SELENBP1 mRNA expression in human fetal and adult tissues. Presented as fold change in comparison to the tissue with the lowest expression level. Panel C, Anti-SELENBP1 Western blot analysis in HT29 (high expression) and SW480 (low expression) colon cancer cell lines. Panel D, Western blot analysis of patient (CII-2 and AII-3) and 4 control fibroblast cell lines. Upper panel: anti-SELENBP1. ▲, SELENBP1; △, non-specific band. Lower panel: loading control (anti-actin). Panel E: Progress curve of MTO activity. ▲, control fibroblast C5120); ■, patient AII-3 ●,control incubation without protein added. At t = 0 MT was added to all samples; the arrow indicates a second addition of MT to the control fibroblasts when the substrate was depleted, resulting in restoration of the initial activity, indicating that the enzyme was still fully functional. Panel F: Lentiviral complementation of patient and control fibroblast using SELENBP1-V5 viruses shows restoration of the MTO activity. As a control GFP-V5 encoding viruses were used. Upper panel: Western blot analysis of SELENBP1 expression (anti-SELENBP1). Middle panel: Western blot analysis of the exogenous expressed V5 tagged proteins (closed arrowhead is SELENBP1-V5, open arrowhead is GFP-V5). Lower panel: loading control (anti-actin). In panels C, D and F the MTO activity of each sample is indicated below the lanes. BG = background, below 0.5 nmol.mg protein^-1.h^-1.

Figure 5. MTO activity and DMS levels in SELENBP1 KO mice

Panel A, MTO activity in erythrocyte extracts from SELENBP1 KO, heterozygous and wild type animals (n=7 for each group). MTO activity in erythrocytes of the SELENBP1 KO animals were at or under the level of detection. Panel B, MTO activities were determined in homogenates of mouse tissues of both SELENBP1 KO and wild type animals (n=3 for each tissue and each genotype). Black symbols are plotted on the left Y-axis, open symbols on the right Y-axis. MTO activities in the muscle and brain of the SELENBP1 KO animals were at or under the level of detection. Panel C, DMS levels in mouse plasma was determined from the SELENBP1 KO (n=5) as well as heterozygous (n=4) and wild type animals (n=3). For panels A, B and C, Boxes indicate the median and the 25 - 75 percentile, whiskers indicate the minimal and maximal values. Statistical analysis was performed with GraphPad Prism using a two-sided unpaired t test with Welch's correction. ***, p<0.001; **, p<0.01; *, p<0.05. Panels D and E, Representative one-dimensional ¹H-NMR spectroscopy of mouse plasma samples measured at pH 2.50. Plasma from a KO animal (D) and a wild type sample (E). The spectra illustrate the increased concentration of DMSO₂ in the SELENBP1 knock out animals.