Molecular cloning and characterization of Hsp27.6: the first reported small heat shock protein from Apis cerana cerana

Abstract

Small heat shock proteins (sHSPs) play an important role in the cellular defense of prokaryotic and eukaryotic organisms against a variety of internal and external stressors. In this study, a cDNA clone encoding a member of the α-crystallin/sHSP family, termed AccHsp27.6, was isolated from Apis cerana cerana. The full-length cDNA is 1,014 bp in length and contains a 708-bp open reading frame encoding a protein of 236 amino acids with a calculated molecular weight of 27.6 kDa and an isoelectric point of 7.53. Seven putative heat shock elements and three NF-κB binding sites were present in the 5'-flanking region, suggesting a possible function in immunity. A semi-quantitative RT-PCR analysis indicated that AccHsp27.6 was expressed in all tested tissues and at different developmental stages. Furthermore, expression of the AccHsp27.6 transcript was induced by exposure to heat shock, H(2)O(2), a number of different chemicals (including SO(2), formaldehyde, alcohol, acetone, chloroform, and the pesticides phoxime and acetamiprid), and the microbes Staphylococcus aureus and Micrococcus luteus. In contrast, the mRNA expression could be repressed by CO(2), the pesticides pyriproxyfen and cyhalothrin, and the microbes Bacillus subtilis and Pseudomonas aeruginosa. Notably, the recombinant AccHsp27.6 protein exhibited significant in vitro molecular chaperone activity and antimicrobial activity. Taken together, these results suggest that AccHsp27.6 might play an important role in the response to abiotic and biotic stresses and in immune reactions.

Fig. 1

Nucleotide sequence of AccHsp27.6 and primers. The cDNA sequence is indicated on the top line, and the names of the primers and their corresponding orientations are shown on the second line

Fig. 2

The tertiary structure of α-crystallin domain residues 67–157

Fig. 3

Amino acid sequence comparison of sHSP homologs from Apis cerana cerana, Macrocentrus cingulum, and Nasonia vitripennis. The asterisks, double dots, and single dots denote fully, strongly, and weakly conserved residues, respectively. The conserved regions are shown in gray. The α-crystallin domain is underlined. The degree of identity relative to AccHsp27.6 is shown at the end of each sequence. The secondary structure assignment of the AccHsp27.6 is shown above the sequence

Fig. 4

The nucleotide sequence and putative transcription factor binding sites of the 5′-flanking region of AccHsp27.6. The transcription and translation start sites are indicated with an arrowhead. The underlined sections indicate the putative NF-κB binding sites, and the boxes indicate the putative HSEs. The TATA-box is highlighted in light gray

Fig. 5

Phylogenetic analysis of sHSP amino acid sequences from different insect species (Diptera, Hymenoptera, and Lepidoptera). The full names of species and the accession numbers of the genes are designated by the following abbreviations: DmHsp26 (Drosophila melanogaster, NM079273), DmHsp27 (Drosophila melanogaster, NM079276), DmCG14207 (Drosophila melanogaster, NM134482), LhHsp20 (Liriomyza huidobrensis, DQ452370), LsHsp21.7 (Liriomyza sativae, DQ452372), McsHsp (Macrocentrus cingulum, EU624206), NvHsp21.7 (Nasonia vitripennis, XP001604512), NvHsp20.6 (Nasonia vitripennis, XM001607625), AccHsp27.6 (Apis cerana cerana, GQ254650), BmHsp20.1 (Bombyx mori, AB195971), BmHsp23.7 (Bombyx mori, AB195973), BmHsp20.4 (Bombyx mori, AF315318), BmHsp22.6 (Bombyx mori, FJ602788), BmHsp21.4 (Bombyx mori, AB195972), AgHsp20.9 (Anopheles gambiae, XM560153), TcHsp21.8 (Tribolium castaneum, XM968592), AmHsp25.6 (Apis mellifera, XM392405), LmHsp20.6 (Locusta miratoria, DQ355964)

Fig. 6

Expression patterns of AccHsp27.6 at different developmental stages and in various tissues. a Expression patterns of AccHsp27.6 at different developmental stages. Total RNA was isolated from larvae (2d second instars, 4d fourth instars, 5d fifth instars, 6d sixth instars), pupae (pw white-eyed pupae, pp pink-eyed pupae, pd dark-eyed pupae), and adults (a1 1 day post-emergence adults, a10 10 day post-emergence adults). The transcripts of AccHsp27.6 were detected throughout the entire development process. b Expression patterns of AccHsp27.6 in various tissues. Total RNA was extracted from the brain, thorax, and abdomen of the adult bees (12 day) at the indicated times, and semi-quantitative RT-PCR was used to examined the transcript level of AccHsp27.6 under different treatments. The β-actin gene was used as an internal control. The histograms indicate the relative expression levels. Values on the y-axis are the normalization units relative to β-actin levels. In addition, the values on the x-axis represent the lanes. The signal intensities (INT/mm²) of the bands were assigned using the Quantity-One™ image analysis software implemented in VersaDoc 4000 (Bio-Rad). Each value is given as the mean (SD) of three replicates

Fig. 7

Expression profiles of AccHsp27.6 under abiotic stress. Total RNA was extracted from adult bees (12 day) treated at the indicated times with heat (a), UV irradiation (b), H₂O₂ (c), and pesticides (d Pyriproxyfen, e Cyhalothrin, f Phoxime, g Acetamiprid) and hazardous chemicals (h). Semi-quantitative RT-PCR was used to examine the transcript levels of AccHsp27.6 under different treatments. FA formaldehyde, alc alcohol, acet acetone, chl chloroform. The β-actin gene was used as an internal control. The line CK indicates the control. The histograms show the relative expression levels. The values on the y-axis are the normalization units relative to β-actin levels. In addition, the values on the x-axis represent the lanes. The signal intensities (INT/mm²) of the bands were assigned using the Quantity-One™ image analysis software implemented in VersaDoc 4000 (Bio-Rad). Each value is given as the mean (SD) of three replicates

Fig. 8

Expression profiles of AccHsp27.6 in response to microbe infection. SA Staphylococcus aureus, ML Micrococcus luteus, BS Bacillus subtilis, PA Pseudomonas aeruginosa. The 3-day-old larvae fed in the 24-well plates were inoculated with microbes and maintained until they were 6 days old. Total RNA was extracted at the indicated times, and semi-quantitative RT-PCR was used to examine the transcript levels of AccHsp27.6. The β-actin gene was used as an internal control. The line CK indicates the control. The histograms show the relative expression levels. The values on the y-axis are the normalization units relative to β-actin levels. In addition, the values on the x-axis represent the lanes. The signal intensities (INT/mm²) of the bands were assigned using the Quantity-One™ image analysis software implemented in VersaDoc 4000 (Bio-Rad). Each value is given as the mean (SD) of three replicates

Fig. 9

Expression in E. coli and purification of the AccHsp27.6 recombinant protein as analyzed by SDS-PAGE. Lane 1 purified AccHsp27.6 recombinant protein; lane 2 non-induced; lane 3 induced; lane M molecular mass standard

Fig. 10

Molecular chaperone activity of AccHsp27.6 shown by the malate dehydrogenase thermal aggregation assay. MDH was heated at 43°C (filled diamond), mixed with AccHsp27.6 in a molar ratio 1:1 (filled square), or mixed with BSA (1:1; filled circle). Absorbance was monitored at 360 nm at regular intervals (10 min) for 1 h

Fig. 11

Antimicrobial activity of AccHsp27.6 shown by the disk diffusion method. aStaphylococcus aureus. bMicrococcus luteus. cBacillus subtilis. dPseudomonas aeruginosa. 1 twofold dilution of the AccHsp27.6 protein solution; 2 fivefold dilution of the AccHsp27.6 protein solution; 3 tenfold dilution of the AccHsp27.6 protein solution; 4 positive control (kanamycin, 250 mg/ml); 5 negative control (the total protein from BL21 (DE3) E. coli, 0.5 mg/ml); 6 PBS; 7 the original concentrated solution of purified AccHsp27.6 protein (0.5 mg/ml)