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 (H 2 S), hydrogen peroxide (H 2 O 2 ) 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 H 2 S and H 2 O 2 . 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.

SEMO‐1, a novel methanethiol oxidase in Caenorhabditis elegans, is a pro‐aging factor conferring selective stress resistance

“…We employed a coupled enzyme assay detecting MTO activity, 17 analyzing two of the three products of the MTO reaction: H 2 S is detected through precipitation as lead sulfide (PbS), while H 2 O 2 formation is assessed fluorimetrically using peroxidase (Figure 1E). Using this assay, we recently measured MTO activity of recombinant human SELENBP1 as well as in lysates from Caco‐2 cells, a human colorectal adenocarcinoma cell line 17 . We here demonstrate that wild type (N2) C. elegans lysates harbor significant MTO activity, whereas production of the MTO product H 2 S is drastically diminished in the semo‐1 knockout strain (Figure 1F).…”

“…To provide direct evidence for MTO activity of SEMO‐1, we cloned the semo‐1 coding sequence from wild type C. elegans (N2) and overexpressed it in E. coli as a recombinant protein. As positive control, human SELENBP1 was produced accordingly and as previously described 17 (Figure 2A). SEMO‐1 showed MTO activity, which was even higher than with its human ortholog (Figure 2B).…”

“…SEMO‐1 is a methanethiol oxidase. (A, C) SELENBP1 and SEMO‐1 as well as two SEMO‐1 mutants (His331Tyr, Gly223Trp) were produced in E. coli as recombinant proteins with a C‐terminal StrepTag 17 . Following SDS‐PAGE, proteins were detected by immunoblotting as well as Coomassie staining.…”

“…SDS‐PAGE and immunoblotting were performed for the detection of purified recombinant proteins as previously described 17 . The following antibodies were used: mouse monoclonal antibody anti‐Strep‐Tag II/StrepMAB (IBA Lifesciences) and horseradish peroxidase (HRP)‐coupled anti‐mouse IgG (Thermo Fisher Scientific).…”

“…17 Bacterial overexpression and isolation of the recombinant proteins also occurred as previously described. 17…”