Identification and Characterization of Aldehyde Oxidase 5 in the Pheromone Gland of the Silkworm (Lepidoptera: Bombycidae)

Abstract

Aldehyde oxidases (AOXs) are a subfamily of cytosolic molybdo-flavoenzymes that play critical roles in the detoxification and degradation of chemicals. Active AOXs, such as AOX1 and AOX2, have been identified and functionally analyzed in insect antennae but are rarely reported in other tissues. This is the first study to isolate and characterize the cDNA that encodes aldehyde oxidase 5 (BmAOX5) in the pheromone gland (PG) of the silkworm, Bombyx mori. The size of BmAOX5 cDNA is 3,741 nucleotides and includes an open reading frame, which encodes a protein of 1,246 amino acid residues. The theoretical molecular weight and isoelectric point of BmAOX5 are approximately 138 kDa and 5.58, respectively. BmAOX5 shares a similar primary structure with BmAOX1 and BmAOX2, containing two [2Fe-2S] redox centers, a FAD-binding domain, and a molybdenum cofactor (MoCo)-binding domain. RT-PCR revealed BmAOX5 to be particularly highly expressed in the PG (including ovipositor) of the female silkworm moth, and the expression was further confirmed by in situ hybridization, AOX activity staining, and anti-BmAOX5 western blotting. Further, BmAOX5 was shown to metabolize aromatic aldehydes, such as benzaldehyde, salicylaldehyde, and vanillic aldehyde, and fatty aldehydes, such as heptaldehyde and propionaldehyde. The maximum reaction rate (Vmax) of benzaldehyde as substrate was 21 mU and Km was 1.745 mmol/liter. These results suggested that BmAOX5 in the PG could metabolize aldehydes in the cytoplasm for detoxification or participate in the degradation of aldehyde pheromone substances and odorant compounds to identify mating partners and locate suitable spawning sites.

Keywords: Bombyx mori; activity staining; aldehyde oxidase; identification; pheromone gland.

Fig. 1.

Sequence alignment analysis of BmAOX5. Overline, [2Fe-2S] redox centers. *, Cysteine residues. Short overline, FAD-binding domain. Dashed overline, MoCo-binding domain. The NAD-binding site typical of XDHs is boxed. The background color of identical residues is black; the background color of positive residues is light gray; the background color of no positive residues is white. BmAOX1, B. mori aldehyde oxidase 1 (NP_001103812); BmAOX2, B. mori aldehyde oxidase 2 (NP_001103811); BmXDH1, B. mori xanthine dehydrogenase 1 (BAA21640); BmXDH2, B. mori xanthine dehydrogenase 2 (BAA24290); BmAOX5, B. mori aldehyde oxidase 5 (AQY62686).

Fig. 2.

Analysis of BmAOX5 expression and localization in the PG of silkworm. (A) Transcription patterns of BmAOX5 detected in female silkworm moth tissues by RT–PCR. M, DL2000; HE & AN, head and antennae; TH & WI, thorax and wings; AB, abdomen; FB, fat body; PG, pheromone gland with ovipositor; OV, ovaries (B) Expression patterns of BmAOX5 detected in the PG during different stages by RT–PCR. M, DL2000; Mne, moth after egg laying without mating; Mme, moth after egg laying with mating; Mm, moth after mating without egg laying; M3, moth day 3 without mating; M2, moth day 2 without mating; M1, moth day 1 without mating; P7, pupa day 7; P6, pupa day 6; P5, pupa day 5. Amplification of the BmActin3 gene was used as an endogenous control. (C) Anatomy of the female silkworm moth. Red arrow indicates the PG. (D) Localization of BmAOX5 transcripts were detected in the PG by in situ hybridization. No RNA probes and BmAOX5 sense RNA probes were used as controls for BmAOX5 ant-sense RNA probes. Scale bar = 300 μm. OT, oviposition tube.

Fig. 3.

Activity determination of BmAOX5 in silkworm PG. (A) Detection of AOX proteins in silkworm PG during different stages by native-PAGE and activity staining. Mne, moth after egg laying without mating; Mme, moth after egg laying with mating; Mm, moth after mating without egg laying; M3, moth day 3 without mating; M2, moth day 2 without mating; M1, moth day 1 without mating; P7, pupa day 7; P6, pupa day 6; P5, pupa day 5. (B) Stability detection of BmAOX in the PG by native-PAGE and activity staining. HA, antenna; PG, pheromone gland (including ovipositor). (C) Detection of BmAOX5 overexpressed in E. coli by SDS–PAGE and CBB staining. (D) Identification of BmAOX5 antibody by SDS–PAGE and western-blotting. M, standard protein marker; pET28a, extracts of E. coli transformed with pET28a empty vector as control; 28a-gene, extracts of E. coli transformed with pET28a vector with overexpression of another BmAOX gene as control; 28a-AOX5, extracts of E. coli transformed with pET28a vector with overexpression of BmAOX5 gene. (E) Activity identification of BmAOX5 in the PG by native-PAGE and activity staining. (F) Protein identification of BmAOX5 by native-PAGE and western-blotting. OV, ovaries as control. Samples for B, E, F were tissues of female silkworm moth day 1 without mating and stored at −80°C before extracting proteins.

Fig. 4.

Activity detection and enzymatic reaction kinetic analysis of BmAOX5. (A) Activity detection of BmAOX5 with different substrates by native-PAGE and AOX activity staining. BzH, benzaldehyde; SIH, salicylaldehyde; Van, vanillic aldehyde; HIH, heptaldehyde; AIH, propionaldehyde; FS, frozen PG. If FS is not marked, it means the PG was fresh, not stored in low temperature. (B) Enzymatic reaction curve of BmAOX5 substrate saturation. (C) Michaelis–Menten equation graph of BmAOX5 catalytic reaction with benzaldehyde as substrate. The formation of insoluble MTT formazan was measured at 560 nm. One OD/min representatives 1 unit of enzyme activity (1U), in the process of catalytic reaction. The concentration of benzaldehyde as a substrate follows: 1, 1.3, 1.6, 2, 2.5, 3.5, 5, 6, 8, and 10 mmol/liter. The OD value of each concentration is the average value of three independent experiments.