Glutathione-conjugated sulfanylalkanols are substrates for ABCC11 and γ-glutamyl transferase 1: a potential new pathway for the formation of odorant precursors in the apocrine sweat gland

Abstract

We have previously shown that precursors of odorous components characteristic of axillary sweat are hardly detectable or undetectable in individuals carrying the 538G > A SNP in the ABCC11 transporter gene. However, it is unclear, whether ABCC11 is directly involved in the transport of these compounds. To approach this question, transport of peptide-conjugated potential precursors of 3-methyl-3-sulfanylhexanol (3M3SH), a key determinant of axillary malodour, was measured using membrane vesicles of Sf9 insect cells overexpressing human ABCC11. Whilst no ABCC11-mediated transport was detected for the dipeptide precursor Cys-Gly-3M3SH, the glutathione conjugate of 3M3SH (SG-3M3SH) was robustly taken up by ABCC11 at a transport rate of 0.47 pmol/mg/min. Collectively, these results illuminate SG-3M3SH as a putative precursor of 3M3SH, which then may undergo intra-vesicular maturation to generate Cys-Gly-3M3SH. Critically, the apocrine sweat gland was demonstrated to express γ-glutamyl transferase 1 (GGT1) protein, which is known to catalyse the deglutamylation of glutathionyl conjugates. Additionally, we provide evidence that recombinant and isolated hepatic human GGT1 is capable of transforming SG-3M3SH to Cys-Gly-3M3SH in vitro. To sum up, we demonstrate that the functionality of ABCC11 is likely to play an important role in the generation of axillary malodour. Furthermore, we identify GGT1 as a key enzyme involved in the biosynthesis of Cys-Gly-3M3SH.

Keywords: ABCC11; apocrine sweat; odour precursors.

Figure 1

Cys-Gly- (a) and SG-3M3SH (b) uptake by ABCC11 vesicles. Time course of ATP-dependent uptake of [³H]-Cys-Gly-3M3SH (a) and [³H]-SG-3M3SH (b) into membrane vesicles prepared from Sf9 cells transfected with ABCC11 (squares) and control Sf9 cells (cross), respectively. Vesicles were incubated at 37°C in uptake buffer containing 100 nm of substrate. The values shown represent means ± SD; each experiment was performed in triplicates.

Figure 2

Inhibition of ABCC11-mediated uptake of SG-3M3SH by MK571. Uptake of SG-3M3SH into ABCC11 vesicles was measured in presence and absence (DMSO vehicle control) of the inhibitor MK571 at a concentration of 10 μm. The values represent means ± SD; each experiment was performed in triplicate. The relative transport of the control without inhibitor was set at 100%.

Figure 3

Immunolocalization of γ-glutamyl transferase 1 (GGT1) protein in apocrine sweat glands. (a) Overview of apocrine sweat gland. The GGT1 signal can be detected in secretory cells, but not in myoepithelial cells or the connective tissue of the dermis. Scale bar corresponds to 100 μm. (b) Magnification of the secretory coil. GGT1 signal is restricted to the apical portion of secretory cells (indicated by arrow). Scale bar corresponds to 25 μm. L: lumen; green signal: specific staining of GGT1; blue signal: DAPI staining of the nuclei.

Figure 4

Mass spectra data showing metabolism of SG-3M3SH by human and bovine γ-glutamyl transferase 1 (GGT1). (a) Sample containing SG-3M3SH and human GGT1 shows Cys-Gly3M3SH formation after reaction time. At the same time, SG-3M3SH is not detectable any more. (b) In the reaction batch containing SG-3M3SH and bovine GGT, generation of Cys-Gly-3M3SH cannot be observed. SG-3M3SH detection is unchanged after the reaction time. (c) In the sample containing SG-3M3SH without enzymes, Cys-Gly-3M3SH cannot be detected whereas SG-3M3SH is still present. Displayed details of the mass spectra are focused on the detection of the specific mass signals for Cys-Gly-3M3SH and SG-3M3H (small boxes included in the respective mass spectra). Cys-Gly-3M3SH (m/z 291.17 [M-H]⁻) was detected in negative ion mode using DMAN as the matrix. The presence of SG-3M3SH was investigated by applying α-CHCA matrix; hence, achieving detection as [M+H]⁺ adduct (m/z 422.19).