Published Ahead of Print 21 September 2012.
2012, 194(23):6410. DOI: 10.1128/JB.01379-12.
Marjan Gucek, David C. H. Yang and Wei-Mei Ching
Amila H. Abeykoon, Chien-Chung Chao, Guanghui Wang,
Methylate Outer Membrane Protein B from
Two Protein Lysine Methyltransferases
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Two Protein Lysine Methyltransferases Methylate Outer Membrane
Protein B from Rickettsia
Amila H. Abeykoon,aChien-Chung Chao,bGuanghui Wang,cMarjan Gucek,cDavid C. H. Yang,aand Wei-Mei Chingb
Department of Chemistry, Georgetown University, Washington, DC, USAa; Viral and Rickettsial Diseases Department, Infectious Diseases Directorate, Naval Medical
Research Center, Silver Spring, Maryland, USAb; and Proteomics Core Facility, NHLBI, Bethesda, Maryland, USAc
Rickettsia prowazekii, the etiologic agent of epidemic typhus, is a potential biological threat agent. Its outer membrane protein B
methyltransferases were synthesized, cloned, and expressed in Escherichia coli, and expressed proteins were purified by nickel-
Rickettsial OmpBs have been shown to participate in adhesion to
mammalian cells in vitro (8, 36). Ectopically expressed rickettsial
lian cells (8, 24). Unlike the ?-helical transmembrane proteins in
plasma membranes, the autotransporter domains of these outer
membrane proteins have structural characteristics of ?-barrel in-
tegral membrane proteins (3, 33). As the immunodominant anti-
genic surface protein, native OmpB induces strong humoral and
cellular immune responses in animal models and in patients (6,
12, 15, 16, 23). Methylation of OmpA of Rickettsia canadensis and
ompB of Rickettsia typhi is known, but whether this is important
for any other pathogenic rickettsia is unknown.
One important but not well understood structural feature of
rickettsial OmpB is the methylation at ε-amino groups of lysine
ylation can potentially alter their structure and function. Bio-
chemical and genetic analyses suggest that methylation of lysine
residues of OmpB correlates with the virulence of Rickettsia
prowazekii (30). OmpBs from the virulent strains Breinl and Evir
are found hypermethylated, while that of the avirulent Madrid E
strain is mostly mono-methylated (11, 29). In addition, hypo-
methylated OmpB from the Madrid E strain elicits less protective
immunity than OmpB from virulent strains of R. prowazekii.
The genes of the avirulent R. prowazekii Madrid E strain have
been compared with those of the virulent revertant Evir strain to
identify the genes that are inactivated in the attenuated Madrid E
strain but not in the virulent Breinl and Evir strains. Genomic
ickettsial OmpBs belong to the outer membrane protein au-
totransporter family and contain large passenger domains.
sequence and transcription analyses showed that the mutation of
RP027/RP028 in the Madrid E strain reverts back to the wild type
(39). The gene encoding one of the putative methyltransferases
RP027/RP028 was found to have a frameshift mutation in the
genes, which are detectable only in avirulent strains (4). The pu-
tative methyltransferases and their native substrates in Rickettsia
have not been identified or characterized, and the mechanism of
pathogenesis of Rickettsia is not well understood.
ification and regulates various functions of diverse proteins (13,
18). The regulatory potential of lysine methylation is greatly ex-
panded because multiple methyl groups can be added to a single
ation of specific residues in histones plays a pivotal role in chro-
matin remodeling that leads to alteration of gene expression and
repression (27). Methylation of membrane proteins has attracted
little attention. OmpB methylation at lysine residues could
conceivably affect bacterium-host cell interactions and inhibit al-
ternative posttranslational modifications, such as acetylation,
Received 31 July 2012 Accepted 11 September 2012
Published ahead of print 21 September 2012
Address correspondence to David C. H. Yang, email@example.com.
Supplemental material for this article may be found at http://jb.asm.org/.
Copyright © 2012, American Society for Microbiology. All Rights Reserved.
jb.asm.org Journal of Bacteriologyp. 6410–6418December 2012 Volume 194 Number 23
on June 10, 2014 by guest
ubiquitination, or sumoylation. Characterization of methyltrans-
the host immune response. In practical clinical applications, en-
zymatic methylation of OmpB in vitro may provide methylated
ettsiae and for developing improved vaccine candidates. As a first
step toward these goals, we searched for methyltransferases from
encoding hypothetical proteins and putative methyltransferases
in the genome of R. prowazekii were examined by bioinformatics.
Recombinant hypothetical proteins and putative methyltrans-
ferases were cloned, purified, and analyzed for methyltransferase
activity. Two rickettsial protein lysine methyltransferases that en-
zymatically methylate recombinant OmpB were found in this
MATERIALS AND METHODS
Materials. Recombinant OmpB fragments, including rOmpB(A) (33 to
273), rOmpB(AN) (33 to 744), and OmpB(K) (745 to 1353) based on the
genomic sequence of R. typhi were prepared as previously described (10).
Briefly, the bacterially expressed proteins in the inclusion body were dis-
solved in 8 M urea and 1% deoxycholate, purified by ion-exchange chro-
matography on DEAE-cellulose in 6 M urea, and refolded. Proteins were
obtained by slow dialysis at stepwise, decreasing concentrations of urea.
SET7 methyltransferase and the histone peptide substrate H3(1–17) were
kii Madrid E and R. prowazekii RP22 in NCBI and annotated encoded
proteins in PIR (Protein Information Resources) were searched and ex-
amined for potential protein methyltransferases. Those methyltrans-
ferases targeting DNA, rRNA, tRNA, mRNA, and small molecule metab-
annotated as hypothetical proteins with potential S-adenosylmethionine
(SAM) binding domains were selected for further analysis. Structural
bioinformatic analysis of the putative methyltransferases was carried out
using the software programs HHpred (34) and 3DLigandSite (37). The
structural templates were selected by the software at respective Web serv-
half of the putative methyltransferases. No known three-dimensional
structures with significant homology to the C-terminal half are available.
The structural models were displayed using the software program Chi-
the Web servers Psipred (19), HHpred (34), and Phyre (20).
Synthesis and cloning of genes encoding putative methyltrans-
ferases from R. prowazekii. Table 1 lists the names and accession num-
bers of genes and encoded proteins of putative methyltransferases.
Codon-optimized single-stranded cDNAs were synthesized by Bioclone
pET28a expression vector (Novagen) containing a 6?His tag. Standard
cloning procedures were performed, and the inserts were verified by re-
striction enzyme digestion and sequencing. The sequences of the cDNAs
for the expression in E. coli are shown in Table S1 in the supplemental
Expression and purification of putative methyltransferases of R.
prowazekii. BL21(DE3) competent cells (Agilent Technologies) were
proteins and putative methyltransferases. Protein expression was carried
out by growing the bacteria from single colonies at 37°C in 2 ml of LB
broth supplemented with 100 ?g/ml kanamycin overnight. The cell cul-
cultures were induced with 0.04 mM isopropyl-?-D-thiogalactopyrano-
side (IPTG) and incubated at 22°C and at 250 rpm overnight. Cells were
harvested by centrifugation and lysed, and the expressed proteins were
purified using nickel-nitrilotriacetic acid (Ni-NTA) column chromatog-
raphy according to the manufacturer’s instructions (GoldBio, St. Louis,
assay and bovine serum albumin as a standard (7).
Standard protein lysine methyltransferase activity assay by incor-
poration of3H-Me. Methyltransferase activity was monitored by the
transfer of3H-methyl (3H-Me) from S-[3H-Me]adenosylmethionine (10
Ci/mmol; Perkin Elmer) to recombinant protein substrates. The purified
recombinant methyltransferases were then examined for protein methyl-
first developed a methyltransferase assay modified from the SET7 meth-
yltransferase assay. The amount of methyl transferred from S-[3H-
Me]adenosylmethionine to histone 3 peptide was determined by What-
bicarbonate (38). Unlike histones, the OmpB fragments are acidic pro-
teins, so Whatman 3MM and 5% trichloroacetic acid (TCA) replacing
S-[3H-Me]adenosylmethionine to histone 3 peptide was found to be the
same as that using the conventional SET7 assay.
The 50-?l reaction mixtures contained 8.3 mM sodium phosphate,
pH 8.0, 160 ?M [3H-Me]SAM (34 mCi/mmol), and 2 ?M rOmpB(AN)
or rOmpB(K). The methylation reaction was initiated by adding limiting
amounts of protein methyltransferase and incubated at 37°C. Aliquots of
the reaction mixture were spotted onto Whatman 3MM cellulose filter
paper discs (Fisher Scientific) at various time points. Reactions were
ice-cold TCA three times, followed by ethanol-ether (1:1 [vol/vol]). The
amounts of acid-precipitable radioactivity were determined by liquid
scintillation counting using a Perkin Elmer Wallace 1410 counter. One
unit of methyltransferase is defined as the amount of enzyme required to
Quantifying radioactivity in proteins extracted from polyacryl-
amide gels. Radioactivity associated with methylated OmpB fragments
separated by SDS gel electrophoresis was determined using the method
previously described with minor modifications (35). To quantitate the
number of methyl groups transferred to each substrate molecule, the ra-
rRP789 or rRP027-028 (6 ?M) in the presence of radioactive SAM (0.16
TABLE 1 Potential protein lysine methyltransferases
Accession no.LocusNo. of amino acids
Hypothetical protein RP789; R. prowazekii Madrid E
Putative methyltransferase RP027-028; R. prowazekii Rp22
Hypothetical protein RP027; R. prowazekii Madrid E
Hypothetical protein RP028; R. prowazekii Madrid E
Hypothetical protein RP527; R. prowazekii Madrid E
Hypothetical protein RP545; R. prowazekii Madrid E
aThe RP027 and RP028 genes are split genes in strain Madrid E of RP027-028 in strain Rp22.
Methylation of Outer Membrane Protein B from Rickettsia
December 2012 Volume 194 Number 23jb.asm.org 6411
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mM with specific activity of 68 mCi/mmol) in 8.3 mM phosphate buffer
(pH 8.0) at 37°C overnight. The reaction was stopped by adding SDS-
sample buffer. Polyacrylamide gels were polymerized with reagents ob-
tained from Bio-Rad Laboratories (Richmond, CA) except that N,N=-
methylenebisacrylamide was replaced with DATD at a ratio of 1 part of
DATD to 10 parts of acrylamide (35). After electrophoresis, the SDS gels
vials, and 0.5 ml of 2% (wt/vol) sodium metaperiodate was added. The
ice for 20 min and then counted by liquid scintillation counting.
Western blot analysis of putative lysine methyltransferase. The en-
at 37°C for 4 h. Protein samples were denatured in standard sample de-
naturing buffer containing 1% SDS and 10 mM mercaptoethanol. The
blot analysis with 250 ?g/ml rabbit anti-trimethyl lysine (Trend Pharma
& Tech Inc., Surrey, Canada) at a 1:2,000 dilution in Odyssey blocking
buffer (phosphate-buffered saline and 0.1% sodium azide) containing
0.1% Tween 20 at 4°C overnight, followed by incubation with IRDye
(800CW)-conjugated anti-rabbit IgG(H?L) (donkey) secondary anti-
body (Rockland Immunochemicals, Inc., Gilbertsville, PA) at a 1:10,000
dilution in blocking buffer for 1 h at room temperature. The signal was
3 times with 1? PBS containing 0.1% Tween 20.
Steady-state enzyme kinetics. The Michaelis-Menten constants and
catalytic constants were obtained by direct fitting of the Michaelis-Men-
using the software program Kaleidagraph.
LC/MS-MS. Ten micrograms each of the fragments rOmpB(AN) and
rOmpB(K) were methylated using 30 ?g of methyltransferase–3.2 mM
S-adenosylmethionine (Perkin Elmer) in 50 ?l of reaction mixture con-
taining 8.3 mM sodium phosphate buffer, pH 8.0. The methylation reac-
tion was carried out at 37°C overnight. The reaction samples were evap-
corresponding AN and K protein bands were cut off from the gel and
subjected to in-gel digestion as described previously (21), with modifica-
acid, followed by the reduction using 10 mM dithiothreitol (DTT). The
protein samples were alkylated with 100 mM iodoacetamide in the dark.
The gel pieces were dehydrated using acetonitrile and rehydrated in 100
mM (NH4)HCO3twice. The protein digestion was carried out using se-
chymotrypsin in 50 mM (NH4)HCO3was mixed with 10 ?g of protein
sample and digested overnight at 25°C. The digested peptides were ex-
acid. The volume was reduced to less than 20 ?l by evaporation, and the
final volume was adjusted to 20 ?l using 1% formic acid. The samples
were purified using Zip-Tip pipette tips with C18resin (Millipore, Biller-
tography-tandem mass spectrometry (LC/MS-MS) analysis. LC/MS-MS
was carried out at the NHLBI Proteomics Core Facility (Bethesda, MD)
using an Eksigent nanoLC-Ultra 2D system (Dublin, CA) coupled to an
LTQ Orbitrap Velos mass spectrometer (Thermo Scientific, San Jose,
analyzed using the Proteome Discoverer v1.3 software program (Thermo
Scientific) and the Mascot search engine running on a six-processor clus-
ter at NIH (http://biospec.nih.gov; version 2.3).
Genes encode putative protein methyltransferases in R. prowa-
kii Madrid E genomic sequence at National Center for Biotech-
nology Information (NCBI). Sixty-two genes encoding known
encode methyltransferases of small molecule metabolites, tRNA,
mRNA, rRNA, and DNA were excluded. All remaining genes that
encode unknown and putative methyltransferases were selected.
from R. prowazekii Madrid E (RP789, RP527, RP545, RP027, and
In order to identify the protein methyltransferase of OmpB in
virulent strains of Rickettsia, we examined the genome of R.
prowazekii Rp22, which is a virulent strain closely related to P.
prowazekii Madrid E. The sequence of another virulent strain,
Breinl, is not currently available. A putative methyltransferase
with the protein locus ADE29537 was found in the R. prowazekii
is referred to as RP027-028 here. The amino acid sequence of
RP027-028 aligns with those of RP028 and RP027 with 100%
identity at its N terminus (1 to 243) and C terminus (283 to 535),
respectively. RP028 and RP027 in Madrid E appear to be split
Table 1 lists the accession number, the protein locus, and the
number of amino acid residues in the encoded protein. None of
these putative methyltransferases have been cloned or biochemi-
cally characterized for any of the rickettsial species.
Structural bioinformatics analyses were carried out. Amino
acid sequence alignment of RP789 and RP027-028 showed 45%
nal domains of RP789 and RP027-028 using 3DLigandSite (37)
gave similar three-dimensional models of the SAM binding do-
mains of RP789 and RP027-028 with bound SAM (Fig. 1), sug-
gesting that both are SAM binding proteins. The C termini of
stantial homology to any proteins in the RCSB Protein Database
suitable for homology modeling.
Proteins homologous to RP789 and RP027-028 were found in
species and strains were compiled. The accession numbers and
identities of these homologous proteins to RP789 and RP027-028
are shown in Table S2 in the supplemental material.
rRP789 and rRP027-028 were expressed and purified. Based
on the gene sequences of the above putative methyltransferases,
we produced the corresponding proteins in E. coli. E. coli codon-
optimized genes that encode potential methyltransferases were
synthesized and inserted into the expression vector pET28a. The
corresponding proteins were expressed in E. coli BL21(DE3) and
purified using Ni-NTA affinity chromatography. The level of ex-
pression and the purity of the proteins were analyzed by SDS-
highly expressed as soluble proteins and successfully purified as
shown in Fig. S1 in the supplemental material. Purified rRP789
and rRP027-028 had molecular masses of 63.5 and 61.5 kDa, re-
spectively, as expected. The overall yields of purified rRP789 and
rRP027-028 were 0.25 and 0.75 mg per 100 ml of culture, respec-
tively. rRP527 was expressed at a lower level than rRP789 or
50 kDa and had an overall yield of 0.15 mg per 100 ml of culture
(see Fig. S1). rRP545 had a very low expression level. The expres-
sion level of rRP545 was not significantly improved by increasing
the concentrations of IPTG to 0.5 or 1 mM, varying lengths of
Abeykoon et al.
jb.asm.org Journal of Bacteriology
on June 10, 2014 by guest
induction time, or changing the host to BL21(DE3) Gold pLysS.
rRP028 and rRP027 were expressed at high levels but were insol-
uble. Purification of rRP028 and rRP027 in the presence of 8 M
urea, followed by refolding by dialyzing against stepwise-decreas-
ing concentrations of urea, did not yield soluble protein.
rRP789 and rRP027-028 catalyzed methylation of rOmpB
fragments. The radioactivity assay was used to examine the enzy-
matic methylation catalyzed by purified recombinant, potential
methyltransferases using recombinant OmpB fragments. Three
rOmpB fragments, AN, K, and A, were used and are referred to as
rOmpB(K). No methyl transfer was detected in the absence of
either rRP789 or the rOmpB fragment. Similarly, as shown in
Fig. 2B, rRP027-028 also catalyzed3H-Me transfer from S-[3H-
Me]adenosylmethionine to rOmpB(AN) and (K). No methyl
transfer was detected in the absence of either rRP027-028 or the
recombinant OmpB fragment. The rate of methyl transfer cata-
lyzed by rRP027-028 to rOmpB(AN) and rOmpB(K), however,
was appreciably slower than those catalyzed by rRP789.
rOmpB(A) was not methylated by either rRP789 or rRP027-028
under the same assay conditions (data not shown). The specific
tial rate of methylation in the presence of excess of rOmpB(AN)
and rOmpB(K). As shown in Table 2, the specific activities of
methyltransferase rRP789 were 56,120 U/mg and 24,500 U/mg
FIG 1 Molecular models of the SAM binding domains of R. prowazekii Madrid E RP789 and R. prowazekii RP22 RP027-028. Molecular models of the putative
and the Web server of 3DLigandSite. The protein is in ribbons, and SAM is in ball and stick models. Figures were generated using Chimera.
rOmpB(AN) (?) or 1 ?M rOmpB(K) (?) in the presence of 0.16 mM [3H]SAM (68 mCi/mmol), 0.2 mM DTT, and 8.3 mM sodium phosphate (pH 8.0). (B)
rRP027-028 catalyzed methylation of rOmpB(AN) (O?O) and rOmpB(K) (O?O) under the same conditions as for panel A. The concentrations of rRP789
and rRP027-028 were equal to the concentration of the rOmpB fragment. Controls containing AN only (--?--), K only (--?--), and methyltransferase enzyme
only ( ) are also shown.
Methylation of Outer Membrane Protein B from Rickettsia
December 2012 Volume 194 Number 23jb.asm.org 6413
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