Identification and characterization of the novel nuclease activity of human phospholipid scramblase 1
© Sivagnanam et al. 2016
Received: 4 March 2016
Accepted: 17 May 2016
Published: 20 May 2016
Human phospholipid scramblase 1 (hPLSCR1) was initially identified as a Ca2+ dependent phospholipid translocator involved in disrupting membrane asymmetry. Recent reports revealed that hPLSCR1 acts as a multifunctional signaling molecule rather than functioning as scramblase. hPLSCR1 is overexpressed in a variety of tumor cells and is known to interact with a number of protein molecules implying diverse functions.
In this study, the nuclease activity of recombinant hPLSCR1 and its biochemical properties have been determined. Point mutations were generated to identify the critical region responsible for the nuclease activity. Recombinant hPLSCR1 exhibits Mg2+ dependent nuclease activity with an optimum pH and temperature of 8.5 and 37 °C respectively. Experiments with amino acid modifying reagents revealed that histidine, cysteine and arginine residues were crucial for its function. hPLSCR1 has five histidine residues and point mutations of histidine residues to alanine in hPLSCR1 resulted in 60 % loss in nuclease activity. Thus histidine residues could play a critical role in the nuclease activity of hPLSCR1.
This is the first report on the novel nuclease activity of the multi-functional hPLSCR1. hPLSCR1 shows a metal dependent nuclease activity which could play a role in key cellular processes that needs to be further investigated.
KeywordsNuclease Histidine Kunitz assay Mg2+ dependent Site-directed mutagenesis Scramblase
Human phospholipid scramblases (hPLSCRs) are type II single pass transmembrane proteins involved in the Ca2+ dependent ATP independent translocation of phospholipids (PLs) across the lipid bilayer . hPLSCR1 was first identified as a 37 kDa integral membrane protein from human erythrocytes, which was capable of bidirectional scrambling of PLs between two leaflets of the lipid bilayer when reconstituted into liposomes. The gene encoding hPLSCR1 was cloned, overexpressed and the recombinant hPLSCR1 exhibited low scrambling activity compared to that of erythrocyte PL scramblase . Five homologs of scramblases (hPLSCR1–5) have been identified so far. hPLSCR1, 3 and 4 were expressed in wide variety of tissues; whereas hPLSCR2 was found only in testis and hPLSCR5 has been shown only at mRNA level [2, 3].
hPLSCR1 possesses a single transmembrane helix but can localize to both plasma membrane (PM) and nucleus. It has been reported that scramblases destroy the lipid asymmetry in PM when intracellular Ca2+ levels were elevated by 1000 fold . Previous studies showed that phosphatidylserine (PS) exposure during apoptotic conditions was directly related to hPLSCR1 expression levels and the amount of PS exposed to the cell surface could be manipulated by modifying the expression levels of hPLSCR1 . Subsequent reports have questioned the role of hPLSCR1 in PS exposure [5–9]. Recently, a member of Ca2+ dependent ion channels, TMEM16F, and members of Xkr protein family were identified to be responsible for phospholipid scrambling in plasma membrane [10–13]. These reports along with other unusual factors for PL translocators such as low molecular weight, single transmembrane domain and slow rate of PL scrambling in-vitro support the argument that hPLSCR1 may not be a true scramblase and can have varied roles within the cell apart from phospholipid scrambling.
hPLSCR1 is a multifunctional protein involved in several other cellular processes including cell signaling, cell proliferation, transcriptional regulation and antiviral defense [14–18]. Overexpression of hPLSCR1 in myeloid cells inhibited tumorigenesis and enhanced cell differentiation [19, 20]. hPLSCR1 interacts with variety of signaling molecules such as epidermal growth factor receptor, c-Abl, Src kinase, Shc and onzins [21–23]. hPLSCR1 also interacts with nuclear proteins including importin-α and topoisomerase II [24, 25] and was shown to exhibit high affinity to chromatin . hPLSCR1 expression is strongly stimulated in response to interferons and viral infections [6, 14]. Interferons are well known to stimulate the expression of several nucleases as a part of antiviral defense . hPLSCR1 inhibits Hepatitis B virus replication partly by activating the Jak/Stat pathway . hPLSCR1 interacts with Human T-cell leukemia virus type-1 (HTLV-1) and represses the Tax-dependent transactivation during HTLV-1 infection .
hPLSCR1 is characterized by five distinct domains conserved across the species: (i) C- terminal helix that spans the plasma membrane and is responsible for activity; (ii) Cysteine palmitoylation motif which helps in anchoring the protein to the membrane; (iii) Nuclear localization signal which helps in nuclear transport; (iv) an EF hand like Ca2+ binding motif and (v) DNA binding domain (M86-E118) . The EF hand like Ca2+ binding motif is vital for Ca2+ binding and scramblase activity of hPLSCR1. Mutations in this motif rendered the protein inactive as the protein lost its ability to bind to calcium . In a previous study, we showed that apart from Ca2+ binding to the EF-hand like motif, hPLSCR1 could also bind to a variety of metal ions including Mg2+ . The DNA binding domain was shown to bind with the promoter of inositol 3-phosphate receptor (IP3R), enhancing the expression of IP3R . Recently we identified a unique N-terminal proline rich domain (PRD) in hPLSCR1 and showed that the PRD is essential for the oligomerization and functional activation of hPLSCR1 .
hPLSCR1 interacts with c-terminal domain (CTD) of topoisomerase IIα (topo IIα) and enhances its decatenation activity . The mechanism of the enhancement of topo IIα catalytic activity by hPLSCR1 is not yet understood. We hypothesize that the mechanism of enhancement of the activity of topo IIα by hPLSCR1 could happen in two ways: (i) hPLSCR1 helps in unwinding or cleaving of catenated substrate for the topo IIα to subsequently decatenate; (ii) hPLSCR1 might have independent nuclease properties that could compliment the decatenation activity of topo IIα.
Based on the ability of hPLSCR1 to bind to DNA and Mg2+, cytoplasmic localization, stimulation by interferons, and enhancement of decatenation activity of topo IIα, we believed that hPLSCR1 could moonlight as a metal ion dependent nuclease. In this study, we have shown that recombinant hPLSCR1 exhibits nuclease properties contributing to a novel feature of this multifunctional protein.
Overexpression and purification of hPLSCR1
hPLSCR1 cDNA clone was obtained from Origene, MD, USA, and was subsequently cloned into pET-28a (+) bacterial expression vector with an N-terminal His tag. This was transformed into E. coli BL-21 (DE3) and grown in LB media containing kanamycin (50 mg/l). Overexpression and purification were performed as described earlier . Briefly, cells were lysed in buffer (20 mM Tris–HCl (pH – 7.4), 200 mM NaCl) by sonication, hPLSCR1 formed inclusion bodies (IB). N- lauroylsarcosine (N-LS) was used to recover native protein from IB followed by dialysis to remove N-LS and the proteins were purified to homogeneity using Ni2+-NTA chromatography. The eluted fractions from Ni2+-NTA chromatography were subjected to anion-exchange chromatography where DEAE sepharose (GE Healthcare, LC, UK) was used. The NaCl eluted protein fractions were again loaded in Ni2+-NTA resin and eluted. The purified protein was further concentrated by Amicon centrifugal filters (10 kDa cut-off) (Millipore, MA, USA) and visualized by silver staining or coomassie staining and confirmed by western blotting using anti-hPLSCR1 monoclonal antibody (Name: PLSCR1 antibody 1E9; catalog no - sc59645; specificity -human; origin - mouse monoclonal IgG1, Santa Cruz Biotechnology, TX, USA).
Generation of point mutant of hPLSCR1
Overlap PCR method was used to generate point mutations in hPLSCR1 where the 5 histidines (H12, H53, H111, H2111, H262) were mutated to alanine and was subsequently cloned in pET28a (+), confirmed by sequencing and named as Mut-hPLSCR1. Overexpression and purification of Mut-hPLSCR1 were done as described earlier. Briefly, mutagenic primers were synthesized to incorporate the specific point mutations (Fig. 8a). Using wild type hPLSCR1 gene as a template, PCR was performed with the following combinations of primers to produce 6 fragments and named as follows. Stage 1 PCR: Fragment 1 - F1, R6; Fragment 2 - F2, R5; Fragment 3 - F3, R4; Fragment 4 - F4, R3; Fragment 5 -F5, R2; Fragment 6 – F6, R1. Stage 2 PCR: Adjacent fragments are used as a template with appropriate primers as follows. Initial 10 cycles of PCR were performed without the primers after which the primers were added; Fragment A - Fragment 1 + Fragment 2 – Primers – F1 and R5; Fragment B - Fragment 3 + Fragment 4 – Primers – F3 and R3; Fragment C – Fragment 5 + Fragment 6 – Primers – F5 and R1; Stage 3 PCR: Fragment I – Fragment A + Fragment B – Primers F1 and R3; Fragment II – Fragment B + Fragment C – Primers F3 and R1. Stage 4 PCR: Mutant hPLSCR1 – Fragment I + Fragment II – Primers F1 and R1.
Decatenation assay was performed using the topo II decatenation assay kit (Topogen, CO, USA) as per manufacturer’s instructions. Briefly, 200 ng of kinetoplast DNA (kDNA) in standard Topo assay buffer (50 mM Tris–HCl (pH - 8.0), 150 mM NaCl, 10 mM MgCl2 and 0.5 mM dithiothreitol) was treated with 1 U of topo IIα pretreated with hPLSCR1 (30 min, 4 °C) and incubated for 15 min at 37 °C. Negative control has only the kDNA but not the enzyme. The products were visualized on a 1 % agarose gel stained with ethidium bromide.
Nuclease activity: gel assay
Nuclease assays were performed with 20 pmol of purified recombinant hPLSCR1, 200 ng of yeast/ human genomic DNA as substrate in the assay buffer containing 50 mM Tris–HCl (pH - 8.0), 150 mM NaCl, 10 mM MgCl2 and 0.5 mM dithiothreitol to a total volume of 20 μl and incubated at 37 °C for 1 h. The reaction products were separated by electrophoresis on 1 % agarose gel with ethidium bromide (0.5 μg/ml); 12 % native PAGE stained with ethidium bromide was also used to visualize the products of the nuclease reaction.
Nuclease activity: Kunitz assay
Amount of DNA : 25 μg (final concentration – 50 μg/ml)
Dilution factor : 20
Amount of protein : 1 pmol/μl
Incubation time : 15 min
Test : DNA treated with hPLSCR1
Control : Untreated DNA
Protease treatment and heat inactivation of hPLSCR1
hPLSCR1 was either heat inactivated by incubating at 65 °C for 15 min or treated with trypsin for 60 min at 37 °C and then used for the nuclease assays and Kunitz assays as described earlier. Mock treated hPLSCR1 was used as a positive control.
Effect of various parameters on nuclease activity
Nuclease assays were performed as described earlier with varying incubation times such as 5, 10, 15, 30, 60, 90 min respectively and were analyzed on a 1 % agarose gel stained with ethidium bromide. Nuclease assay was also performed at various enzyme concentrations and visualized on a 1 % agarose gel as described earlier. Various substrates such as RNA, linear dsDNA, ssDNA and plasmid DNA were used for nuclease assay to identify the specificity of nuclease activity of hPLSCR1. The effect of temperature on nuclease activity was observed by performing assays at 25 °C, 30 °C, 37 °C, 40 °C, 45 °C and 50 °C and incubated for 60 min for nuclease assay or 15 min for Kunitz assay. Nuclease assays were performed at various pH ranging from 4.5 to 9.5 with pH 8 as the reference pH and were quantified using Kunitz assay or visualized on 1 % agarose gel as described earlier. Effect of metal ions on nuclease activity was studied by performing nuclease assays using an assay buffer without MgCl2 and adding 10 mM of MgCl2, 10 mM of CaCl2 and 10 mM of ZnCl2 respectively. Dose dependent studies were also performed for Ca2+, Mg2+ and Zn2+ (5 mM, 10 mM, 15 mM, and 20 mM). Nuclease assays and Kunitz assays were set up in the presence and absence of 4 mM of different amino acid modifiers namely, N-ethylmaleimide (NEM), 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF), diethyl pyrocarbonate (DEPC), phenyl glyoxal (PG). The reaction mixtures were incubated at 37 °C for 60 min and analyzed on a 1 % agarose gel. Dose dependent Kunitz assays were performed for hPLSCR1 with 2 mM, 4 mM, 8 mM and 16 mM of DEPC.
Circular Dichroism studies
Circular Dichroism (CD) studies were performed with a JASCO J-810 spectropolarimeter (Jasco, MD, USA). Far UV-CD spectra were recorded at 25 °C with a thermostat cell holder. 10 μM of protein in assay buffer was used for the scan with increasing concentrations of MgCl2. Samples were scanned using 1 mm path length cuvettes from 250 to 190 nm.
Data represented was statistically analyzed using Student’s t-test and p < 0.01 was considered to be significant.
Overexpression and purification of recombinant hPLSCR1
Decatenation assay for topo IIα in the presence of hPLSCR1
Nuclease activity of hPLSCR1
Parameters affecting nuclease activity
Effect of protein modifying reagents on nuclease activity
Role of histidine residues in nuclease activity of hPLSCR1
hPLSCR1 is a multifunctional protein involved in several key cellular pathways such as cell cycle, apoptosis, Ca2+ homeostasis. In this study, we demonstrated that hPLSCR1 possesses novel function of nuclease activity for the first time. Finding the physiological implications of the nuclease activity of hPLSCR1 could pave way to identify new roles for hPLSCR1 in several cellular processes. Even though hPLSCR1 was reported to enhance the decatenation activity of topo IIα, the underlying mechanism is not yet deciphered. hPLSCR1 could either (i) alter the structure of topo IIα thereby leading to an increase in the catalytic activity; (ii) help in unwinding or cleaving of the DNA substrate bound to topo IIα; (iii) possess independent nuclease properties that aid in decatenation activity of topo IIα. To investigate this, the decatenation assay was performed with topo IIα pretreated with 2 pmol and 20 pmol of hPLSCR1. At low amounts of hPLSCR1, we observed enhancement of decatenation activity similar to previous report . When 20 pmol of hPLSCR1 was used in the decatenation assay, a distinct band was observed along with the decatenation products of topo IIα. The appearance of this unique band was further confirmed by performing the decatenation assay with hPLSCR1 in the absence of topo IIα explaining that apart from enhancing the decatenation activity of topoisomerases, hPLSCR1 could independently possess nuclease properties which could aid in the enhancing the catalytic activity of topo IIα. Topo IIα is known to interact with CAD nuclease and is involved in chromatic condensation. The CAD nuclease apart from digesting DNA during apoptotic execution, it also interacts with topo IIα and enhances the decatenation activity of topo IIα in vitro . In another report, topo IIβ interacts with an extracellular nuclease in mammalian spermatozoa and the interaction enhances the decatenation activity of topo IIβ . Similarly, we believed that hPLSCR1 could have nuclease properties, which could be the reason for enhancement of decatenation activity of topo IIα in vitro. To support this hypothesis, sequence alignment of hPLSCR1 with known nucleases with bioinformatics tools revealed some key findings. PRD of hPLSCR1 has vast similarities with the PRD of TREX1, a mammalian member of DnaQ-like 3′–5′ exonuclease family . TREX1 exonuclease activity is supported by a metal ion catalyzed phosphoryl transfer mechanism. TREX1 constitutes about 45 prolines and its PRD contains about 6 prolines, which are involved in interactions with various protein complexes [38, 39]. Sequence analysis showed that about 16 out of 29 prolines in PRD of hPLSCR1 were aligned and 4 prolines of TREX1 PRD exactly corresponded with PRD of hPLSCR1 (Data not shown). The Mg2+ binding site (176 ATEDMDCLT184) in human FLAP endonuclease I (FEN1), a protein involved in DNA replication and repair was also conserved in hPLSCR1 (142 AAEDTDCCT150). The bold characters in the sequence indicate the residues involved in Mg2+ binding in FEN1, which are conserved in hPLSCR1. Hence, these striking similarities along with various other factors in the literature such as Mg2+ binding, up regulation by interferons, involvement during apoptosis, interaction and enhancement of decatenation activity of topo IIα and processing of kDNA even in absence of topo IIα intrigued us to elucidate the unique property of hPLSCR1 as a nuclease.
Purified recombinant hPLSCR1 exhibited a dose dependent nuclease activity when incubated with yeast genomic DNA and human genomic DNA at 37 °C. Overexpression and purification was repeated for E. coli cells transformed with pET 28a (+) vector without any insert (vector-purified protein). No distinct bands were revealed in SDS-PAGE analysis of the vector-purified protein and the samples did not show any nuclease activity (data not shown). A random 37 kDa protein that was purified by the same protocol as mentioned in ‘Methods’ was also tested as a negative control to eradicate the possibilities of a co-purified contaminating host nuclease because of the purification protocol (data not shown). Absence of nuclease activity in vector-purified sample and in random protein clearly states that the nuclease activity is borne only by hPLSCR1 and not by other host contaminating nucleases. hPLSCR1 was active at temperatures between 25 and 45 °C (Fig. 5a), where a temperature of 37 °C was found to be optimum for its activity, which was similar for many other human nucleases such as TREX1, FLAP endonuclease 1 (FEN 1) and DNase 1. The optimal pH for nuclease activity was found to be between pH 8.0 and 9.0 (Fig. 5b) suggesting that hPLSCR1 probably acts as an alkaline nuclease. Most of the nonspecific nucleases such as TREX1 and FEN1 had highest activity at 7.5 and 8.0 respectively [38, 40]. This was further confirmed by assaying nuclease activity with various substrates such as ssDNA, dsDNA and RNA. hPLSCR1 exhibited nuclease activity towards dsDNA and RNA but not ssDNA. hPLSCR1 exhibited nicking activity towards plasmid DNA which could explain that hPLSCR1 might be a nicking endonuclease which needs to be further investigated. TREX-1 digests ssDNA and dsDNA with mismatched 3′ termini . These findings suggest that hPLSCR1 is a non-specific alkaline nuclease.
Majority of nucleases require metal ions such as Mg2+, Ca2+ and Zn2+ for their activity. Metal ions in nucleases are found to play a dual role: (i) enhancing the affinity of the substrate to the enzyme which could be sequence or structure specific and (ii) directly involved in catalysis where the phosphate oxygen bond is cleaved . To verify the specificity of metal ions for nuclease activity of hPLSCR1, assays were performed with Ca2+, Zn2+ and Mg2+ (Fig. 6). Ca2+ did not induce nuclease activity, which was astonishing as Ca2+ binding is vital for the PL translocation by hPLSCR1 across the lipid bilayer. It was found that Mg2+ and Zn2+ stimulated nuclease activity and might possibly play a role in stabilizing the enzyme structure apart from catalysis. hPLSCR1 has an EF hand like Ca2+ binding motif and point mutations in the D275 resulted in loss of calcium binding and scramblase activity . We also reported that Mg2+ binds to the EF hand like Ca2+ binding motif in hPLSCR1 (D273 – D284) . Hence to understand the metal binding properties of hPLSCR1, D275A point mutant of hPLSCR1 was generated and checked for scramblase activity and nuclease activity. The mutant D275A-hPLSCR1 showed a complete loss of scramblase activity, but the nuclease activity was not affected (data not shown). This could possibly explain that Mg2+ could have a binding site other than the EF-hand like motif which remains to be investigated. Further studies should be performed to understand the mechanism of Mg2+ interaction with hPLSCR1.
Protein modification experiments revealed that histidine, cysteine and arginine residues in hPLSCR1 were important for nuclease activity. Histidines and cysteines are the most commonly occurring active site residues in nucleases. In a similar study, DEPC modification of CAD nuclease inhibited the nuclease activity. CAD nuclease has two histidine residues at its active site that is essential for its nuclease activity. The active site of CAD possesses two histidine residues and DEPC modification inactivated the enzyme . Based on our results, we generated a histidine point mutant (Mut-hPLSCR1) where all the histidine residues in hPLSCR1 were mutated to alanine. Mut-hPLSCR1 showed a threefold decrease in nuclease activity only when assayed at 20-60 pmol and showed statistically insignificant activity at lower concentrations (2-10 pmol). This confirms that the nuclease activity is only by hPLSCR1 and eliminates the possibility of co-purification of a host contaminating nuclease. Histidine could be involved either in the catalytic site or for stabilizing the enzyme substrate complex which needs to be further investigated.
The mechanism and the physiological role of the nuclease property of hPLSCR1 in normal and pathological conditions remain to be investigated. In a recent report, hPLSCR1 was shown to be induced and localized to PM and periplasmic region upon dsDNA transfection in normal immortalized ovarian surface epithelial cells . The physiological relevance of this marked induction is not yet identified. Since it is already well documented that hPLSCR1 is a part of interferon mediated anti-viral defense, the induced hPLSCR1 during dsDNA transfection might have a role as a nuclease in defense against foreign DNA.
In summary, this is the first report showing a novel Mg2+ dependent activity of hPLSCR1. The nuclease activity was strictly metal dependent and could act on dsDNA, RNA but not ssDNA as substrates. DEPC treated hPLSCR1 exhibited reduced nuclease activity and further mutation of histidines resulted in a 60 % loss of activity. Histidines could play a critical role in nuclease activity of hPLSCR1. Further studies are required to ascertain the exact mechanism of nuclease activity, critical residues involved in catalysis, Mg2+ binding, substrate binding and the physiological relevance for nuclease activity of hPLSCR1.
4-(2- aminoethyl) benzenesulfonyl fluoride hydrochloride
double stranded DNA
Flap endonuclease 1
human phospholipid scramblase 1
Proline rich domain
single stranded DNA
Authors would like to acknowledge DST-FIST, Department of Science and Technology, Government of India (GoI) for CD studies. US would like to acknowledge Ministry of Human Resources Development (MHRD), GoI for fellowship.
This work was supported by research grant from Council of Scientific and Industrial Research, Government of India.
Availability of data and material
SNG devised and coordinated the study. SNG and US designed the experiments. US performed most of the experiments. SNM performed the experiments shown in Figs. 5, 6 and 7. SNG and US interpreted the data and wrote the manuscript. All authors read and approved the final manuscript.
The authors declare that they have no competing interest.
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