Protein acyltransferase function of purified calreticulin. Part 1: Characterization of propionylation of protein utilizing propoxycoumarin as the propionyl group donor

Article abstract of DOI:10.1093/jb/mvq002

We have earlier reported that an endoplasmic reticulum luminal protein calreticulin (CR) mediated the acetylation of certain receptor proteins such as glutathione S-transferase (GST) by polyphenolic acetates, leading to irreversible inhibition. This function of calreticulin was termed calreticulin transacetylase. In this communication, we have demonstrated for the first time the ability of the purified recombinant calreticulin of a parasitic nematode Haemonchus contortus to transfer propionyl group from 7,8-Dipropoxy-4- methylcoumarin (DPMC) to recombinant Schistosoma japonicum glutathione S-transferase (rGST). Calreticulin transacetylase exhibited hyperbolic kinetics and yielded Km (140 μM) and Vmax (105 units) when the concentration of DPMC was varied keeping the concentration of rGST constant. rGST thus propionylated was found to positively interact with anti-acetyl lysine antibody. Also, the nanoscale LC-MS/MS analysis identified the propionylation sites on three lysine residues: Lys-11, -180 and -181 of rGST. These results highlight the transacylase function of calreticulin (CRTAase). The Authors 2009. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

Full text of DOI:10.1093/jb/mvq002

J. Biochem. 2010;147(5):625–632 doi:10.1093/jb/mvq002
Protein acyltransferase function of purified calreticulin. Part 1:
characterization of propionylation of protein utilizing
propoxycoumarin as the propionyl group donor
Received August 22, 2009; accepted November 8, 2009; published online January 12, 2010
Prabhjot Singh1, Prija Ponnan1,2
Shibu Krishnan3, Tapesh Kumar Tyagi1,
,
DAB, diaminobenzidene; DAMC, 7,8-diacetoxy-4-
methylcoumarin; DHMC, 7,8-dihydroxy-4-
methylcoumarin; DPMC, 7,8-dipropoxy-4-
Nivedita Priya1, Seema Bansal1,2
,
Domenica Scumaci3, Marco Gaspari3,
Giovanni Cuda3, Paritosh Joshi4,
Jasvinder Kaur Gambhir5, Daman Saluja6,
Ashok Kumar Prasad2, Luciano Saso7,
Ramesh Chandra Rastogi2,
methylcoumarin; GSH, reduced glutathione; GST,
glutathione S-transferase; HAT, histone acetyltrans-
ferase; NOS, nitric oxide synthase; PA, polyphenolic
acetates; 7-PMC, 7-propoxy-4-methylcoumarin;
PVDF, polyvinylidene difluoride; rGST, recombinant
glutathione S-transferase; rhCRTAase, recombinant
Haemonchus contortus calreticulin transacylase;
SDS—PAGE, sodium dodecylsulpha-
Virinder Singh Parmar2 and
Hanumantharao Guru Raj1,*
te—polyacrylamide gel electrophoresis.
1Department of Biochemistry, V.P. Chest Institute; 2Department of
Chemistry, University of Delhi, Delhi 110007, India; Dipartimento
3
di Medicina Sperimentale e Clinica, Universita di Catanzaro ‘Magna
Græcia’ Campus ‘S. Venuta’, Loc. Germaneto 88100, Catanzaro,
Italy; 4Division of Biochemistry, Indian Veterinary Research
Institute, Izatnagar 243 122; 5Department of Biochemistry,
University College of Medical Sciences, Shahdra, Delhi 110095;
6Dr B.R. Ambedkar Centre for Biomedical Research, University of
Delhi, Delhi 1100 07, India; and 7Department of Human Physiology
and Pharmacology ‘Vittorio Erspamer’ Sapienza University of
Rome, P.le Aldo Moro 500185, Rome, Italy
Post-translational modifications plays pivotal role in
regulation of cell processes. One of the major post
translational modifications is lysine modification by
way of methylation, phosphorylation and acetylation.
Recently, propionylation and butyrylation of lysine is
reported in histones mediated by histone acetyltrans-
ferase (HAT) such as p300/CBP (1).
*Hanumantharao Guru Raj, Professor and Head, Department of
Biochemistry, V.P. Chest Institute, University of Delhi, Delhi 1100
07, India. Tel: þ91 11 27667497, Fax: þ91 11 27667497,
E-mail: rajhg@yahoo.com
Our laboratory has concentrated on the novel role of
calreticulin (CR) in mediating the acetylation of cer-
tain functional proteins such as cytochrome P-450,
cytochrome P-450 reductase, nitric oxide synthase
(NOS) and glutathione S-transferase (GST) utilizing
polyphenolic acetates (PA) as the acetyl group
donors (2—6). We delineated certain biological effects
such as the anti-mutagenic action (7), enhancement
of intracellular levels of nitric oxide (NO), inhibition
of ADP-induced platelet aggregation and inhibition of
protein kinase C in the asthmatic patients arising out
of the transacetylase action of CR on PA (8, 9).
In the present communication, we have attempted to
study acyltransferase function of CR utilizing propox-
ycoumarin as a propionyl group donor. Recently, we
have reported the placental CR mediated acetylation
of neuronal NOS by DAMC and the concomitant
autoacetylation of CR by using mass spectrometry
(5, 10). The analysis of post-translational modifica-
tions of protein by mass spectrometry has gained
popularity because of its sensitivity and rapid charac-
terization (11, 12). We have here observed CR
mediated propionylation of the receptor protein, a
recombinant GST of Schistosoma japonicum (rGST)
by 7,8-Dipropoxy-4-methylcoumarin (DPMC) using
nanoscale-tandem mass spectrometry (MS/MS).
Hence, calreticulin protein acetyltransferase is now
assigned the generalized term calreticulin transacylase
(CRTAase).
We have earlier reported that an endoplasmic reticulum
luminal protein calreticulin (CR) mediated the acetyla-
tion of certain receptor proteins such as glutathione
S-transferase (GST) by polyphenolic acetates, leading
to irreversible inhibition. This function of calreticulin
was termed calreticulin transacetylase. In this commu-
nication, we have demonstrated for the first time
the ability of the purified recombinant calreticulin of
a parasitic nematode Haemonchus contortus to transfer
propionyl group from 7,8-Dipropoxy-4-methylcoumarin
(DPMC) to recombinant Schistosoma japonicum
glutathione S-transferase (rGST). Calreticulin transa-
cetylase exhibited hyperbolic kinetics and yielded Km
(140 kM) and Vmax (105 units) when the concentration
of DPMC was varied keeping the concentration of
rGST constant. rGST thus propionylated was found
to positively interact with anti-acetyl lysine antibody.
Also, the nanoscale LC-MS/MS analysis identified the
propionylation sites on three lysine residues: Lys-11,
-180 and -181 of rGST. These results highlight the
transacylase function of calreticulin (CRTAase).
Keywords: anti-acetyl lysine antibody/calreticulin/
Haemonchus contortus/propoxycoumarins/protein
acylation.
Abbreviations: CDNB, 1-chloro-2,4-dinitrobenzene;
CR, calreticulin; CRTAase, calreticulin transacylase;
ß The Authors 2010. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved
625

P. Singh et al.
digested according to Schevchenko et al. (15). Briefly, sliced gel
pieces were washed with 100 mM NH4HCO3 and HPLC-grade acet-
onitrile (Sigma-Aldrich) (1 : 1, v/v) (buffer A). Then, the protein was
in-gel reduced, and subsequently alkylated with iodoacetamide
(Sigma). After a washing step with buffer A, the gel pieces were
dried in a vacuum centrifuge, and then rehydrated at 4ꢀC in diges-
tion buffer (50 mM NH4HCO3, 5 mM CaCl2) containing 25 ng/ml
trypsin. After overnight incubation, the peptides were extracted
from the gel using three separate washings with a mixture of
acetonitrile/water/formic acid 70/25/5 (v/v/v). The extracts were
combined and dried down in a vacuum centrifuge.
Chromatography was performed on an Ultimate nano LC system
from Dionex (Sunnyvale, CA), using a valveless setup (16, 17). The
peptide extracts were redissolved in 40 ml of loading pump solvent
(see below) and 10 ml were loaded onto an in-house packed 100 mm
i.d. Integra FritTM (New Objective, Cambridge, MA) trapping
column (packing bed length 1.5 cm) at 10 ml/min flow of loading
pump solvent, consisting of H2O/acetonitrile/trifluoroacetic acid
(TFA) 97.95:2:0.05 (v/v/v). After 4 min of column washing, the trap-
ping column was switched on-line to the analytical column: an in-
house packed 50 mm i.d. Pico FritTM column (New Objective), filled
with the same stationary phase used for the trapping column pack-
ing: 3 mm C18 silica particles (Dr Maisch, Entringen, Germany).
Peptide separation started at 100 nl/min using a binary gradient.
Materials and Methods
Cloning, expression and purification of recombinant calreticulin
transacylase (rhCRTAase) from H. contortus
rhCRTAase was cloned in pPRO EX TM expression system. The
clone was bulk cultured and the bacterial cell lysate were prepared.
rhCRTAase was purified to homogeneity as described earlier in
detail (13).
Assay of rhCRTAase
rhCRTAase was assayed (14) using DAMC/DPMC and rGST as the
substrates as per the details given in our earlier report (4). The assay
mixture consisted of 0.25 M potassium phosphate buffer (pH 6.5),
rhCRTAase (5 mg protein), DAMC/DPMC (100 mM) added in 50 ml
DMSO, purified rGST (5 mg protein) and water to make up 0.8 ml.
The contents of the tube, scaled up as per requirement were prein-
cubated at 37ꢀC for various periods. The aliquots were removed
periodically into a spectrophotometer cuvette containing CDNB
and GSH to make up their concentration (1 mM) in a total
volume of 1 ml and the progress of the rGST activity was followed
at 340 nm using a Cary spectrophotometer (Cary Bio100). Reactions
wherein substrate i.e. DAMC/DPMC or rhCRTAase were omitted,
respectively, were used as controls. The unit of TAase was expressed
in terms of percentage inhibition of rGST under the conditions of the
assay and ensured that the reaction was linear with respect to
enzyme concentration and incubation time.
Mobile phase
A
was H2O/acetonitrile/formic acid/TFA
97.9:2:0.09:0.01 (v/v/v/v); mobile phase B was H2O/acetonitrile/
formic acid/TFA 29.9:70:0.09:0.01 (v/v/v/v). Gradient was from 5
to 45% B in 40 min. After 10 min at 95% B, the column was
re-equilibrated at 5% B for 20 min before the following injection.
MS detection was performed on a QSTAR XL hybrid LC-MS/
MS from Applied Biosystems (Foster City, CA) operating in positive
ion mode, with nESI potential at 1,300 V, curtain gas at 15 units,
CAD gas at 3 units. Information-dependent acquisition (IDA) was
performed by selecting the two most abundant peaks for MS/MS
analysis after a full TOF-MS scan from 400 to 1,600 m/z lasting 2 s.
Both MS/MS analyses were performed in enhanced mode (2 s/scan).
Threshold value for peak selection for MS/MS was 30 counts.
Measurement of kinetic constants for rhCRTAase utilizing
acyloxycoumarins
The enzyme was assayed as per the procedure described above. The
concentrations of DAMC and DPMC were varied from 10 to
200 mM keeping the concentration of rGST constant during preincu-
bation with rhCRTAase for 10 min followed by the addition of GSH
and CDNB for GST assay. The reciprocal of the initial rate of
CRTAase expressed in terms of % inhibition of GST under condi-
tion of assay was plotted (Lineweaver—Burk) against the reciprocal
of the concentration of DPMC/DAMC to deduce the Km and Vmax
values.
Data analysis
Demonstration of rhCRTAase catalyzed acylation of rGST by
DPMC/DAMC (western blot)
MS/MS data were converted to Mascot Generic Format (mgf) by
the Analyst software 1.1 (Applied Biosystems). Data were searched
on the Mascot search engine (www.matrixscience.com), version 1.9
(18), against the MSDB protein database (accessed on October 2007)
using the following parameters: MS tolerance 20 ppm; MS/MS
tolerance 0.3 Da; fixed modifications carbamidomethyl cysteine;
variable modifications methionine oxidized, propionyl (K); enzyme
trypsin; max. missed cleavages 2 (19).
Polyclonal anti-acetyl lysine antibody was used for the recognition of
propionylated lysine residues of receptor protein. Purified rhCRTAa
se (10 mg) was incubated with rGST (30 mg), DPMC/DAMC
(100 mM) in 10 mM potassium phosphate buffer (pH 7.2) incubated
for 30 min at 37ꢀC in a shaking water bath. After the completion of
reaction, sample buffer (loading dye) was added in the reaction mix-
ture to stop the reaction. This reaction mixture was used to detect
the propionylation of rGST by western blot technique. The reaction
mixture devoid of rhCRTAase was chosen as an appropriate control
to elucidate the role of CRTAase in catalyzing the enzymatic
propionylation of the receptor protein. After electrophoresis, the
separated protein on the gel was transblotted to a polyvinylidene
difluoride membrane (PVDF) at 300 mA for 3.5 h at 4ꢀC. PVDF
membranes were kept in blocking reagent (5% blotto) to block the
non-specific sites. The primary antibody was diluted (1:1,000) in tris-
buffer saline with 0.05% Tween-20 detergent (TBST) containing 1%
BSA and was incubated with the blot for 1 h at 4ꢀC with moderate
agitation. The PVDF membranes were extensively washed with
TBST, followed by incubation with secondary antibody (goat
anti-rabbit HRP conjugate) for same time duration at room temper-
ature. The membrane was washed extensively with TBST/TBS and
the transblotted acylated protein band was visualized by treating
with diaminobenzidene (DAB) and hydrogen peroxide.
Autoacylated rhCRTAase as a stable intermediate
For the preparation of autopropionylated/autoacetylated rhCRTAa
se, the protein (100 mg) was incubated separately with DPMC/
DAMC (100 mM) for 30 min and run on Native-PAGE under
non-denaturing condition at 4ꢀC. Negative staining was done to
locate the bands of autopropionylated/autoacetylated rhCRTAase,
the bands were cut and protein was eluted by crushing the gel in
PBS. For the proper elution of protein, the crushed gel pieces were
kept in PBS for overnight at 4ꢀC. The autopropionylated/autoace-
tylated rhCRTAase so obtained was incubated with purified rGST
separately for 30 min at 37ꢀC. Further, western blotting was per-
formed as described above to identify acylated rGST.
Results
Demonstration of propionylation of rGST by nanoscale
LC-MS/MS
Immunoblot identification of rhCR and rGST
The rhCR and rGST purified (Figs 1A and 2A) to
homogeneity were identified as evident from immunor-
eactivity of the former with anti-calreticulin antibody
(Fig. 1B) (1:1,000, v/v; Stressgen) and the latter with
anti-GST peroxidase antibody (Fig. 2B) (1:2,500, v/v;
Sigma).
In test sample purified rhCRTAase (10 mg) was incubated with rGST
(30 mg), DPMC (100 mM) in 10 mM potassium phosphate buffer
(pH 7.2) incubated for 30 min at 37ꢀC in a shaking water bath.
The sample devoid of purified rhCRTAase served as control.
These mixtures were then boiled in denaturing buffer prior to
SDS—PAGE. After electrophoretic separation, the gel was stained
with coomassie brilliant blue dye. Gel bands corresponding to the
expected rGST molecular weight (26,000 Mr) were excised and
626

Calreticulin transacylase
1
2
0.09
0.08
0.07
0.06
0.05
0.04
0.03
0.02
0.01
0
Lane
Km = 90
M
B
A
A
V
max = 143 units
117
Lane
1
2
117
85
85
rhCRTAase
(60 kDa)
rhCRTAase
(60 kDa)
48
34
48
34
– 0.02
0
0.02
0.04
0.06
0.08
0.1
M
0.12
–0.01
26
19
26
19
–1
0.14
0.12
0.1
Fig. 1 Purity of rhCR and its immunological identification using
anti-calreticulin antibody. (A) SDS—PAGE analysis of purified rhCR.
Lanes: 1, Purified rhCR (60,000 Mr); lane 2, Mr standards
(b-galactosidase 117,000 Mr, bovine serum albumin 85,000 Mr,
ovalbumin 48,000 Mr, carbonic anhydrase 34,000 Mr,
B
Km = 140
max = 105 units
V
0.08
0.06
0.04
0.02
0
b-lactoglobulin 26,000 Mr, lysozyme 19,000 Mr). (B) Western blot
was carried out using anti-calreticulin antibody (1: 1,000, v/v;
Stressgen). Lane 1, Mr standards (b-galactosidase 117,000 Mr,
bovine serum albumin 85,000 Mr, ovalbumin 48,000 Mr, carbonic
anhydrate 34,000 Mr, b-lactoglobulin 26,000 Mr, lysozyme 19,000
Mr); lane 2, rhCR (10 mg).
– 0.05
–0.02
0
0.05
0.1
0.15
–1
B
A
Lane
2
1
Lane
1
2
Fig. 3 CRTAase assay. Substrates DAMC and DPMC were sepa-
rately preincubated (37ꢀC, 10 min) with rhCRTAase 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 CRTAase were determined at varying substrate
concentrations (10—200 mM). (A) Lineweaver—Burk plot of initial
velocities in the presence of varying DAMC concentrations. (B)
Lineweaver—Burk plot of initial velocities in the presence of varying
DPMC concentrations.
117
117
85
85
48
34
48
34
rGST
(26 kDa)
rGST
(26 kDa)
26
19
26
19
of acetoxy and propoxycoumarins in CRTAase
activity, we investigated the kinetic parameters of the
rhCRTAase in more detail. Initial reaction velocities
were determined at varying substrate concentra-
tions (10—200 mM), and Lineweaver—Burk (double-
reciprocal) plots were produced and the Km and Vmax
values were subsequently determined for each
substrate. The Km and Vmax obtained for DAMC
were 90 mM and 143 units, respectively (Fig. 3A)
while Km and Vmax values for DPMC were 140 M
and 105 units, respectively (Fig. 3B).
Fig. 2 Purity of rGST and its immunological identification using
anti-GST peroxidase antibody. (A) SDS—PAGE analysis of purified
rGST. Lanes: 1, Purified rGST (26,000 Mr); lane 2, Mr standards
(b-galactosidase 117,000 Mr, bovine serum albumin 85,000 Mr,
ovalbumin 48,000 Mr, carbonic anhydrase 34,000 Mr,
b-lactoglobulin 26,000 Mr, lysozyme 19,000 Mr). (B) Western blot
was carried out using anti-GST peroxidase antibody (1: 2,500, v/v;
Sigma). Lane 1, Mr standards (b-galactosidase 117,000 Mr, bovine
serum albumin 85,000 Mr, ovalbumin 48,000 Mr, carbonic anhydrate
34,000 Mr, b-lactoglobulin 26,000 Mr, lysozyme 19,000 Mr); lane 2,
rGST (15 mg).
Immunoblot identification of CRTAase catalyzed
acylation of rGST by DAMC/ DPMC
Receptor protein like rGST was used as the substrate
for CRTAase catalyzed acylation (acetylation and pro-
pionylation) by DAMC and DPMC. The rGST was
separately preincubated with the substrates and
rhCRTAase followed by western blot using anti-acetyl
lysine antibody. Figure 4 (lanes 1 and 6) clearly shows
acylated rGST bands as well as the autoacylation of
CRTAase when either of the acyl group donors
(DAMC and DPMC) were used as substrates.
Specificities of transacylase to propoxy derivative of
coumarins
The transacylase activity of rhCR (recombinant
H. contortus calreticulin) was assayed by the method
of Raj et al. (4) similar to the assay of transacetylase.
The relative specificities of propoxy and acetoxy deriv-
ative of 4-methylcoumarins were studied to compare
the ability of acyloxy homologues to act as donor for
CRTAase mediated protein acylation. For this pur-
pose, DAMC and DPMC were preincubated with
CRTAase and rGST followed by assay of GST as
described earlier (4) in order to measure the
CRTAase activity. The time dependent preincubation
of reaction mixture showed a linear enhancement in
CRTAase activity. To further understand the effect
Autoacylated CRTAase as the stable intermediate in
the protein acyltransferase reaction
The autoacylated CRTAase prepared by incubation
with DAMC and DPMC was isolated on Native Gel.
The isolated autoacylated CRTAase (Fig. 5, lanes 1
627

P. Singh et al.
and 5) was found capable of acylating rGST indepen-
dent of acyl group donors DAMC/DPMC as demon-
strated by the immunoreactivity of the acylated rGST
with anti-acetyl lysine antibody (Fig. 5, lanes 2 and 4).
Further, by immunoreactivity with anti-acetyl lysine
antibody it is evident that the autoacylated
rhCRTAase upon interaction with rGST resulted in
the transfer of acyl groups as revealed by faint band
intensity of the autoacylated rhCRTAase during west-
ern blot (Fig. 5, lanes 2 and 4).
MS analysis as described in the MATERIALS AND
METHODS section. The protein was identified with a
Mascot score of over 300 in all samples, and an overall
sequence coverage of 28%. Interestingly, two peptides
bearing in total three covalent propionylations on
three different lysines were identified by MS/MS data-
base search. The overall list of identified peptides, with
special emphasis on modified ones, is reported in
Table I.
Propionylated peptides were only detectable in test
sample and not in the control sample. This could be
appreciated by drawing selected ion chromatograms
(SICs) for each of the two propionylated peptides
detected. Figure 6 shows SICs for one of the two pep-
tides, as an example. It can be appreciated that the
intense peak identified by data-dependent MS/MS as
being, propionylated IKGLVQPTR (retention time
30 min, m/z 534.3) could not be found back in the
SICs referring to the control sample. This indicated
that the specific modifications detected by MS/MS
were only present in the incubation mixture containing
the propionyl donor and the corresponding enzyme.
Figure 7A and B reports the MS/MS spectra of the
two precursor ions which allowed for the identification
of three propionylation sites. Propionylation is not
expected to produce any intense neutral loss fragment,
similarly to what recently reported for lysine acetyla-
tion. In fact, peptide sequences and the covalent
Nanoscale LC-MS/MS
In order to confirm covalent propionylation of the
target protein and to pinpoint as many sites of mod-
ification as possible, the incubation mixtures were
separated by SDS—PAGE. The protein bands corre-
sponding to rGST protein in the control sample and
in the putatively propionylated sample were subjected
to in-gel digestion by trypsin and nanoscale LC-MS/
4
5
1
2
3
6
117
85
48
1
2
3
5
4
85
34
26
48
34
26
19
Fig. 4 rhCRTAase catalyzed acetylation and propionylation of rGST
by DAMC/DPMC: western blot analysis using anti-acetyl lysine
antibody. Lane 1, rhCRTAase þ DPMC (100 mM) þ rGST; lane 2,
DPMC (100 mM) þ rGST (control); lane 3, Mr standards
(b-galactosidase 117,000 Mr, bovine serum albumin 85,000 Mr,
ovalbumin 48,000 Mr, carbonic anhydrase 34,000 Mr,
b-lactoglobulin 26,000 Mr, lysozyme 19,000 Mr); lane 4, rhCRTAase
þ DMSO þ GST (control); lane 5, DAMC (100 mM) þ GST; lane 6,
rhCRTAase þ DAMC (100 mM) þ GST. Details are given under the
MATERIALS AND METHODS section.
Fig. 5 Autoacylated rhCRTAase catalyzed acylation of rGST.
Autoacylated rhCRTAse isolated from Native PAGE (as described
under the MATERIALS AND METHODS section) was incubated with rGST
and western blot was performed using anti-acetyl lysine antibody.
Lane 1, isolated autoacetylated rhCRTAase; lane 2, autoacetylated
rhCRTAase þ rGST; lane 3, Mr standards (bovine serum albumin
85,000 Mr, ovalbumin 48,000 Mr, carbonic anhydrase 34,000 Mr,
b-lactoglobulin 26,000 Mr); lane 4, autopropionylated rhCRTAase
þ rGST; 5, isolated autopropionylated rhCRTAase. Details are
given under the MATERIALS AND METHODS section.
Table I. Peptides from rGST of Schistosoma japonicum identified by nanoscale LC-MS/MS and Mascot database search.
Observed
Mw
Mw
Delta
Missed
Mascot
Peptide sequence
513.79
516.79
531.81
591.85
657.85
534.33
1025.58
1031.57
1061.6
1181.68
1313.69
1066.64
1025.58
1031.58
1061.61
1181.68
1313.69
1066,65
0.00
ꢁ0.01
0.00
0.00
0.00
0
0
2
1
1
1
59
55
47
49
67
40
IEAIPQIDK
LTQSMAIIR
LVCFKKR þ2 Propionyl
RIEAIPQIDK
IAYSKDFETLK
IKGLVQPTR þ Propionyl
ꢁ0.01
Peptide fragments with high Mascot scores are provided here (adequate explanations are given in the RESULTS section). Modified lysines
(propionylated) are underlined and in bold.
628

Calreticulin transacylase
found to be bimolecular in nature and the formation
of 7,8-dihydroxy-4-methyl coumarin (DHMC),
a
deacetylated product of the CRTAase catalyzed reac-
tion was identified by us (4, 22). Our earlier studies
established that CR isolated from rat liver and
human placenta catalyzed acetylation of receptor
protein GST 3-3 (21, 23) by DAMC as observed by
interaction of acetylated GST 3-3 with anti-acetyl
lysine antibody. Further, we have also demonstrated
CRTAase catalyzed acetylation of GST and NOS by
DAMC using mass spectrometry. Since we have
observed that GST 3-3 and GST from S. japonicum
share 460% amino acid sequence similarity, we
are now routinely using recombinant GST from
S. japonicum (rGST) as the receptor protein. In this
communication, we have extended the investigations
to explore the ability to transfer propionyl group to
the receptor protein rGST by rhCRTAase utilizing
propoxycoumarin as the propionyl group donor.
By the application of nanoscale LC-MS/MS, the local-
ization of N-e-propionylated lysine residues in rGST
could be examined. Accordingly, the modified lysine
residues were surface exposed and distributed ran-
domly over the sequence of rGST of S. japonicum.
The propionylated rGST could be identified using
polyclonal anti-acetyl lysine antibody as acetylated
and propionylated lysines share structural similarity.
Accordingly, Zhao et al. demonstrated the HAT cata-
lyzed propionylated histones utilizing propionyl CoA
using anti-acetyl lysine antibody (1). It is worth men-
tioning that this observation pertains to the in vivo
acylation of protein wherein the receptor protein
(Histone) underwent acetylation as well as propionyla-
tion by the endogenous acetyl group and propionyl
group donors’ namely acetyl-CoA and propionyl-
CoA, respectively. In the present work, we have uti-
lized propoxycoumarin as the exclusive propionyl
group donor and the product of CR-catalyzed propio-
nylation of rGST was shown to interact with anti-
acetyl lysine antibody for the first time. In addition,
the product of propionylation mainly the propiony-
lated rGST was also identified by mass spectrometry.
It is pertinent to state that HAT such as HAT1, CBP
and p300 were also found to accommodate higher acyl
group donors such as propionyl CoA and butryl CoA
without steric hindrance to mediate the transfer of acyl
group to histones (1). Although the acyloxycoumarin
was found to be effectively acted upon by transacylase,
the thioacetates such as acetyl CoA and propionyl
CoA, the biological acyl group donors, were found
to be the poor substrates (unpublished data). This
observation is reminiscent of the enzymatic acetylation
of arylamine utilizing p-nitrophenylacetate as the sub-
strate for the arylamine N-acetyltransferase which
exhibited a pretty high Vmax (140-fold higher) as com-
pared to acetyl CoA when used as the acetyl group
donor (24). The LC-MS/MS results confirmed the
identity of propionylated rGST from S. japonicum.
The spectra obtained when analyzed by proteome
search engine MASCOT (18), indicated three distinct
peptide with significant score as propionylated on
lysine residues (Table I). Three lysine residues
Lys-11, -180 and -181, were found to be propionylated
Fig. 6 Selected ion chromatograms for m/z 534.3. Chromatographic
peak identified in test sample, as propionylated IKGLVQPTR
(bottom panel) is absent in the control sample (top panel).
modification sites were assigned based on strong ion
scores obtained by intact b and y ions observable in the
spectra. High Mascot scores for the MS/MS spectra
strongly support the covalent propionylation of
amino acid sequence. These data fully supports the
propionylation of three lysine residues of rGST,
Lys-11, -180 and -181.
Discussion
During the course of our persistent investigations on the
biochemical action of substituted 4-methylcoumarins,
the role of liver microsomes in mediating the acetyla-
tion of some functional proteins by acetoxycoumarins
such as DAMC, resulting in the modification of their
activities was postulated. The existence of a transace-
tylase (TAase) capable of transferring the acetyl group
from DAMC to a receptor protein such as GST culmi-
nating in the inhibition of glutathione conjugation
activity was envisaged (4, 20—22). The irreversible inhi-
bition of cytosolic GST caused by incubation
of DAMC with rat liver microsomes served as a
convenient procedure for the assay of TAase (4).
TAase was purified to homogeneity utilizing this
assay procedure and was identified as an endoplasmic
reticulum luminal protein calreticulin (CR). The
protein acetyltransferase activity of CR was termed
CRTAase (21). CRTAase catalyzed reaction was
629

P. Singh et al.
Fig. 7 MS/MS spectra of propionylated peptides. Assignment of the site of propionylation was confirmed by corresponding b and y ions. (A) b2
and y8 ions confirms propionylation of Lys-11. (B) y3 and y2 ions confirms propionylation of Lys-180 and -181, respectively.
(Chart 1). The fragment ions corresponding to b and y
ions of the peptides as shown in Fig. 7A and B clearly
indicates propionylated lysine residues.
CRTAase-catalyzed protein acetylation by DAMC
was found to be accompanied by autoacetylation of
CRTAase (10, 23). Likewise, the autopropionylation
of CRTAase was also encountered (Fig. 4, lane 1).
The autoacylated CRTAase was found to transfer
acyl groups to the receptor protein rGST (Fig. 5).
We have earlier observed an increased net negative
charge in autoacetylated CR (10) in comparison with
native CR. The acetylation of lysine e-NH3þ groups in
a-amylase was reported to offer stability by increase in
net negative charge leading to decrease in rate of
unfolding by anionic surfactant and also diminished
protein aggregation (25). Accordingly, the autoacy-
lated CR can be expected to be a stable intermediate
in the protein acyltransferase function of CR as
reported here (Fig. 5). Several acetyltransferases such
as HATs and transcription factors (TFIIB) are known
630

Calreticulin transacylase
O
OH
O
O
HO
O
O
O
O
O
rhCRTAase
rGST
+
rGST
O
+
Acetylated
GST*
DHMC
DAMC
O
OH
O
O
HO
O
O
O
O
O
rhCRTAase
+
rGST
rGST
O
+
Propionylated
GST*
DHMC
DPMC
* Represents lysine N-epsilon acylation (Details given in the text)
Scheme 1 Protein transacylase function of calreticulin.
1 mspilgywki kglvqptrll leyleekyee hlyerdegdk wrnkkfelgl efpnlpyyid
61 gdvkltqsma iiryiadkhn mlggcpkera eismlegavl dirygvsria yskdfetlkv
121 dflsklpeml kmfedrlchk tylngdhvth pdfmlydald vvlymdpmcl dafpklvcfk
181 krieaipqid kylksskyia wplqgwqatf gggdhppk
Conflict of interest
None declared.
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