An intrinsically disordered radish vacuolar calcium-binding protein (RVCaB) showed cryoprotective activity for lactate dehydrogenase with its hydrophobic region

Honami Osuda b, Yui Sunano b, Masakazu Hara a,b,c,⁎
a Research Institute of Green Science and Technology, Shizuoka University, 836 Ohya, Shizuoka, Shizuoka 422-8529, Japan
b Graduate School of Integrated Science and Technology, Shizuoka University, 836 Ohya, Shizuoka, Shizuoka 422-8529, Japan
c Graduate School of Science and Technology, Shizuoka University, 836 Ohya, Shizuoka, Shizuoka 422-8529, Japan

a b s t r a c t

A soluble protein fraction from radish (Raphanus sativus L.) taproot had cryoprotective activity for lactate dehydrogenase (LDH). The activity was found mainly in the heat-stable fractions of soluble proteins. The cryopro- tective protein, whose molecular mass was 43 kDa in sodium dodecyl sulfate polyacrylamide gel electrophoresis, was obtained by successive chromatographies on TOYOPEARL SuperQ and TOYOPEARL DEAE. MALDI-TOF MS/MS analysis indicated that the purified protein was a radish vacuolar calcium-binding protein (RVCaB), which is reportedly related to calcium storage in the vacuoles of radish taproot. The purified RVCaB inhibited the cryoinactivation, cryodenaturation, and cryoaggregation of LDH. RVCaB had greater cryoprotective activity than general cryoprotectants. When RVCaB was divided into 15 segments (Seg01 to Seg15, 15 amino acids each), Seg03, which had a high hydrophobicity scale, showed remarkable cryoprotective activity. This indicated that RVCaB protected LDH from freezing and thawing damage presumably through a specific hydrophobic area (i.e., Seg03).

Keywords: Cryoprotection Dehydrin
Intrinsically disordered proteins
Raphanus sativus L.
Vacuolar calcium-binding protein

1. Introduction

The cold tolerance of plants is a crucial factor in determining plant production, vegetation formation, seed longevity, and so on [1,2]. Knowledge of the molecular basis of cold tolerance has been applied to various technologies, such as postharvest storage and seed banks [3,4]. The production of cryoprotectants, which can prevent damage to cells and biomolecules due to freezing, is involved in the cold responses of plants [5]. Compatible solutes such as betaine, proline, and sugars can act as cryoprotectants [6]. In addition, late embryogenesis abundant (LEA) proteins and cryoprotectins are known to be proteinous cryopro- tectants [7,8].
LEA proteins were produced in the late stage of seed maturation and in growing plants exposed to various stresses, including cold [9–11]. Ge- netic analyses and transgenic studies demonstrated that the accumula- tion of LEA proteins was correlated with seed longevity and stress tolerance in plants [9–12]. Although LEA proteins have been known to prevent the freeze denaturation of cold-sensitive enzymes [13], there are few reports of plant proteins other than LEA proteins showing cryo- protective activities for such enzymes.
Group 2 LEA proteins, also called dehydrins, are plant-specific and intrinsically disordered [9,10]. Dehydrins are among the most- characterized LEA proteins. It has been repeatedly demonstrated that dehydrins are multifunctional: they protect freezing-sensitive enzymes, and bind to various molecules (such as lipids, water, ions, and nucleic acids) [14–16]. Among these functions, cryoprotective activity is thought to be a major role of dehydrins, as many studies have previ- ously reported the issue (e.g., [17–20]). Genetic and transgenic ap- proaches have found that the expression of dehydrin genes reduced cold damage to plants [21–25]. The in vitro and in vivo evidence sup- ported that dehydrins are related to establishing cold tolerance in plants.
Previously, we found that taproot of radish (Raphanus sativus L.) contained dehydrin, which was detected by an antibody against a KS- type dehydrin of Arabidopsis thaliana (AtHIRD11) [26]. The radish dehydrin existed mostly in the high-salt extract of cell debris that had been pelleted by centrifugation of radish taproot homogenates. After purification, the radish dehydrin designated as RsDHN (R. sativus dehydrin) showed cryoprotective activity for malate dehydrogenase [27]. Thereafter, it was revealed that the soluble fraction of the taproot homogenate had considerable cryoprotective activity, whereas the sol- uble fraction contained a small quantity of antigens for the anti-AtHIRD11 antibody. This suggested that one or more cryoprotective proteins that are likely different from dehydrins might exist in the solu- ble fraction of the taproot homogenate.
In this paper, we report the purification of a cryoprotective protein from the soluble fraction of the taproot homogenate. The purified pro- tein was identified as a radish vacuolar calcium-binding protein (RVCaB), which was proposed to be related to the sequestration of cal- cium in the vacuole. The protein was previously isolated from the vacu- olar membranes of the radish taproot via ion exchange chromatography and gel filtration chromatography [28]. Here, we proposed a simple pu- rification protocol with a higher yield of RVCaB. In addition, we found that the cryoprotective site was located near the N-terminus of RVCaB. The putative mechanisms underlying the cryoprotective activity of RVCaB and its physiological roles in radish were discussed.

2. Materials and methods

2.1. Chemicals

Anion exchange resins, TOYOPEARL SuperQ-650M and TOYOPEARL DEAE-650M, were purchased from Tosoh (Tokyo, Japan). Lactate dehy- drogenase (LDH, rabbit muscle, recombinant) and nicotine adenine di- nucleotide (NADH) were obtained from Oriental Yeast (Tokyo, Japan). 8-Anilino-1-naphthalene sulfonic acid (ANS) and dithiothreitol (DTT) were purchased from Sigma (Tokyo, Japan) and Wako (Osaka, Japan), respectively.

2.2. Peptides

An automated solid phase peptide synthesizer (Tetras, Advanced ChemTech, Louisville, KY, USA) was used to prepare peptides (Seg01 to Seg15). The peptides were purified by an ultrafast liquid chromato- graph (UFLC-20AB, Shimadzu, Kyoto, Japan) with a C18 reversed- phase column (Altima™ 4.6 × 250 mm). A linear gradient of acetonitrile (5–95%) in 0.05% trifluoroacetic acid solution was performed for 25 min. The synthesized peptides were identified by mass spectrometry (LCMS- 2020, Shimadzu) and lyophilized. When the peptides were dissolved in solution for use, the peptide concentrations were determined from the dry weight.

2.3. Purification of RVCaB

European red radish (R. sativus L.), obtained from a local food market in Shizuoka, Japan, was used for the purification of RVCaB. Radish taproot (26 g fresh weight) was ground by a food grater on ice. The squeezed juice, obtained by passing the ground root through double gauze, was centrifuged at 10,000 g for 10 min at 4 °C. The super- natant was collected in a 50-ml centrifuge tube (TPP, Trasadingen, Switzerland) and then DTT was added to reach a concentration of 1 mM. This sample was considered the crude extract (20.5 mL). The crude extract was placed in an aluminum block heater (dry thermos unit DTU\\1B, TAITEC, Saitama, Japan) which was controlled at 100 °C for 40 min. After cooling for 10 min on ice, the formed aggregates were precipitated by centrifugation (at 10,000 g for 10 min at 4 °C). The supernatant (20 mL, heat-stable fraction) was loaded onto the TOYOPEARL SuperQ-650 M column (45 mm × 1.5 mm ID) at a flow rate of 1.2 mL min−1. The column was washed with 10 mM Tris-HCl buffer (pH 7.5) containing 1 mM DTT (running buffer). Bound proteins were eluted with a linear gradient of NaCl (0 to 500 mM) in the running buffer by an Econo Gradient pump (Bio-Rad, Tokyo, Japan) at 1.5 mL min−1 for 25 min. The fraction size was approximately 3.3 mL.
The cryoprotective fractions were combined (9.9 mL) and desalted by a gel filtration column (NAP-25, GE Healthcare, Tokyo, Japan) equili- brated with the running buffer. The sample was applied to the TOYOPEARL DEAE-650 M column (40 mm × 1.5 mm ID) at a flow rate of 1.2 mL min−1. After the column was washed with the running buffer, linear gradient elution was performed as described above except that the change in NaCl concentration was from 0 to 250 mM at a flow rate of 1.0 mL min−1. The active fractions were combined, desalted, and stored at −20 °C until use. The cryoprotective activity of the purified protein was stable under this storage condition.
The amount of protein was determined from the band intensities in sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS- PAGE). The gel was stained with Coomassie brilliant blue (Bio-Safe, Bio-Rad). The data for the electrophoretogram were obtained by the Fu- sion Solo S imaging system (Vilber Lourmat, Collégien, France). ImageJ software (https://imagej.nih.gov/ij/) was applied to quantify the pro- tein bands. Bovine serum albumin was used as a standard.

2.4. Protein identification

The purified protein was identified by using matrix-assisted laser desorption ionization time-of-flight mass spectrometry tandem mass spectrometry (MALDI-TOF MS/MS) with the oMALDI-Qq-TOF MS/MS QSTAR Pulsar i system (Applied Biosystems, Foster, CA, USA). PEAKS de novo sequencing software was applied to predict the amino acid se- quence of the protein’s fragments. A peak whose m/z was 2302 was matched to ATADVEQVTPAAAEHVEVTPPK (acetylated at the N- terminus). A Mascot search (http://www.matrixscience.com) and a BLAST analysis (https://blast.ncbi.nlm.nih.gov/Blast.cgi) indicated that the sequence was matched only to a radish vacuolar calcium-binding protein (RVCaB, accession AB035900).

2.5. Cryoprotective activities

Cryoprotective activities were evaluated by the inhibition of cryoinactivation of LDH. In some cases, the inhibition of cryodenaturation and the inhibition of cryoaggregation of the enzyme were also tested. The following experiments were performed according to our previous methods [29] with some modifications.
The inhibitory activity of LDH cryoinactivation was determined as follows. In brief, the test solutions (30 μL) were mixed with the LDH so- lution (20 μL, 0.34 μM as a monomer in 10 mM Tris-HCl buffer pH 7.5) in 1.5-mL plastic tubes. In the case of peptides (Seg01-Seg15), the test so- lutions contained the peptides at a concentration of 275 μg mL−1. Thus, the peptide concentration in all the tubes was 165 μg mL−1. The tubes were immersed in liquid N2 for 1 min and then in a water bath at 25 ± 2 °C for 3 min. This freezing and thawing process was done three times, and then the LDH activity was measured. The samples (4 μL) were added to the reaction solutions (196 μL), i.e., 9.5 mM Tris- HCl pH 7.5, 0.58 mM sodium pyruvate, and 60 μM NADH, in a 96-well microplate. Changes in absorbance at 340 nm were recorded at 25 °C by using a microplate reader (Varioskan Flash, Thermo Fisher Scientific, Tokyo, Japan). In most cases, the LDH activities were decreased to ap- proximately 20% of the initial activities by the freezing and thawing treatment. To evaluate the cryoprotective activity, a value of relative cryoprotective activity was used. The range of decrease in LDH activity after freezing and thawing was standardized as 100% of relative cryoinactivation. For example, if a sample recovered 70% of relative cryoinactivation, the relative cryoprotection value was 70%. This type of data representation was applied to Figs. 1C, 2B, and 5. Besides that, when the inhibition of LDH cryoinactivation by different concentrations of RVCaB was tested, the initial enzymatic activity of LDH (before freez- ing and thawing) was relativized to 100%. This representation was done in Figs. 4A and 6A.
LDH cryodenaturation was assessed by an ANS fluorescence probe, which can quantify hydrophobicity on the protein surface. Mixtures containing ANS (10 μM), LDH (4 μM), RVCaB (corresponding concentra- tions), and 10 mM sodium phosphate buffer pH 7.0 were prepared in a total volume of 250 μL in 1.5-ml plastic tubes. After the three freezing and thawing cycles, fluorescence was detected in the 96-well plates (Ex 350 nm and Em 470 nm, Varioskan Flash). The increment of fluorescence enhanced by freezing and thawing in the sample contain- ing no RVCaB was standardized as 100% (Figs. 4B and 6B).
The cryoaggregation of LDH was measured by turbidity. Test solu- tions (250 μL) consisting of 4 μM LDH, corresponding concentrations of RVCaB, and 10 mM Tris-HCl buffer pH 7.5 were prepared in 1.5-ml plastic tubes. The solution was treated with the three freezing−thawing cycles described above, after which turbidity was determined in a 96- well microplate (415 nm, Bio-Rad iMark). The increment of absorbance by freezing and thawing in the sample without RVCaB was standardized as 100% (Figs. 4C and 6C).

3. Results

Cryoprotective activities were determined by the inhibition of cryoinactivation of lactate dehydrogenase (LDH), a model of cryosensitive enzymes [30]. After the homogenates from the radish tap- root were centrifuged, the soluble fraction was analyzed by SDS-PAGE (Fig. 1A, lane Sf). This fraction contained various proteins and showed apparent cryoprotective activity (Fig. 1C, Sf). Since cryoprotective pro- teins (e.g., dehydrins) were known to be highly hydrophilic and thus heat stable, we decided to prepare the heat-stable fraction from the sol- uble fraction to obtain the cryoprotective proteins. Consequently, most of cryoprotective activity was maintained (approximately 87% of the ac- tivity in the soluble fraction) in the heat-stable fraction (Fig. 1C, Hs). SDS-PAGE analysis indicated that the heat-stable fraction contained a major protein whose molecular mass was approximately 43 kDa (Fig. 1A, lane Hs, an arrowhead).
Subsequently, an anion exchange chromatography (TOYOPEARL uperQ) was applied. Proteins were eluted with a linear gradient of NaCl (0.0–0.5 M) (Fig. 1B). Fraction number 7, which showed the highest cryoprotective activity among the eluate fractions (Fig. 1C), pri- marily contained the 43 kDa protein (Fig. 1B, an arrowhead). The 43 kDa protein was also found in the 6th and 8th fractions, in which consider- able cryoprotective activities were detected. On the other hand, fraction numbers 4, 5, and 9–13 showed cryoprotective activities even though they did not contain the 43 kDa protein, suggesting that proteins other than the 43 kDa protein and/or nonprotein molecules might con- tribute to the cryoprotective activity of the heat-stable fraction. How- ever, we focused on the 43 kDa protein because its presence was predominant and reproducibly observed in the cryoprotective fractions. Fraction numbers 6, 7, and 8 were combined for the subsequent chro- matography (TOYOPEARL DEAE). The results demonstrated that the amounts of the 43 kDa protein were well correlated with the cryopro- tective activities through the eluate fractions (Fig. 2). The purity of the 43 kDa protein was 94.7%, and the yield on the basis of cryoprotective activity was 10% (Table 1).
A MALDI-TOF MS/MS analysis with a de novo sequencing method revealed that the 43 kDa protein contained the amino acid sequence ATADVEQVTPAAAEHVEVTPPK. According to a BLAST search, the sequence totally matched that of the correspond- ing site (A2TADVEQVTPAAAEHVEVTPPK23) of a radish vacuolar calcium-binding protein (RVCaB, accession AB035900) (Fig. 3). Considering a previous report that detected RVCaB at the size of 43 kDa in SDS-PAGE [28], we confirmed that RVCaB was the cryo- protective protein purified from radish taproot. RVCaB was first isolated from the vacuoles of radish taproot [28]. The theoretical molecular weight of RVCaB was 27,094 and the isoelectric point was calculated as 4.1 (Supplementary Fig. 1A). A previous report described that RVCaB in SDS-PAGE (43 kDa) was larger than the theoretical molecular mass (27 kDa), because generally acidic pro- teins slowly migrated in SDS-PAGE [28]. The protein had small amounts of hydrophobic residues and neither aromatic nor cyste- ine residues (Supplementary Fig. 1B). Analysis by using secondary structure prediction software suggested that RVCaB had a primar- ily coiled structure (Supplementary Fig. 1C). Indeed, RVCaB was demonstrated to be an intrinsically disordered protein (IDP) in the previous study [31].
The concentration dependence of the cryoprotective activities of RVCaB was analyzed (Fig. 4). Although the LDH activity was reduced to approximately 20% of the initial activity after the freezing and thawing treatment, this reduction was mitigated by RVCaB in a concentration- dependent manner (Fig. 4A). The protection dose 50% (PD50) value was 1.8 μM. Cryodenaturation was recorded by the fluorescence of ANS, which is a hydrophobicity indicator during the denaturation pro- cess of proteins. Cryoaggregation was determined by the turbidity of the protein solution. RVCaB inhibited the cryodenaturation and cryoaggregation of LDH (Fig. 4B, C). The PD50 values for cryodenaturation and cryoaggregation were 1.5 and 1.4 μM, respectively.
Finally, cryoprotective sites of RVCaB were investigated. RVCaB had two repeat sequences, from E91 to E120 and from E121 to E150, which were totally identical (Fig. 3, represented by yellow highlights). In this work, the sequence was divided into 15 segments designated Seg01 to Seg15 (15 amino acids each). Seg07 and Seg08 covered the repeat sequences. The inhibitory activities of these segments for the cryoinactivation of LDH were determined (Fig. 5). As a result, four seg- ments, Seg02, Seg03, Seg06, and Seg14, showed cryoprotective activities, with Seg03 being the most potent. This suggests that Seg03 was a major cryoprotective site of RVCaB. Seg03 inhibited the cryoinactivation, cryodenaturation, and cryoaggregation of LDH (Fig. 6). However, higher concentrations were needed in Seg03 than in RVCaB in order to examine the cryoprotective activities. The PD50 values for cryoinactivation, cryodenaturation, and cryoaggregation were 21, 17, and 21 μM, respectively.

4. Discussion

Here we report that a cryoprotective protein was purified from radish taproot and identified as RVCaB, a radish vacuolar calcium-binding pro- tein. The protein was obtained from the soluble fraction of taproot via heat treatment and two-step anion exchange chromatographies. The pu- rity of the final sample was approximately 95% (Table 1). Previous purifi- cation procedures required the isolation of vacuolar membranes, ion exchange chromatography, and gel filtration chromatography [28], indi- cating that the present purification method was simpler than the previous one. In a previous report, 100 μg of RVCaB was prepared from 2 kg of tap- root [28]. In our method, on the other hand, 11 mg of RVCaB was purified from 26 g of taproot. This indicates that the purification efficiency of the present method was at least 4000 times higher than that of the previous method. Radish is a Brassicaceae vegetable that has been widely produced in Asia and Europe [32]. Thus, using our purification procedure, RVCaB can be prepared as a cryoprotectant from excessively produced radish. Circular dichroism (CD) analysis previously determined that the RVCaB structure was disordered [31]. RVCaB purified in the present study was also shown to be disordered by CD (Supplementary Fig. 2A), suggesting that our purification procedures did not affect the structural characteris- tics of RVCaB.
A comparison of our results against those of previous studies reveals that the cryoprotective characteristics of RVCaB were similar to those of dehydrins, except that the two amino acid sequences were totally distinct from one another. It is worth mentioning that both RVCaB and dehydrins are IDPs [16,31], which are highly disordered in solution and hence heat stable. Both proteins were rich in hydrophilic amino acids, although some hydrophobic amino acids were found. RVCaB and dehydrins inhibited the cryoinactivation of LDH. Fig. 4A shows that the protection dose 50% (PD50) value of RVCaB was approximately 1.8 μM (49 mg L−1, calculated from a molecular weight of 27,110). On the other hand, the corresponding value of AtHIRD11 (an Arabidopsis dehydrin) was approximately 2.6 μM (28 mg L−1, calculated from a mo- lecular weight 10,796) [27], indicating that the two proteins inhibited the LDH cryoinactivation to similar degrees. Moreover, their cryoprotec- tive activities were remarkably high, because the cryoprotection of common cryoprotectants such as trehalose, proline, and glycine betaine occurred at concentrations of around 10 g L−1 [26]. Taken together, the present and previous results suggest that similar mechanisms, which might be related to the length of the disordered region, underlie the cryoprotective activities of RVCaB and dehydrins.
We found that Seg03 (V31AAAVVADSAPAPVT45) was the major cryoprotective site of RVCaB (Fig. 5). Although the whole range of the amino acid sequence of RVCaB was predicted to be highly disordered (Supplementary Fig. 3A, IUpred2A software [33]), some hydrophobic areas were localized in the sequence (Supplementary Fig. 3B, ProtScale software [34]). Intriguingly, the region corresponding to Seg03 was pre- dicted to be the most hydrophobic area through the sequence. The CD analysis demonstrated that the structure of Seg03 was disordered (Supplementary Fig. 2B), suggesting that the hydrophobic amino acids of Seg03 were likely to have been exposed to the solution. Seg02, Seg06, and Seg14, which had low but significant cryoprotective activities, tended to show hydrophobicity. Also, AtHIRD11 (98 amino acids in length) had two cryoprotective segments, AtHIRD11_NK1 (M1AGLINKIGDALHIG15) and AtHIRD11_Kseg (H41KEGIVDKIKDKIHG55), in which most hydropho- bic amino acids of the dehydrin were located [29]. AtHIRD11_Kseg was AtHIRD11′s K-segment, which is a conserved sequence in all dehydrins. It was demonstrated that hydrophobic amino acids were required for the cryoprotective activity of the K-segment [35]. Thus, hydrophobic amino acids may be related to the cryoprotective activities of RVCaB and dehydrins.
Here, a putative mechanism for the cryoprotection of LDH by RVCaB was represented (Fig. 7). It has been reported that, during the freezing and thawing process, hydrophobic areas (i.e., hydrophobic patches) were exposed on the surface of LDH. The hydrophobic patches may also have been formed during the deconstruction of tetrameric LDH due to freezing and thawing. After that, LDH aggregated via the hydro- phobic patches [36] (Fig. 7). The hydrophobic Seg03 of RVCaB may hinder the hydrophobic self-association with cryo-damaged LDH. How- ever, it is likely that the hydrophobic effect by Seg03 was not the only factor that determined the cryoprotective activity of RVCaB, because the PD50 value of Seg03 (21 μM) was approximately 12 times higher than that of RVCaB. A large hydrodynamic radius due to the disordered
Protection by RVCaB via hydrophobic attraction nature of the structure might contribute to the cryoprotective activity of RVCaB. On this point, RVCaB might stabilize LDH on the basis of a pref- erential exclusion mechanism [37,38] and an extended molecular shield mechanism [39,40] as well, both of which have been established as mechanisms of protein protection. In the case of dehydrins, the cryopro- tective activities were attributed basically to the large hydrodynamic ra- dius [41,42], whereas transient hydrophobic interaction without binding is needed to facilitate the cryoprotective activities [29]. In addi- tion, LEA proteins and small heat shock proteins, both of which prevent protein denaturation, have been known to possess disordered regions [43]. Taken together, the previous and present results suggested that the disordered nature is a crucial factor for protective IDPs, including RVCaB.
Finally, a physiological role of RVCaB in radish was discussed. RVCaB was found mainly in the taproot of radish and was little detected in leaves [44]. Since RVCaB has been found in the vacuolar lumen of the taproot, it has been suggested that RVCaB contributed to the sequestra- tion of calcium ion to the interior of vacuoles [28,45]. Here, we added cryoprotective activity to RVCaB’s functions. Generally, radish is grown in autumn and harvested in winter. Since the radish taproot mainly con- sists of parenchymal cells whose interiors are filled with vacuoles, preventing damage to vacuoles is important for the taproot. Moreover, taproot is a crucial organ for the storage of nutrients for the following spring. The cryoprotective protein RVCaB may be produced to reduce damage to taproot from the cold of winter.

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