Fig 1.
Functional classification of the total rat nasal mucus.
The 211 proteins identified with more than one peptide were classified into 13 functional categories. For each category, a percentage is indicated. Each protein is counted in only one category.
Fig 2.
Sequence alignment of the mu class of rat GSTs.
The numbering of the amino acid residues is based on the GSTM5 sequence. The sequences were aligned with Bioedit software. The amino acids are colored based on their physicochemical properties. The catalytic Tyr residue is indicated in black and highlighted in yellow. The accession numbers for GSTM1, 2, 3, 4, 5 and 6 are P04905, P08010, P08009, B29231, Q9Z1B2 and A0A0G2K6L4, respectively.
Table 1.
Identification of rat nasal mucus enzymes involved in detoxification.
Table 2.
Amino acid sequence identity within the GSTs of the mu class.
Fig 3.
Western blot analysis of GSTM2 in rat OE.
(A) GSTs of the mu class were detected in two fractions of the supernatant of the OE lysate (extract S1 for lane 1 and extract S2 for lane 2) and in the pellet of the OE lysate (extract C for lane 3). Lanes 4 and 5 correspond to the nasal mucus and the purified recombinant GSTM2 (100 ng) from the rat, respectively. The molecular weight markers are indicated on the left of the gels in kDa. (B) Western blot analysis using the GSTM2 antibody shows the absence of the detection of recombinant GSTA1 (100 ng) and GSTP1 (100 ng) in lanes 1 and 2 but the detection of human recombinant GSTM1 (100 ng) and rat recombinant GSTM2 (100 ng) in lanes 3 and 4, respectively.
Fig 4.
Expression analysis of rat GSTs of the mu and alpha classes.
(A) Western blot analysis of rat GST expression using antibodies against GSTM1, (B) GSTM4 and (C) GSTA1. The different blots include two fractions of the supernatant of the OE lysate (extract S1 for lane 1 and extract S2 for lane 2, all panels), the pellet of the OE lysate (extract C for lane 3, all panels), the nasal mucus (lane 4) and the purified recombinant rat GSTM2 (100 ng) (lane 5, panel A and B) or the purified recombinant human GSTA1 (100 ng) (lane 5, panel A). The molecular weight markers are indicated on the left of the gels in kDa.
Fig 5.
Expression of GSTs of the mu class in the rat nasal cavity.
Distribution of the mu class GST immunoreactivity using an anti-GSTM2 antibody. The two sections of the (A) frontal and (B) transverse views of the rat nasal cavity are represented on the rat schema. Ectoturbinates (Ec), septum (S) and endoturbinates (En) are indicated. The scale bar is 2300 μm.
Fig 6.
Immunohistochemistry analysis of mu class GST expression.
Distribution of the mu class GST immunoreactivity using an anti-GSTM2 antibody. (A, B, C and D) Different details showing the olfactory (OR) and respiratory epithelium (RE) and different cell types and structures, including the olfactory sensory neurons (OSN), sustentacular cells (SC), basal cells (BC), nerve bundle (NB), Bowman’s glands (BG), and Bowman’s gland duct (BGD). Panel D is a higher magnification of panel C, showing the nasal olfactory mucus (OM). The scale bar is 50 μm for panels A, B and F, 100 μm for panel E, 200 μm for panel D and 500 μm for panel C.
Fig 7.
Immunohistochemistry analysis of the expression of the mu and alpha class GSTs.
Histological expression pattern of mu class GSTs using anti-GSTM1 (A), GSTM4 (B) and GSTA1 (C) antibodies. The scale bar is 2300 μm for the three panels.
Fig 8.
Measure of the inhibition rates of odorant molecules toward GSTM2 activity.
Inhibition of recombinant rat GSTM2 by 20 odorant molecules. The inhibition rates for rat GSTM2 acting on CDNB were calculated at a concentration of 1 mM CDNB and glutathione in the presence of 10 μM or 100 μM odorant (light and dark gray colors, respectively). The error bars show the standard deviation and were calculated from three independent experiments. Statistical analysis was performed using a bilateral Student’s t-test. Asterisks indicate statistical significance (*P < 0.05 and **P < 0.01). The best inhibitors are indicated by the highest inhibition rates.
Fig 9.
Formation of the odorant glutathione conjugation.
Enzymatic glutathione conjugation was measured using high-performance liquid chromatography in the presence of different odorants, glutathione and the recombinant rat GSTM2. To determine the enzymatic glutathione conjugation, the non-enzymatic conjugation was subtracted. The error bars show the standard deviation and were calculated from three independent experiments. Statistical analysis was performed using a bilateral Student’s t-test. Asterisks indicate statistical significance (*P < 0.05 and **P < 0.01).
Table 3.
Enzymatic and spontaneous glutathione conjugation of odorants.