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Biol Res
2018 Mar 27;511:8. doi: 10.1186/s40659-018-0156-9.
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HSP70 from the Antarctic sea urchin Sterechinus neumayeri: molecular characterization and expression in response to heat stress.
González-Aravena M
,
Calfio C
,
Mercado L
,
Morales-Lange B
,
Bethke J
,
De Lorgeril J
,
Cárdenas CA
.
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BACKGROUND: Heat stress proteins are implicated in stabilizing and refolding denatured proteins in vertebrates and invertebrates. Members of the Hsp70 gene family comprise the cognate heat shock protein (Hsc70) and inducible heat shock protein (Hsp70). However, the cDNA sequence and the expression of Hsp70 in the Antarctic sea urchin are unknown.
METHODS: We amplified and cloned a transcript sequence of 1991 bp from the Antarctic sea urchin Sterechinus neumayeri, experimentally exposed to heat stress (5 and 10 °C for 1, 24 and 48 h). RACE-PCR and qPCR were employed to determine Hsp70 gene expression, while western blot and ELISA methods were used to determine protein expression.
RESULTS: The sequence obtained from S. neumayeri showed high identity with Hsp70 members. Several Hsp70 family features were identified in the deduced amino acid sequence and they indicate that the isolated Hsp70 is related to the cognate heat shock protein type. The corresponding 70 kDa protein, called Sn-Hsp70, was immune detected in the coelomocytes and the digestive tract of S. neumayeri using a monospecific polyclonal antibody. We showed that S. neumayeri do not respond to acute heat stress by up-regulation of Sn-Hsp70 at transcript and protein level. Furthermore, the Sn-Hsp70 protein expression was not induced in the digestive tract.
CONCLUSIONS: Our results provide the first molecular evidence that Sn-Hsp70 is expressed constitutively and is non-induced by heat stress in S. neumayeri.
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29587857
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1131001 Fondo Nacional de Desarrollo Científico y Tecnológico
Fig. 1. Schematic representation of cognate heat shock protein and multiple alignment of the Sn-Hsp70 with those other cognate and inducible Hsp70 sequences. Blue boxes represent different signatures of Hsp70. Green box represent the ATP/GTP binding site. Red box represent the nuclear localization signal. Grey box represent a multiple tetrapeptide GGMP motif. Purple box represent an EEVD motif. Sequences highlighted in gray indicate conserved residues. Accession Numbers: Strongylocentrotus purpuratus: XP 802057; Oncorhynchus mykiss: P08108; Bos taurus: P19120; Crassostrea ariakensis: AAO41703; Crassostrea gigas: AAD31042; Mus musculus: AAA37859; Bos Taurus: NP776769; Crassostrea virginica: CAB89802; Crassostrea gigas: BAD15286
Fig. 2. Phylogenetic relationships among Hsp70 family members. The tree was constructed by Neighbour-Joining method. Numbers at each branch indicate the percentage of bootstrap values after 1000 replications. Scale bar represent the amino acid substitutions per site for a unit branch length
Fig. 3. Expression analysis of Sn-Hsp70 mRNA. a Expression of Hsp70 in tissue by RT-PCR. b
Sn-Hsp70 mRNA expression was measured by qPRC in coelomocytes following heat stress. Results are mean ± SE of three independent experiments carried out on a pool of four sea urchins from control group at 0.5 °C (black) and thermal stressed exposed to 5 °C (white) and 10 °C (grey) during 1, 24 and 48 h. The housekeeping gene used in the qPCR was the 28S rRNA. The primers efficiency was 112 and 109% for 28S rRNA and Sn-Hsp70, respectively
Fig. 4. Protein detection of Sn-Hsp70 in S. neumayeri. a Validation of anti-Hsp70. Line 1: SDS-PAGE proteins profile of coelomocytes lysates stained with coomassie blue. Line 2: SDS-PAGE proteins profile stained with silver. Line 3: western blot for antibody validation show only a specific band (arrow) in the expected molecular weight (70 kDa), the antibody not present nonspecific recognition in other molecular weights. b Western blot to detect Sn-Hsp-70 in coelomocytes during the acute heat stress. c Indirect ELISA detection by Optical density to 450 nm of Sn-Hsp-70 protein in coelomocytes and digestive tract. Differences in Sn-Hsp70 detection are not significant by ELISA (P < 0.05)
Fig. 5. Confocal images of Sn-Hsp70 proteins in coelomocytes. a Coelomocytes labeled for Hsp70 proteins were expressed in large coelomocytes with an eccentric nucleus. Bars = 50 μm. b The positive coelomocytes have intense labeling of the cytoplasm. Bars = 10 μm. Circulating coelomocytes were labeled for DNA (red) and Hsp70 (green)
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