Site-directed mutagenesis in the P-domain of calreticulin transacylase identifies Lys-207 as the active site residue

Abstract

In silico-docking studies from previous work have suggested that Lys-206 and lys-207 of calreticulin (CR) play a pivotal key role in its well-established transacetylation activity. To experimentally validate this prediction, we introduced three mutations at lysine residues of P-domain of CR: K → A, P^mut−1 (K -206, -209), P^mut−2 (K -206, -207) and P^mut−3 (K -207, -209) and analyzed their immunoreactivity and acetylation potential. The clones of wild-type P-domain (P^wt) and three mutated P-domain (P^mut−1, P^mut−2 and P^mut−3) were expressed in pTrcHis C vector and the recombinant P^wt, P^mut−1, P^mut−2 and P^mut−3 proteins were purified by Ni–NTA affinity chromatography. Screening of the transacylase activity (TAase) by the Glutathione S Transferase (GST) assay revealed that the TAase activity was associated with the P^wt and P^mut−1 while P^mut−2 and P^mut−3 did not show any activity. The immune-reactivity to anti-lysine antibody was also retained only by the P^mut−1in which the Lys-207 was intact. Retention of the TAase activity and immunoreactivity of P^mut−1 with mutations introduced at Lys-206, Lys-209, while its loss with a mutation at Lys-207 residue indicated that lysine-207 of P-domain constitutes the active site residue controlling TAase activity.

Supplementary Information

The online version contains supplementary material available at 10.1007/s13205-021-02659-1.

Introduction

Calreticulin (CR) is an ER luminal protein that binds Ca²⁺ ions and is a eukaryotic cell molecular chaperone (Michalak et al. 2009). CR is split into three structural and functional domains. The amino terminal (N-domain) is highly conserved and composed of eight anti-parallel β-strands with a single disulfide bridge, and a globular structure. The N-domain has a binding site for carbohydrates and a binding site for Zn²⁺ ions. Proline-rich P- domain binds Ca²⁺ ions with high affinity (Michalak et al. 1999, 2009). The P- domain structure as predicted by NMR studies reveals that it contains an extended region stabilized by three antiparallel β-sheets and interacts in the ER lumen with other chaperones (ERp57) (Ellgaard et al. 2001) The carboxy terminal (C- domain) is a highly acidic cluster of aspartate and glutamate residues and possesses high-binding capacity for Ca²⁺ ions (Kd =  ~ 1–2 mM and ~ 25–50 mol Ca²⁺/mol protein) (Michalak et al. 1999, 2009). Calreticulin is a versatile multifunctional protein and plays a role in a wide range of pathways and biological systems including protein folding, Ca²⁺ homeostatis regulation, transcription pathway modulation, cell adhesion, apoptosis and embryonic development (Michalak et al. 2009; Hebert et al. 2007).

We have earlier established a novel role of CR in mediating the acetylation of certain functional proteins such as cytochrome P-450, cytochrome P-450 reductase, protein kinase C (PKC), nitric oxide synthase (NOS) and glutathione S-transferase (GST) using polyphenolic acetates (PA) as the donor of the acetyl group (Raj et al. 1998, 1999,2000; Bansal et al. 2008; Kohli et al. 2004) We delineated certain biological effects of CR mediated protein acetylation such as anti-mutagenic effects (Raj et al. 2001) enhancement of intracellular nitric oxide (NO) levels, inhibition of ADP-induced platelet aggregation and inhibition of protein kinase C in asthmatic patients resulting from CR’s transacetylase action on PA (Khurana et al. 2006; Gulati et al. 2007) Ectopic expression of human CR in cultured cells has been found to enhance endogenous levels of protein acetylation as well as modify the cytotoxicity of acetylated polyphenols, suggesting its role in protein acetylation and stress response, besides regulating calcium homeostasis in cells (Verma et al. 2011). Calreticulin is a multi-domain protein and we have been interested in identifying the protein acetyltransferase function with specific CR domains.

Haemonchus contortus blood-sucking nematode has been recently characterized by the production of the recombinant full-length CR and its three domains (Suchitra et al. 2005) Analysis of amino acid sequence from CR from H. contortus (hCR) shows a sequence identity of > 70 percent with human CR (Singh et al. 2009) Studies using clones of N, C and P domains of H. contortus have shown that the acylation of Schistosoma japonicum recombinant GST (rGST) using acyloxycoumarin/acetyl-CoA as donors of the acyl group is associated with the P – domain and comparable to the full-length hCR (Singh et al. 2009, 2011) Immuno-probe (acetylated lysine antibody) and LC–MS/MS analysis identified the acetylation of several lysine residues on rGST catalyzed by the P- domain using acetoxy coumarin/acetyl CoA as an acetyl group donor (Singh et al. 2011).

We earlier established that lysine residues -206, -207, -209 and -238 of the human placental calreticulin (CR) P – domain undergo autoacetylation during the CRTAase reaction progress (Bansal et al. 2009). Further, computational blind docking studies have predicted the putative site of binding acyloxycoumarins with CR in the protein acyltransferase function of CR that involves two P- domain lysine residues Lys- 206 and -207 (Ponnan et al. 2014).

To validate the predictions from the docking studies, we investigated the process to find out the active site residue of CRTAase by introducing mutation at the lysine residues of P-domain of CR, where lysine was replaced with alanine (K → A) using directed mutagenesis: P^mut−1 (K -206, -209), P^mut−2 (K -206,-207) and P^mut−3 (K -207,-209). Cloning in TA vector and then subcloning into pTrc HisC vector followed by purification using Ni–NTA affinity column and analysis of TAase activity in all three mutants showed that the lysine-207 of P-domain constitutes the active site residue controlling TAase activity.

Materials and methods

Cloning, expression and purification of recombinant P^wt and P^{mut−1,2 and 3} of CR

Primer designing

Human Calreticulin cDNA cloned at EcoRI site in pblue script was a kind gift from Dr. Robert Clark, University of Texas Health Sciences Centre, San Antonio. TX. USA (Fig. S1). All the mutants for P-domain (P^mut−1,2,3) as well as wild type P-domain (P^wt) clones were generated using this clone as the template and the following primers:

PF1: 5′-CTCGAGCCTATGAGGTGAAGATTGACAA-3′ Tm = 59.9, Xho I
PR1: 5′-GAATTCGATCTGCCGGGGCTTCCACT-3′ Tm = 62.6, EcoR I
PMR1: 5′-AGGATCCGCTATCTTCGCGGGTGGCAGGAAGTC-3′ Tm = 69.3
PMR2: 5′-AGGATCCTTTATCGCCGCAGGTGGCAGGAAGTC-3′ Tm = 68.1
PMR3: 5′-AGGATCCGCTATCGCCTTGGGTGGCAGGAAGTC-3′ Tm = 69.3

*Mutated oligonucleotides are underlined and shown in bold, where the mutation is introduced for replacing lysine by alanine. Restriction site of Xho I in forward primer and EcoR I in reverse primer is highlighted in bold.

The P-domain of CR was amplified by PCR reaction using suitable primers with restriction sites Xho I in the forward (PF1) and EcoR I in the reverse primer (PR1). The cDNA of CR was used as the template. The conditions for amplification were: an initial denaturation step at 95 °C for 5 min followed by 30 cycles of 30 s denaturation at 95 °C, 1 min annealing 60 °C and extension of 30 s at 72 °C. The eluted 310 bp amplicon was first cloned in pGEM-T Easy vector and transformed in E. Coli (DH5α). The recombinant gene was excised and then ligated to pTrc His-C expression vector at Xho I and EcoR I site. The sequence and the reading frame of the positive clone (pTrc His-C-P^wt) were confirmed by sequencing using the facility at M/s Macrogen, Seoul, Korea.

Cloning of mutated primers for mutants of P-domain of CR

To clone the mutants of the P-domain of CR, three primers were designed containing mutations at desired sites, to replace lysine with alanine. The following PCR reactions were carried out to generate mega primers (MPs): (i) PF1 and PMR1 were used to synthesize Mega Primer-1 (MP-1), where (K → A, -206,-209), (ii) PF1 and PMR2 were used to synthesize Mega Primer-2 (MP-2), where (K → A, -206,-207) and (iii) PF1 and PMR3 were used to synthesize Mega Primer-3 (MP-3), where (K → A, -207,-209). The conditions for amplification were: an initial denaturation step at 95 °C for 5 min followed by 30 cycles of 30 s denaturation at 95 °C, 1 min annealing at 65 °C with the extension of 30 s at 72 °C. The amplicons of MP-1, -2 and -3 were eluted from the gel and purified using a gel extraction kit from M/s Qiagen, USA.

Cloning of P^mut−1,2,3 of CR

The mutants of P-domain were amplified by PCR reaction using suitable MPs with restriction sites Xho I as the forward primer (MP-1,2,3) and PR1 as the reverse primer with EcoR I site. The cDNA of P^wt cloned above was used as the template. The wild type P-domain (P^wt) and the three mutants of P-domain (P^mut−1,2,3) were successfully cloned first into pGEM-T Easy vector and then sub-cloned into pTrcHis C expression vector. The mutated mega primers (MP-1,2,3) with desired mutations were synthesized using the following pairs of primers: (i) PF1 and PMR1 for Mega Primer-1 (MP-1), where K → A mutation at -206,-209 amino acid residues was introduced. (ii) PF1 and PMR2 for Mega Primer-2 (MP-2), where K → A mutation at -206,-207 was introduced and (iii) PF1 and PMR3 for Mega Primer-3 (MP-3), involving K → A mutation at -207,-209 residue. A 100 bp amplicon of MP-1, 2 and 3 obtained was excised and eluted from gel to use as forward primers for introducing mutations. The mutants of P-domain were amplified by using a combination of MP-1, 2 and 3 as forward primer separately with PR1 as the reverse primer, resulting into three mutated amplicons of P^mut−1,2,3 of 310 bp each. The 310 bp amplicons of P^wt and P^mut−1,2,3 were excised and eluted from the gel and successfully cloned into pGEM-T Easy vector. The white colonies obtained on X-gal plate after blue-white screening were cultured for each clone and isolated plasmids were digested with Xho I and EcoR I, a fall out of 310 bp in each case confirmed the cloning of positive clones which was further confirmed by sequencing. The pop-out of 310 bp from Xho I and EcoR I digested clones of TA-P^wt and P^mut−1,2,3 were eluted and sub-cloned into pTrc HisC expression vector. The positive clones obtained after digestion of pTrc HisC-P^wt/P^mut−1,2,3 with Xho I and EcoR I resulting in a fall out of 310 bp (Figure S2) were sequenced further to confirm the desired cloning of mutants. The reading frame as well as the mutation of the positive clones (pTrc HisC-P^mut−1/2/3) was confirmed by DNA sequencing using a facility at M/s Macrogen, Seoul, Korea.

Expression, purification and immuno-identification of the proteins from pTrc HisC-P^{wt/mut−1/2/3}

E. coli BL21 cells were transformed in pTrcHisC vector (pTrcHisC-P^{wt/mut−1,/2/3}) with a construct containing the wild type and mutated P- domains coding sequence. Transformed cells were cultured in 5 mL LB medium supplemented with ampicillin (50 µg/mL) and IPTG for 6 h at 37 °C. Cell pellets from 200 ml culture were frozen with 4 volumes of lysis buffer (20 mM sodium phosphate (pH 7.8), 1 M NaCl and 1 mg/mL lysozyme), thawed and incubated in ice for 30 min. It was then sonicated at 10 Hz for 1 min in ice (repeated twice with 1 min interval) and the suspension was centrifuged at 10,000xg for 15 min at 4 °C. The supernatant was mixed with Nickel-agarose resin (1 mL) for 30 min at 4 °C and rotated end over end and then packed in a column with a buffer consisting of 20 mM sodium phosphate (pH 7.8) and 1 M NaCl. Elution was performed with 5, 50 and 500 mM imidazole (pH 7.8). The expressed recombinant proteins P^wt/P^mut−1,2,3 (Figure S3) were purified by Ni–NTA column with 50 mM imidazole. The fractions containing the proteins were extensively dialyzed against 10 mM phosphate buffer (pH 7.4). All the purified proteins (P^{wt/mut−1/2/3}) were run on SDS-PAGE and bands were transferred to nitrocellulose membrane and probed with anti-His antibody (1:1000, v/v; Sigma Chemical Co., USA).

Protein acyltransferase activity of P^{wt/mut−1/2/3}

DAMC/DPMC/7 –AMC (7- acetoxy-4-methylcoumarin)/7 –PMC (7 – propanoyloxy-4-methylcoumarin)/acetyl-CoA/propanoyl CoA and rGST were used as substrates as described above.¹⁵ The assay mixture consisted of 0.25 M potassium phosphate buffer (pH 6.5), P^{wt/mut−1/2/3} (5 µg protein), DAMC/DPMC/7-AMC /acetyl –CoA/propanoyl-CoA (100 µM) added in 50 µL DMSO, purified rGST (5 µg protein) and water to make up the final volume to 0.8 mL. The contents of the tube were pre-incubated at 37 °C for different periods. The aliquots were periodically removed into a spectrophotometer cuvette containing 1-chloro 2,4-dinitrobenzene (CDNB) and reduced glutathione (GSH) to form their concentration (1 mM) in a total volume of 1 mL and the progress of rGST activity was followed at 340 nm using Cary spectrophotometer ( Cary Bio 100). The reactions in which DAMC/DPMC/7 –AMC/7-PMC/acetyl-CoA/propanoyl CoA or P^{wt/mut−1/2/3} have been omitted were used as controls respectively. Bi-substrate kinetics of the recombinant P^wt protein was used to explore the catalytic mechanism. The fixed concentrations of DAMC (5–25 μM) were incubated with varying concentrations of rGST (0.02–0.12 nM) in the above-described assay mixture. The double reciprocal plots between 1/v₀ v/s 1/[rGST] at different fixed concentrations of substrate i.e. DAMC were drawn. The data were analyzed using the kinetics of Cleland (Cleland 1999).

Demonstration of P^{wt/mut−1/2/3} catalyzed acylation of rGST by acyloxycoumarins (western blot)

Purified proteins (P^{wt/mut−1/2/3}) (10 µg) was separately incubated in potassium phosphate buffer (10 mM, pH 7.2) with rGST (30 µg), acyloxycoumarin (100 µM) for 30 min at 37° C in a shaking water bath followed by immunoblot analysis with anti-acetyl lysine antibody as described above (Singh et al. 2009).

Results and discussion

Expression, purification and immuno-identification of recombinant proteins (P^wt/P^mut−1,2,3)

The purified recombinant proteins when probed with anti-His antibody gave a prominent band of 19 kDa (Fig. 1). The molecular weights of the recombinant proteins (P^wt/P^mut−1,2,3) were found to be similar since the mutation is a point mutation only and has not affected the molecular mass.

Fig. 1.

Western blotting of recombinant P^wt/ P^mut−1,2,3 proteins using anti-His antibody. Purified recombinant P^wt/P^mut−1,2,3 proteins (15 µg) were run on SDS-PAGE and the bands were transblotted to PVDF and probed with anti-His antibody (1:1000, v/v; Sigma). Lane 1: rec. P^wt protein, lane 2: Prestain Protein marker, lane 3: rec. P^mut−1 protein, lane 4: rec. P^mut−2 protein, lane 5: rec. P^mut−3 protein and lane 6: Control (BSA). (P^wt: Recombinant Wild type P- Domain protein, P^mut−1: Recombinant P- Domain protein with a mutation at (K -206, -209), P^mut−2: Recombinant P- Domain protein with a mutation at (K -206,-207) and P^mut−3: Recombinant P- Domain protein with a mutation at (K -207,-209)

Protein acyltransferase activity of recombinant P^{wt/mut−1/2/3}

Analysis of the purified recombinant P^{wt/mut−1/2/3} for their transacylase activity (as described earlier)⁷ showed that P^wt and P^mut−1 could transfer the acyl moiety from acyloxycoumarins as well as from acyl CoA to the rGST, while P^mut−2 and P^mut−3 were devoid of the transacylase activity (Table 1). The kinetics of P^wt and P^mut−1 catalyzed transacylation revealed that the activity of the P^mut−1 was comparable to that of the P^wt (Table 1). These results together with an earlier demonstration of CR/P-domain of CR as a stable intermediate in acyltransferase activity (Singh et al. 2011), suggested the ping-pong mechanism of catalysis by the CR/P-domain catalyzed acylation of the receptor protein. To test this prediction, the bi-substrate kinetic analysis of P-domain (P^wt) of CR was carried out. We determined the steady-state kinetic parameters for rGST at various concentrations of DAMC, a potent donor of acyl group (Raj et al. 2000; Bansal et al. 2008; Kohli et al. 2004). Double reciprocal plots of initial velocities obtained from these experiments (Fig. 2) show a parallel line pattern that is suggestive of a “ping-pong” kinetic mechanism according to the algorithms of Cleland (v = V_max [A][B]/K_ma[B] + K_mb[A] + [A][B]). The kinetic parameters obtained fit to a “ping-pong” mechanism and yielded K_m values 0.15 ± 0.02 nM and 47 ± 1.8 µM for the rGST and DAMC as substrates, respectively; k_cat was found to be 1.21 ± 0.03 min⁻¹.

Table 1.

Comparison of specificities of rec. P^wt/P^mut−1 (P^wt: Recombinant Wild type P- Domain protein, P^mut−1: Recombinant P- Domain protein with a mutation at (K -206, -209) for acyloxycoumarins and acyl-CoA

Substrates	Pwt		Pmut−1
	Km (µM)	Vmax (units)	Km (µM)	Vmax (units)
DAMC	47 ± 1.52	165 ± 1.74	58 ± 1.15	134 ± 1.72
DPMC	70 ± 1.67	130 ± 1.23	91 ± 1.27	108 ± 1.56
7-AMC	64 ± 1.3	135 ± 1.87	83 ± 1.19	97 ± 1.56
7-PMC	93 ± 1.91	118 ± 1.47	117 ± 1.21	98 ± 1.37
Acetyl-CoA	152 ± 2.14	92 ± 1.31	128 ± 1.83	75 ± 1.62
Propanoyl-CoA	232 ± 3.24	68 ± 2.15	252 ± 3.83	52 ± 2.52

Substrates acyloxycoumarins/acyl CoA were separately preincubated (37 °C, 10 min) with P^wt/P^mut−1 and rGST in potassium phosphate buffer (pH 6.5) followed by the addition of GSH and CDNB. The absorbance was measured at 340 nm. Initial reaction velocities of TAase were determined at varying substrate concentrations (10–250 μM). Lineweaver–Burk plot of initial velocities in the presence of varying substrate concentrations were plotted and kinetic constants were computed

Fig. 2.

Bi-substrate kinetic analysis of P-domain of CR. Bi-substrate kinetics of the P^wt (recombinant wild type P-Domain protein) of CR is used to investigate the mechanism of catalysis. Initial velocity pattern is shown in the double reciprocal plot in which 1/velocity is plotted against 1/[rGST]. The fixed concentration of DAMC were 5 µM (●), 10 µM (▲), 15 µM (■) and 25 µM (♦). The best fit to the data (in triplicates) displayed a parallel line pattern consistent with a ping-pong kinetic mechanism

Recombinant P^{wt/mut−1/2/3} catalyzed acylation of rGST by acyloxycoumarins

To find out as to which lysine residue was involved in the acylation process, the purified recombinant P^wt/ P^mut−1,2,3 proteins were separately preincubated with rGST and 7,8-dipropanoyloxy-4-methylcoumarin, an acyl group donating substrate followed by Western blotting with anti-acetyl lysine antibody, which detects post-translationally modified proteins in epsilon-amine groups of the lysine residues. Blots clearly revealed that both the P^wt and P^mut−1protein have their transacylase activity (Fig. 3a, lane 1 and 5) intact while transacylase activity of P^mut−2 and P^mut−3 was abolished (Fig. 3a, lane 3 and 4). A parallel SDS-PAGE (Fig. 3b) was run to show the presence and purity of all the proteins (P^wt/P^mut−1,2,3 and rGST). Retention of the TAase activity when lysine residue -207 was not mutated highlighted the important role of lysine -207 of P-domain in the TAase activity.

Fig. 3.

Western Blotting analysis of recombinant P^wt/ P^mut−1,2,3 catalyzed propionylation of rGST. a Immunobloting with anti-acetyl lysine antibody. b SDS-PAGE. Purified recombinant P^wt/P^mut−1,2,3 proteins (10 µg) were incubated with rGST (30 µg) and acyloxycoumarin were run on SDS-PAGE and the bands were transblotted to PVDF and probed with anti-acetyl lysine antibody. (P^wt: Recombinant Wild type P-Domain protein, P^mut−1: Recombinant P- Domain protein with a mutation at (K -206, -209), P^mut−2: Recombinant P- Domain protein with a mutation at (K -206,-207) and P^mut−3: Recombinant P- Domain protein with a mutation at (K -207,-209). Lane 1: rec. P^wt + rGST + DPMC (100 µM), lane 2: Prestained protein marker, lane 3: rec. P^mut−1 + rGST + DPMC (100 µM), lane 4: rec. P^mut−2 + rGST + DPMC (100 µM), lane 5: rec. P^mut−3 + rGST + DPMC (100 µM)

The physiological relevance of protein acetylation mediated by CRTase has been well established (Raj et al. 2000; Bansal et al. 2008; Kohli et al. 2004; Verma et al. 2011; Khurana et al. 2006; Gulati et al. 2007). Identification of Lys-207 as the critical residue in the active site of CRTase from the present studies has provided opportunities for developing pharmacological agents that can target this site (and the residue) that can either up-regulate or down-regulate CRTase activity. This can be exploited for modulating the functioning of several proteins including physiologically relevant (and their changes implicated in pathologic conditions) enzymes whose activity is linked to their acetylation status. Future efforts should be focused on the development of these agents that specifically target Lys-207 in the P-domain of CR.

Conclusion

The transacetylation potential of CR in acetylating target proteins using acyloxycoumarins/acyl CoA as donors of the acyl group has been well-established earlier, with an active site localized to the P-domain of CR. Results of the present study using site-directed mutagenesis have established that the lysine residue 207 in the P domain of CR is critical for the TAase activity.

Supplementary Information

Below is the link to the electronic supplementary material.

Abbreviations

7-AMC

7-Acetoxy-4-methylcoumarin

CR

Calreticulin

CDNB

1-Chloro-2,4-dinitrobenzene

CRTAase

Calreticulin transacylase

DAB

Diaminobenzidene

DAMC

7,8-Diacetoxy-4-methylcoumarin

DHMC

7,8-Dihydroxy-4-methylcoumarin

DPMC

7,8-Dipropanoyloxy-4-methylcoumarin

GSH

Reduced glutathione

GST

Glutathione S-transferase

HAT

Histone acetyltransferase

PA

Polyphenolic acetates

P^mut−1,2,3

P-domain mutant-1,2,3

P^wt

Wild type P-domain

NO

Nitric oxide

NOS

Nitric oxide synthase

7-PMC

7-Propanoyloxy-4-methylcoumarin

MP

Mega Primer

PVDF

Polyvinylidene difluoride

rGST

Recombinant glutathione S-transferase

SDS-PAGE

Sodium dodecylsulphate-polyacrylamide gel electrophoresis

TAase

Transacylase activity

Author contributions

Rini Joshi and Prabhjot Singh were involved in the planning and executing the entire experimental work and preparing the initial draft of the manuscript. Naresh K. Sharma was helpful in purifying the recombinant proteins. Prija Ponnan was involved in docking studies. Jasvinder K. Gambhir, Diwan S. Rawat and Bilkeri S. Dwarakanath were helpful in the preparation, finalization and proofreading of the manuscript. Virinder S. Parmar and Ashok K. Prasad provided all the coumarins used in these studies. Daman Saluja planned the entire procedure of the experiments and guided through the entire experiments and was involved in the preparation and proofreading of the manuscript. The initial work was carried out in the Laboratories of (Late) Professor Hanumantharao G. Raj under his supervision.

Compliance with ethical standards

Conflicts of interest

All authors consent to this submission and declare no conflicts of interest.

Ethical statement

The authors bear all the ethical responsibilities for the work included in this manuscript. They declare that the research was conducted in the absence of any commercial or financial relationships and that it does not include any animal and/or human trials.