A coupled enzyme assay for detection of selenium-binding protein 1 (SELENBP1) methanethiol oxidase (MTO) activity in mature enterocytes

Abstract

Methanethiol, a gas with the characteristic smell of rotten cabbage, is a product of microbial methionine degradation. In the human body, methanethiol originates primarily from bacteria residing in the lumen of the large intestine. Selenium-binding protein 1 (SELENBP1), a marker protein of mature enterocytes, has recently been identified as a methanethiol oxidase (MTO). It catalyzes the conversion of methanethiol to hydrogen sulfide (H₂S), hydrogen peroxide (H₂O₂) and formaldehyde. Here, human Caco-2 intestinal epithelial cells were subjected to enterocyte-like differentiation, followed by analysis of SELENBP1 levels and MTO activity. To that end, we established a novel coupled assay to assess MTO activity mimicking the proximity of microbiome and intestinal epithelial cells in vivo. The assay is based on in situ-generation of methanethiol as catalyzed by a bacterial recombinant l-methionine gamma-lyase (MGL), followed by detection of H₂S and H₂O₂. Applying this assay, we verified the loss and impairment of MTO function in SELENBP1 variants (His329Tyr; Gly225Trp) previously identified in individuals with familial extraoral halitosis. MTO activity was strongly enhanced in Caco-2 cells upon enterocyte differentiation, in parallel with increased SELENBP1 levels. This suggests that mature enterocytes located at the tip of colonic crypts are capable of eliminating microbiome-derived methanethiol.

Keywords: Caco-2 cells; Hydrogen peroxide; Hydrogen sulfide; Methanethiol oxidase; Selenium-binding protein 1.

Graphical abstract

Fig. 1

Coupled MTO assay, with in situ-generation of methanethiol through MGL-catalyzed methionine degradation. (A) MGL from Brevibacterium aurantiacum was epitope-tagged, expressed in E. coli and analyzed by immunoblotting (0.5 μg protein/lane) and staining of SDS-PAGE gels (2 μg protein/lane), respectively. (B) Reaction scheme of the coupled MTO assay. (C) Schematic layout of the MTO assay established here. (D) The MGL reaction releases methanethiol, as demonstrated by GC-MS-analysis. Extracted ion chromatogram (EIC) from the mass range 47.5–48.5, which corresponds to the molecular ion of methanethiol. Blue: reaction mixture containing MGL and Met (and PLP); red: reaction mixture without methionine. AU: arbitrary units. Bottom panel: corresponding mass spectrum of the MGL/Met reaction, which fits to the reported mass spectrum from the NIST-library (version 2016). (E) Methanethiol availability in MTO reaction wells, as assessed using DTNB. Blue: reaction mixture containing MGL and Met; red: reaction mixture without MGL. Data given as means ± SD of three independent measurements. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 2

MTO activity of SELENBP1 and two variants found in individuals with extraoral halitosis. (A) SELENBP1 (wild-type, WT) as well as SELENBP1(Gly225Trp) and SELENBP1(His329Tyr) were epitope-tagged, expressed in E. coli and analyzed by immunoblotting (0.5 μg protein/lane) and SDS-PAGE (2 μg protein/lane), respectively. (B) Detection of H₂S released during MTO reaction catalyzed by SELENBP1 (or its variants) as precipitates of PbS after interaction of H₂S with lead (II) acetate. Data shown are representative of three independent experiments. (C) H₂O₂ released during 3 h of SELENBP1-catalyzed methanethiol oxidation as detected through HRP-catalyzed oxidation of a fluorescent probe. Values were normalized for content of the respective SELENBP1 variant and expressed as relative values, with data for WT-SELENBP1 set to 1. Inset: H₂O₂ concentrations increase linearly for at least 3 h during the assay. Data are given as means ± SD of three independent measurements (inset, 30 min: two measurements ± maximum deviation).

Fig. 3

Both SELENBP1 levels and MTO activity increase in Caco-2 intestinal epithelial cells during differentiation. Caco-2 cells were cultured until reaching confluence (day 0) or until day 14 post confluence, followed by cell lysis. (A) SELENBP1, E-cadherin (as control for differentiation) and the housekeeping protein GAPDH (as control for equal loading) were detected in Caco-2 cell lysates by immunoblotting. (B, C) For the MTO assay, the soluble protein fraction of cell lysate (1 mg/ml) was incubated in the presence or absence of 1 mM of the catalase inhibitor 3-amino-1,2,4-triazole (AT). Samples were incubated with or without methionine (Met) as MGL substrate. (B) H₂S was detected through precipitation as lead sulfide (PbS) upon interaction with lead (II) acetate. (C) H₂O₂ was quantitated in an enzymatic assay using a fluorescent probe, with data given as means +SD of three biological replicates. Values were normalized relative to the respective methionine negative control. Right panel: Data are differences between MTO and Control treatments, normalized to the treatment at day 0 without AT. Data are significantly different from each other if no labeling letter (a, b, or c) is shared between groups (p < 0.05 as determined by ANOVA, followed by Student-Newman-Keuls test).

Fig. 4

Schematic representation of the proposed role of SELENBP1 as a methanethiol oxidase (MTO) in the colon. SELENBP1 (blue protein symbol) expression in intestinal epithelial cells increases along with their differentiation. Highest SELENBP1 levels are found in the epithelial cells closest to the colonic lumen [13] and the microbiome, a major source of methanethiol. Upon diffusion into the epithelial cells, methanethiol is converted by SELENBP1 to hydrogen peroxide, hydrogen sulfide and formaldehyde. The reaction products, in turn, may affect cellular proliferation and differentiation by acting as redox signaling molecules. Scheme created with BioRender.com. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)