Post-diapause synthesis of ArHsp40-2, a type 2 J-domain protein from Artemia franciscana, is developmentally regulated and induced by stress

Post-diapause cysts of Artemia franciscana undergo a well-defined developmental process whereby internal differentiation leads to rupture of the cyst shell, release of membrane-enclosed nauplii and hatching to yield swimming larvae. The post-diapause development of A. franciscana has been examined at biochemical and molecular levels, yet little is known about molecular chaperone function during this process. In addressing this we recently described ArHsp40, a type 1 J-domain protein in post-diapause A. franciscana cysts and larvae. The current report describes ArHsp40-2, a second J-domain protein from A. franciscana. ArHsp40-2 is a type 2 J-domain protein, lacking a zinc binding domain but containing other domains characteristic of these proteins. Notably, ArHsp40-2 possesses a double barrel β-domain structure in its substrate binding region, as does ArHsp40. qPCR revealed a relatively low amount of ArHsp40-2 mRNA in 0 h cysts which increased significantly until the E1 stage, most likely as a result of enhanced transcription, after which it declined. An antibody specific to ArHsp40-2 was produced and used to show that like its mRNA, ArHsp40-2 accumulated until the E1 stage and then decreased to amounts lower than those in 0 h cysts. The synthesis of ArHsp40-2 was induced by heat shock indicating that ArHsp40-2 is involved in stress resistance in cysts and nauplii. Accumulation in cysts during early post-diapause development followed by its sharp decline suggests a role in protein disaggregation/refolding, a function of Hsp40s from other organisms, where ArHsp40-2 assists in the rescue of proteins sequestered during diapause by p26, an abundant small heat shock protein (sHsp) in A. franciscana cysts.


Introduction
Fertilized eggs of the extremophile crustacean, Artemia franciscana develop ovoviviparously in the female's brood sac and at five days post-fertilization swimming nauplii (larvae) are released [1,2]. Under optimal conditions larvae molt several times and reach sexual maturity in four to five weeks. With changing parameters such as photoperiod and temperature, oviparous incubated at room temperature in 1 μm filtered, UV treated and autoclaved 33.0 ppt saltwater from Halifax Harbor, NS, Canada, hereafter termed seawater. Animals were collected for mRNA and protein preparation following hydration (0 h cysts), after 5 h at 27˚C (5 h cysts), after 10 h at 27 o C (10 h cysts/E1 nauplii) and as completely emerged, membrane-enclosed E2/ E3 nauplii (Fig 1). After the hatching of E2/E3 nauplii, 1 st instar larvae were collected by photo-taxis [29,30] and either homogenized immediately or incubated at 27˚C for either 16 h or 26 h to respectively generate early and late 2 nd instar larvae (Fig 1). The research described in this paper was performed in accordance with the ethical guidelines provided by the Canadian Council on Animal Care (CCAC). The University Committee on Laboratory Animals (UCLA) of Dalhousie University approved the research and assigned the Protocol Number 117-36.

Cloning and sequencing of A. franciscana type 2 Hsp40 cDNA
One mg of cysts developed for 5 h was harvested from seawater on 5 μm nylon mesh filters (Spectrum Labs Inc., Rancho Dominguez, CA, USA), flash frozen in liquid nitrogen and then homogenized in 500 μl of TRIzol1 (Invitrogen, Burlington, ON, Canada). RNA was extracted according to manufacturer's instructions. Single-strand cDNA was synthesized with the Super-Script1 III First-Strand Synthesis System for RT-PCR (Invitrogen, Burlington, ON, Canada). RNA was quantified by measuring the absorbance at 260 nm and 0.1 μg mRNA was used as template for synthesizing cDNA with SuperScript1 III First-Strand Synthesis Kit for RT-PCR (Invitrogen, Burlington, ON, Canada) and oligo dT 20 following the manufacturer's instructions. All amplifications were also performed without the addition of reverse transcriptase to confirm the absence of genomic DNA.

Preparation of antibody to ArHsp40-2
A polyclonal antibody against ArHsp40-2 peptide 277-DALCGTKVDVPTLSGE-292 (Anti40-type 2) was raised in rabbits (Abiocode, Agoura Hills, CA, USA) and tested for specificity with Hsp40s produced in Escherichia coli and in protein extracts from A. franciscana cysts and nauplii.
For the production of full length recombinant ArHsp40-2, cDNA for the protein was generated by PCR using Platinum Taq Polymerase (Invitrogen, Burlington, ON, Canada), 0.2 mM primers containing restriction enzyme sites for BamH1 and HindIII (Table 1) and 0.5 μg of cDNA from A. franciscana cysts developed for 5 h as template using the reaction: 5 min at 94˚C, 30 cycles of 30 s at 94˚C, 30 s at 54˚C, and 1 min at 72˚C, followed by 10 min at 72˚C. The cDNA was digested with BamH1 and HindIII at 37˚C overnight and then purified with Wizard1 SV PCR Clean-Up System (Promega, Madison, WI, USA). The digested cDNA was ligated into the His-tagged prokaryotic expression vector pRSET C (Invitrogen) that had been digested with BamH1 and HindIII and the recombinant plasmids were transformed into E. coli BL21(DE3) pLysS (Invitrogen) for purification. The synthesis of ArHsp40 and ArHsp40-2 was induced with 1 mM isopropyl thio-ß-D-galactoside (IPTG) for 6 h at 37˚C and the recombinant Hsp40s were purified from cell free extracts of E. coli with the MagneHis™ Protein Purification System (Promega, Madison, WI, USA). ArHsp40 was obtained in the same manner using a full-length cDNA prepared previously [29].
To prepare protein extracts from shrimp, 100 mg of 0 h A. franciscana cysts and 1 st instar nauplii, recovered separately from seawater and frozen as described above, were homogenized on ice in 100 μl of Pipes buffer (100 mM Pipes, 1 mM MgCl 2 , 1 mM EGTA, pH 7.4) containing protease inhibitors (Halt Protease Cocktail, #87,786, Pierce Biotechnology, Rockford, IL, USA) in a 1:100 ratio (v/v) with homogenate and centrifuged at 12,000g for 10 min at 4˚C. After determination of protein concentration by the Bradford assay [31] samples of extracts from 0 h cysts and 1 st instar nauplii and His-tagged recombinant ArHsp40 purified as described above were diluted in 4 X treatment buffer (250 mM Tris, 280 mM SDS, 40% (v/v) glycerol, 20% (v/v) ß-mercaptoethanol, 0.2% (w/v) bromophenol blue, pH 6.8), placed in a boiling water bath for 5 min and centrifuged at 10,000g for 10 min at 4˚C.
To test antibody specificity 10 μg of A. franciscana cell free protein extract, 1 μg of each purified recombinant Hsp40 and 5 μl Pink Plus Pre-stained Protein Ladder (FroggaBio Inc., Toronto, ON, Canada) were resolved in 12.5% SDS polyacrylamide gels, transferred to 0.2 μm nitrocellulose membranes (BioRad, Mississauga, ON, Canada) over night at 100 mAmps and blocked for 1 h at room temperature in 5% (w/v) Carnation low fat milk powder in TBS (10 mM Tris, 140 mM NaCl, pH 7.4). Blocked membranes were then probed for 15 min at room temperature with the antibodies to A. franciscana Hsp40s diluted 1:1,000 in TBS. Membranes were washed after incubation in primary antibody for 1, 2, 3 and 4 min in TBS-T (10 mM Tris, 140 mM NaCl, 0.1% (V/V) Tween-20, pH 7.4). Subsequent to washing, membranes were incubated for 20 min at room temperature with HRP-conjugated goat anti-rabbit IgG antibody (Sigma-Aldrich, Oakville, ON, Canada) diluted 1:10,000 in TBS and then washed for 1, 2, 4 and 4 min in TBS-T followed by a final 3 min wash in TBS. Antibody-reactive proteins were visualized with Clarity ECL TM Western Blotting Substrate (BioRad, Mississauga, ON, Canada) and a MF-Chemi-BIS 3.2 gel documentation system (DNR Bio-Imaging Systems, Neve, Israel).

Quantization of ArHsp40-2 mRNA in post-diapause A. franciscana
One mg of each of the seven different A. franciscana life history stages examined (Fig 1) was harvested and flash frozen as described above prior to homogenization in 500 μl of TRIzol1 (Invitrogen) for RNA extraction. RNA was quantified as described before and 0.1 μg mRNA was used as template for synthesizing cDNA with the SuperScript1 III First-Strand Synthesis Kit for RT-PCR (Invitrogen) as described above.
For qPCR, forward and reverse primers (Table 1) were used to amplify an ArHsp40-2 cDNA fragment of 144 bp, and an α-tubulin cDNA fragment of 276 bp, the latter as internal standard. Primers were at 1 mM and 0.5 μl of cDNA was employed as template using the following reaction: 5 min at 94˚C, 30 cycles of 30 s at 94˚C, 30 s at 54˚C for ArHsp40-2 (49˚C for α-tubulin), and 1 min at 72˚C, followed by 10 min at 72˚C in a Rotor-Gene RG-3000 (Corbett Research, Sydney, NSW, Australia). qPCR was conducted with a QuantiFast1 SYBR1 Green PCR Kit (Qiagen, Mississauga, ON, Canada). Melt curve analysis were calculated by Rotor-Gene 6 Software. qPCR experiments with an efficiency of 90% or greater were accepted for analysis (Corbett Research, Sydney, NSW, Australia).
The mRNA copy numbers for ArHsp40-2 and α-tubulin were calculated using standard curves, R 2 >0.99 [9]. Standard curves were generated by PCR using 0.5 μl cDNA with Platinum PCR supermix (Qiagen) and 0.4 mM primers for ArHsp40-2 and α-tubulin (Table 1). The PCR product concentrations were determined by measuring the absorbance at 260 nm, and the copy number calculated based on the length of the PCR products using a base pair mass of 650 Da (http://cels.uri.edu/gsc/cdna.html). The cDNA standards were diluted in a 10-fold series with TE buffer (10 mM Tris, 1 mM EDTA, pH 8.0), and 0.5 μl of each dilution was used as template with the QuantiTect1 SYBR1 Green PCR Kit (Qiagen). All cDNA samples were assayed in duplicate. The Ct values employed to create the standard curves were fitted by linear regression for ArHsp40-2 and α-tubulin mRNAs.

Quantization of ArHsp40-2 in post-diapause A. franciscana
One-hundred mg of A. franciscana at the 7 life history stages examined (Fig 1) was harvested and flash frozen before homogenization on ice in 100 μl of Pipes buffer and centrifugation as described above. After determination of protein concentration by the Bradford assay [31] samples were diluted in 4 X treatment buffer, placed in a boiling water bath for 5 min and centrifuged at 10,000g for 10 min at 4˚C.

Heat induction of ArHsp40-2 in A. franciscana larvae
First instar A. franciscana nauplii grown at 27˚C in seawater were collected 4 h after hatching when they contained reduced amounts of ArHsp40-2, and then incubated in 20 ml seawater in Corex tubes at 39˚C for 1 h in a programmable water bath (VWR International LLC, Mississauga, ON, Canada) [1]. Heat shocked animals were harvested on nylon mesh filters, frozen and either homogenized on ice with protease inhibitors in Pipes buffer as described before or incubated at 27˚C for 2, 4, 6, and 8 h for recovery and then collected and homogenized. Forty μg of each cell-free protein homogenate was resolved in SDS polyacrylamide gels, transferred to nitrocellulose, blocked and probed with either Anti40-type2 or Anti-Y prior to washing and imaging as described above.

Image analysis
Images of developing animals were captured using a Nikon AZ100 microscope. Images and figures were prepared for publication using Photoshop (Adobe Creative Cloud; Adobe Systems Inc). Bands of immunoreactive proteins on western blots were quantified with Image Studio Software (Li-Core Biosciences, Lincoln, NE, USA), and band intensities for ArHsp40-2 were compared to band intensities for tyrosinated α-tubulin at each development stage examined.

Statistical analysis
One-way ANOVA followed by a Dunnett's test was carried out in order to detect significant difference between all means from control means. All data were plotted as means +/-SE unless otherwise stated. Analyses were carried out using GraphPad Prism 5 software (GraphPad Software Inc., La Jolla, CA, USA).

ArHsp40-2 cDNA
ArHsp40-2 cDNA (Accession number MH138022) from 5 h cysts cloned by RT-PCR consisted of 1065 bp which encoded a 354-amino acid polypeptide with a predicted molecular mass of 38.9 kDa and a theoretical pI of 8.99 (Fig 2). ArHsp40-2 possessed a conserved N-terminal Jdomain with a HPD motif, followed by a Gly/Phe (G/F) rich region with a conserved DIF motif and then a carboxyl-terminal domain. ArHsp40-2 lacked a zinc binding domain containing CXXCXGXG motifs and it shared significant sequence similarity, except in the G/F rich region, with type 2 J-domain proteins from several animals (Fig 3 and Table 2). ArHsp40 [29] and ArHsp40-2 possessed 64% similarity and 36% identity (Fig 4) with ArHsp40-2 having a deletion of 53 amino acid residues at its carboxyl terminus as compared to ArHsp40. The carboxyl-terminal domain contained a double barrel β-domain followed by an α-helix which forms a dimerization domain similar to that found in ArHsp40 and other J-domain proteins (Fig 5).

ArHsp40-2 synthesis was induced by heat shock in A. franciscana larvae
ArHsp40-2 increased almost 10-fold in 1 st instar nauplii heat shocked at 39˚C for 1 h and remained elevated for 6 h of recovery before declining (Fig 9)

Discussion
Diapausing cysts of A. franciscana survive long periods of time in an essentially ametabolic state where they are resistant to stresses such as heat, cold, anoxia, oxidation and desiccation [5,[33][34][35][36][37]. In preparation for diapause, embryos of A. franciscana up-regulate synthesis of the sHsp p26 [1,8], which is thought to sequester denaturing proteins when ATP concentration is low and ATP-dependent molecular chaperones are unable to fold proteins. RNAi experiments confirm that p26 has an important role in protecting diapause cysts during stress [9], but because this sHsp lacks foldase activity other molecular chaperones including Hsp110, Hsp90 and Hsp70, and their accessory proteins such as the Hsp40s, are required to refold proteins  Table 2. The domains of ArHsp40-2 are over-lined in black and labeled in red. SBD, substrate binding domain. Identical amino acid residues are indicated by an asterisk, similar residues by a colon and semi-conserved residues by a period. The number of amino acid residues in each sequence is indicated on the right. https://doi.org/10.1371/journal.pone.0201477.g003 ArHsp40-2, a type 2 J-domain protein from Artemia franciscana   ArHsp40-2, a type 2 J-domain protein from Artemia franciscana sequestered by p26. Hsp70 and Hsp90 are synthesized in A. franciscana and A. sinica embryos that are destined to enter diapause and they are available to maintain protein homoeostasis during embryogenesis and post-diapause development [6,35,[38][39][40][41]. Additionally, embryos fated to become nauplii up-regulate mRNA for Hsp60, Hsp70, Hsp90 and Hsp110 [39] and Hsp70 increases in nauplii under heat stress where it can play a role in resisting infection by Vibrio campbellii [42,43].
Little is known about Hsp40s in A. franciscana and other crustaceans undergoing diapause, but they are up-regulated in insects during diapause. For example, in early diapause of the flesh fly, Sarcophaga crassipalpis Hsp40 in the brain is up-regulated and hyper-phosphorylated [44,45], but how this contributes to diapause in uncertain. Hsp40 mRNA increases upon diapause entry and metabolic depression in the silk worm Bombyx mori, as is true for Hsp70 and Hsc70 mRNAs [46]. The synthesis of Hsp40 mRNA is also stimulated in insects by stress. As a case in point, Hsp40 mRNA is induced by heat in the leaf miner Liriomyza sativa and by cold in Drosophila melanogaster [47][48][49]. Larvae of the gall fly Eurosta solidaginis synthesize Hsp40 when exposed to freezing [50] and Hsp40 mRNA is up-regulated in S. crassipalpis and D. melanogaster adults under oxidative stress [51,52].
We characterized an A. franciscana J-domain protein named ArHsp40 and showed that it is likely to play a role in stress tolerance during diapause and to influence post-diapause development [29]. To expand the study of Hsp40s in A. franciscana, a second J-domain protein termed ArHsp40-2 has been identified and it is described herein. Unlike ArHsp40, ArHsp40-2 lacks a zinc binding domain, although the two proteins share significant sequence similarity and structural elements, demonstrating that they are related. Multi-sequence alignment showed that ArHsp40-2 and type 2 Hsp40s from divergent species are similar, other than in the GFrich region. ArHsp40-2 mRNA, present in relatively low amounts in 0 h cysts, increased approximately eight-fold during the first five hours of post-diapause development and remained elevated until cyst shells cracked and membrane-enclosed nauplii emerged. The augmentation in mRNA indicates transcriptional regulation of the ArHsp40-2 gene during early post-diapause development in A. franciscana, although other possibilities such as an increase in ArHsp40-2 mRNA stability cannot be ruled out at this time. The amount of ArHsp40-2 mRNA underwent a significant drop in E2/E3 nauplii and declined even further in instar II nauplii, implying that ArHsp40-2, if it followed the same trend as the mRNA, functions mainly during early post-diapause development, a time when proteins sequestered by p26 are required by the developing embryo. To determine the level of ArHsp40-2 during post-diapause development of A. franciscana, an antibody shown to react with purified recombinant ArHsp40-2 and a single protein tubulin was determined. Lane 1, 0 h cysts; 2, 5 h cysts; 3, 10 h cysts/E1; 4, E2/E3; 5, instar 1 nauplii; 6, early instar 2 nauplii; 7, late instar 2 nauplii. The amount of ArHsp40-2 in life history stages indicated by bars labeled with asterisks are significantly different from the amount of ArHsp40-2 in 0 h cysts, ÃÃÃ , P<0.005. Error bars represent standard error of 3 replicates per experiment.
https://doi.org/10.1371/journal.pone.0201477.g008 ArHsp40-2, a type 2 J-domain protein from Artemia franciscana in cell free extracts of cysts and nauplii, but not with purified recombinant ArHsp40, was used. The early post-diapause increase in ArHsp40-2 relative to that in 0 hour cysts was less than that seen for its mRNA but the pattern of accumulation was very similar, again indicative of ArHsp40-2 function during early post-diapause development when p26 is releasing sequestered proteins for refolding. Additionally, ArHsp40-2, like ArHsp40, was induced by heat where it had the potential to contribute to stress tolerance by transferring proteins to Hsp70 which then remain bound because ATP concentration is low.

Conclusions
A cDNA, termed ArHsp40-2 was cloned from A. franciscana and shown to encode a type 2 Jdomain protein which lacked a zinc binding domain. ArHsp40 and ArHsp40-2 shared sequence and structural similarities, and the synthesis of ArHsp40-2 was, like ArHsp40, developmentally regulated, but their patterns of synthesis were distinct indicating dissimilar functions. Both proteins were induced by heat in nauplii. The results suggest a role for ArHsp40-2 in diapausing cysts of A. franciscana and during early post-diapause development where it assists in the rescue of proteins sequestered on p26, an abundant sHsp specific to diapause.