S-Nitrosogluthathione reductase activity of amphioxus ADH3: insights into the nitric oxide metabolism

Abstract

Nitric oxide (NO) is a signalling molecule involved in many physiological functions. An important via of NO action is through the S-nitrosylation of proteins, a post-translational modification that regulates the activity of enzymes, protein-protein interactions and signal transduction pathways. Alcohol dehydrogenase class III (ADH3) recognises S-nitrosoglutathione (GSNO), the main reservoir of non-protein S-nitrosothiol, and functions as an effective GSNO reductase (GSNOR) and as a safeguard against nitrosative stress. To investigate the evolutionary conservation of this metabolic role, we have produced recombinant Branchiostoma floridae ADH3. Pure preparations of ADH3 showed 2-fold higher activity as GSNOR than as formaldehyde dehydrogenase, the previously assumed main role for ADH3. To correlate ADH3 expression in the gut with areas of NO production, we analysed the tissue distribution of the nitric oxide synthase (NOS) enzyme in amphioxus larvae. Immunostaining of the NOS enzyme revealed expression in the gut and in the dorsal region of the club-shaped gland. Co-localization in the gut supports the ADH3 and NOS joint contribution to the NO/SNO homeostasis.

Figure 1

Amphioxus total protein homogenate (1.0 mg) resolved on non-denaturing 7.5% polyacrylamide gel electrophoresis and stained for formaldehyde dehydrogenase (lane 1) and GSNO reductase (lane 2) activities. Coomassie stained SDS/PAGE of samples corresponding to the different purification steps of recombinant B. floridae ADH3: 40 μg of total protein homogenate (soluble fraction) from an E. coli expressing B. floridae ADH3 (lane 3); 16 μg of partially purified ADH3 eluted form a Talon Metal Affinity Resin (BD Biosciences Clontech, USA) after enterokinase cleavage (lane 4), and 2.3 μg of pure recombinant enzyme after Superdex200 FPLC column (lane 5). Pure recombinant protein (2.0 μg) was loaded onto non-denaturing 7.5% polyacrylamide gel electrophoresis and stained for formaldehyde dehydrogenase (lane 6) and GSNO reductase (lane 7) activity

Figure 2

(A). Comparison of the deduced amino acid sequences of B. floriade NOS (Bf NOS) with human (Hs nNOS, iNOS and eNOS) and Drosophila (Dm NOS) forms. Strictly conserved and highly conserved residues (>60%) are shown in black background, and similar residues in gray. Defined binding domains (as in 27) are overlined in red: cofactor-binding sites for heme (1), calmodulin (2), FMN (3), FAD pyrophosphate (4), FAD isoalloxazine (5), NADPH ribose (6), NADPH adenine (7) and the C-terminal conserved NADPH binding sequence (8). The sequence used for phylogenetic analyses begins at position D352 of human nNOS (*). (B) Phylogenetic reconstruction of NOS enzymes. Figures at nodes are the scores from 1000-bootstraps resampling of the data (NJ, in black) or quartet puzzling supports values (ML, in red). Ac, Aplysia californica, California sea hare; As, Anopheles stephensi; Bm, Bombix mori; Cc, Cyprinus carpio, carp; Dr, Danio rerio, zebrafish; Dm, Drosophila melanogaster; Gg, Gallus gallus; Hs, Homo sapiens; Ls, Lymnaea stagnalis, great pond snail; Ms, Manduca sexta, tobacco hornworm; Mm, Mus musculus; Om, Oncorhynchus mykiss, rainbow trout; Rr, Rattus norvergicus, Rp, Rhodnius prolixus, insect; Sp, Strongylocentrotus purpuratus, sea urchin, Tp, Takifugu poecilonotus; Xt, Xenopus tropicalis.

Figure 3

Whole-mount immunostaining of 48 h B. floridae larvae with an anti-NOS antibody. (A) Right-sided view. NOS signalling in the developing midgut and hindgut region separated by a non stained area, the ilio-colon ring (arrow), and in the dorsal region of the club-shaped gland (arrowhead). (B) Left-sided view. NOS signalling in the gut shows two non stained regions, which correspond to the ilio-colon ring (arrow) and to the lateral ciliated tract (double arrowhead). (C-E) Magnified view of the confocal sections of the right side, light and left side of the gut, respectively, showing left-right asymmetry in NOS localization. (F) Detail of the NOS staining in the dorsal region of the club-shaped gland (arrowhead). (G)Western blot analysis of amphioxus protein extracts. A total of 1.0 mg of soluble protein extract were resolved by electrophoresis on a SDS-7.5% polyacryla-mide gel, transferred to a polyvinylidene difluoride (PVDF) membrane and incubated with an anti-NOS antibody. Positions of molecular mass markers (in kDa) are shown on the left. (H) Adh3 expression visualized by whole-mount in situ hybridization as reported in .