Human METTL7B is an alkyl thiol methyltransferase that metabolizes hydrogen sulfide and captopril

Abstract

Methylation of alkyl thiols is a biotransformation pathway designed to reduce thiol reactivity and potential toxicity, yet the gene and protein responsible for human alkyl thiol methyltransferase (TMT) activity remain unknown. Here we demonstrate with a range of experimental approaches using cell lines, in vitro systems, and recombinantly expressed enzyme, that human methyltransferase-like protein 7B (METTL7B) catalyzes the transfer of a methyl group from S-adenosyl-L-methionine (AdoMet) to hydrogen sulfide (H₂S) and other exogenous thiol small molecules. METTL7B gene modulation experiments, including knockdown in HepG2 cells and overexpression in HeLa cells, directly alter the methylation of the drug captopril, a historic probe substrate for TMT activity. Furthermore, recombinantly expressed and purified wild-type METTL7B methylates several thiol compounds, including H₂S, 7α-thiospironolactone, L-penicillamine, and captopril, in a time- and concentration-dependent manner. Typical for AdoMet-dependent small molecule methyltransferases, S-adenosyl-L-homocysteine (AdoHcy) inhibited METTL7B activity in a competitive fashion. Similarly, mutating a conserved aspartate residue, proposed to anchor AdoMet into the active site, to an alanine (D98A) abolished methylation activity. Endogenous thiols such as glutathione and cysteine, or classic substrates for other known small molecule S-, N-, and O-methyltransferases, were not substrates for METTL7B. Our results confirm, for the first time, that METTL7B, a gene implicated in multiple disease states including rheumatoid arthritis and breast cancer, encodes a protein that methylates small molecule alkyl thiols. Identifying the catalytic function of METTL7B will enable future pharmacological research in disease pathophysiology where altered METTL7B expression and, potentially H₂S levels, can disrupt cell growth and redox state.

Figure 1

Knockdown of METTL7B gene expression in HepG2 cells: (A) RT-PCR data measuring METTL7B gene expression following treatment with METTL7B siRNA for 72 h compared to scramble treated control. (B) HepG2 S-methyl captopril formation following 24 h incubation in cells treated with METTL7B siRNA (72 h) compared to scramble control. All data is presented as the mean ± s.d. Individual data points are plotted from two (A) or three (B) separate experiments. Significance was determined using unpaired two-tailed t test. ****P < 0.0001.

Figure 2

Overexpression of the METTL7B gene in HeLa cells: (A) RT-PCR data measuring METTL7B gene expression in HeLa cells treated with a METTL7B overexpression plasmid compared to control cells transfected with an empty expression vector. (B) S-methyl captopril measured 24 h following the addition of captopril (500 µM) to HeLa cells treated with METTL7B overexpression plasmid compared to control cells. (C) FLAG-tagged METTL7B expression is only observed in HeLa cells treated with the METTL7B overexpression plasmid and not in controls. Western blot using an anti-FLAG antibody confirmed the expression of METTL7B in HeLa cells. Full gel images are available in Supplementary Fig. 1. All data is presented as the mean ± s.d. Individual data points are plotted from two separate experiments. Significance was determined using unpaired two-tailed t test. ****P < 0.0001.

Figure 3

Purified His-GST-METTL7B: (Lane 1) SDS-PAGE silver stain of a representative gel showing purified His-GST-METTL7B (A). Gel lanes were loaded with a 1 µg total protein as determined by A_280. (Lane 2) Anti-GST antibody western blot of purified His-GST-METTL7B (1 µg total protein). (Lane 3) Western blot using anti-METTL7B antibody of purified His-GST-METTL7B (A) using 0.1 µg total protein. Molecular weight markers are shown to the left. The lower molecular bands, marked by letters B and C, are fusion protein fragments containing the dual His-GST affinity tag.

Figure 4

SDS-PAGE and western blot analysis of purified His-GST-METTL7B-D98A: (Lane 1) SDS-PAGE silver stain of a representative gel showing purified His-GST-METTL7B-D98A (A) (4.2 µg total protein) (Lane 2) Anti-GST antibody western blot of purified His-GST-METTL7B-D98A (3 µg total protein). (Lane 3) Anti-METTL7B antibody western blot of purified His-GST-METTL7B-D98A (4.3 µg total protein). Molecular weight markers are shown to the left. Upper bands, above 55 kDa, are co-purifying chaperone proteins as determined by proteomic analysis (data not shown). Lower molecular bands, marked by letters B and C, are fusion protein fragments containing the dual His-GST affinity tag.

Figure 5

Thiol methyltransferase activity of purified His-GST-METTL7B and His-GST-METTL7B-D98A: Peak area of S-methyl captopril normalized to internal standard (PAR) at two substrate concentrations for both wild-type METTL7B (His-GST-METTL7B) and METTL7B containing a D98A point mutation (His-GST-METTL7B-D98A). Samples were protein-normalized to 0.15 mg/mL via A_280. Data is presented as the mean ± s.d. Significance was determined using unpaired two-tailed t test. ****P < 0.0001.

Figure 6

Captopril methylation by His-GST-METTL7B at various AdoMet concentrations in the presence or absence of AdoHcy: (A) His-GST-METTL7B catalyzed AdoMet-dependent methylation of captopril exhibits saturable Michaelis–Menten kinetics. Peak area ratio of methyl captopril to internal standard (PAR) versus concentration of AdoMet. (B) Inhibition of His-GST-METTL7B captopril methylation by AdoHcy. PAR versus AdoMet concentration curve at varying concentrations of AdoHcy. Data presented as mean ± s.d. n = 3 for (A) and average ± error (n = 2) for (B).

Figure 7

Semi-quantitative screening of select small molecule methyltransferase probe substrates. Multiple methyltransferase probe substrates were incubated for 1 h with His-GST-METTL7B. Formation of AdoHcy (indication of methyl transfer) was measured using the Promega MTaseGlo kit. Activity was normalized to dopamine, a catechol O-methyltransferase probe that showed no activity. All data is presented as the mean ± s.d. Significance was determined using unpaired two-tailed t test. ****P < 0.0001. ***P < 0.001. **P < 0.01.

Figure 8

Rate of thiol methylation for His-GST-METTL7B with multiple substrates: (A) Luminescence versus concentration curves for 7α-thiospironolactone methylation as measured by AdoHcy formation. (B) Luminescence versus concentration curve for dithiothreitol methylation as measured by AdoHcy formation. (C) Peak area ratio (PAR) versus concentration curve for hydrogen sulfide methylation as measured by formation of methylsulfide. All data is presented as the mean ± s.d.