The ycaC-related protein from the amphioxus
Branchiostoma belcheri (BbycaCR) interacts with
creatine kinase
Shengjuan Jiang
1,2,
*, Xuemei Sun
1,
* and Shicui Zhang
1
1 Department of Marine Biology, Ocean University of China, Qingdao, China
2 College of Life Science, Anhui Science and Technology University, Fengyang, China
Isochorismatase catalyzes the conversion of isochoris-
mate, in the presence of H
2
O, into 2,3-dihydro-2,3-di-
hydroxybenzoate and pyruvate, via hydrolysis of a
vinyl ether, an uncommon reaction in biological sys-
tems [1,2]. The isochorismatase superfamily has been
divided by the Structural Classification of Proteins
(SCOP) database into five subfamilies: nicotinamidase
(EC 3.5.1.19); nicotinamidase-related enzyme; N-carba-
moylsarcosine amidohydrolase (EC 3.5.1.59); isochoris-
matase (EC 3.3.2.1); and a subfamily of bacterial
sequences of unknown function exemplified by the
Escherichia coli ycaC gene product [3]. The tertiary
structures observed for these proteins share a common
fold, and the key features of the active site are highly
conserved, despite low sequence identity (20%). For
example, all structures determined to date from this
superfamily display a rare nonproline cis-peptide bond
at the active site, which helps to position the substrate-
binding residues appropriately [4]. There is not, how-
ever, universal conservation of the catalytic triad
Asp19, Arg(Lys)84 and Cys118 (Protein Data Bank
entry 1YAC, numbering as in E. coli) across the multi-
ple subfamilies.
The ycaC gene comprises a 621-bp ORF in E. coli,
which was initially identified when Bilous et al. [5].
sequenced the dimethylsulfoxide reductase (dmsABC)
operon and surrounding regions of the genome.
The ycaC gene product has been predicted to be a
homo-octameric hydrolase with unknown substrate
specificity, catalyzing isochorismatase-like reactions [6],
Keywords
amphioxus; Branchiostoma; creatine kinase;
isochorismatase superfamily; ycaC-related
protein
Correspondence
S. Zhang, Department of Marine Biology,
Ocean University of China, Qingdao 266003,
China
Fax: +86 532 82032787
Tel: +86 532 82032787
E-mail: sczhang@ouc.edu.cn
*These authors contributed equally to this
work
(Received 29 May 2007, revised 10 July
2008, accepted 18 July 2008)
doi:10.1111/j.1742-4658.2008.06602.x
The ycaC-related gene, ycaCR, is uncharacterized, and has no assigned
function to date. Here we clearly showed that the ycaC-related gene from
the amphioxus Branchiostoma belcheri,BbycaCR, coded for a novel mem-
ber of the isochorismatase superfamily, which is mainly localized in the
mitochondrial fraction. Both pull-down and reverse pull-down analyses
revealed that BbycaCR was able to interact with creatine kinase, an
enzyme involved in energy transduction, in addition to binding to native
ycaCR, forming a homopolymer. Surprisingly, neither isochorismatase,
nicotinamidase nor N-carbamoylsarcosine amidohydrolase activity was
detected for BbycaCR, although it possessed the putative catalytic triad of
Asp19, Arg(Lys)84 and Cys118 that is found in ycaC proteins. Both tissue
section in situ hybridization and immunohistochemistry showed that
BbycaCR was ubiquitously expressed in amphioxus, although at different
expression levels, suggesting that BbycaCR plays a conserved fundamental
cellular role in amphioxus. It is proposed that BbycaCR may be indirectly
involved in energy transduction.
Abbreviations
BbCK, Branchiostoma belcheri creatine kinase; Dig, digoxigenin; NCBI, National Center for Biotechnology Information.
FEBS Journal 275 (2008) 4597–4605 ª2008 The Authors Journal compilation ª2008 FEBS 4597
although biochemical and functional characterization
is still lacking. The ycaC genes have recently been
isolated from other bacteria such as Bacillus
cereus (GenBank accession number: NP_832865),
Salmonella typhimurium (NP_459926), Bradyrhizobium
japonicum (NP_770892), Sinorhizobium meliloti
(NP_436249), and Burkholderia fungorum (ZP_00031917).
Interestingly, ycaC gene homologs have also been iden-
tified in both vertebrates such as human (AAH08367),
monkey (XP_001088057), rat (XP_001059344), dog
(XP_850761), and frog (Q5PQ71), and invertebrates,
including fruit fly (NP_651869) and silkworm
(NP_001040541). They all possess a ycaC-related
domain, known as the isochorismatase domain, and
are therefore called ycaC-related (ycaCR) genes.
However, the ycaCR product has not yet been
characterized, and its function remains unknown.
From the gut cDNA library of the amphioxus
Branchiostoma belcheri, a protochordate bridging the
gap between invertebrates and vertebrates [7], we iso-
lated a cDNA clone encoding a ycaC-related homolog,
BbycaCR (AAT39420). The aims of this study were
thus to examine the expression of BbycaCR and to
characterize the biochemical properties of the encoded
protein.
Results
Sequence and phylogeny of BbycaCR
BbycaCR cDNA obtained from the gut cDNA library
of B. belcheri was 891 bp long, and its longest ORF
coded for a deduced protein of 201 amino acids with a
predicted molecular mass of approximately 22 kDa and
an calculated isoelectric point of 7.2 (editseq 5.01;
DNASTAR Inc). The 5¢-UTR was 84 bp long and had
two in-frame termination codons (at positions )12 and
)51), and the 3¢-UTR was 201 bp long. and had a poly-
adenylation tail. A blastp search at the National Center
for Biotechnology Information (NCBI) showed that
BbycaCR had the ycaC-related domain at positions 17–
172, which is typical of the isochorismatase superfamily.
Moreover, prediction by the swiss-model program
(http://swissmodel.expasy.org/) demonstrated that it
possessed a three-layer a–b–asandwich topology struc-
ture, which is characteristic of E. coli ycaC protein [6].
Furthermore, an alignment of ycaC and ycaCR proteins
showed that they all had the putative catalytic triad
Asp19, Arg(Lys)84 and Cys118 (Protein Data Bank
entry 1YAC, numbering as in E. coli) and a conserved,
rare, nonproline cis-peptide bond, suggesting that
ycaCRs, including BbycaCR, are a group of highly con-
served proteins with structural features similar to those
of ycaC. However, the phylogenetic tree constructed by
the neighbor-joining method using the sequences of
ycaCR proteins and representative members of all five
subfamilies of the isochorismatase superfamily showed
that amphioxus ycaCR and ycaCR proteins from other
species were clustered together, separate from the other
five subfamilies, indicating that ycaCRs, including
BbycaCR, may form a novel subfamily of the isochori-
smatase superfamily (data not shown). In addition,
prediction by mitoprot II software [8], showed that
BbycaCR had a mitochondrial targeting sequence
MSGLVRRLGKVGVDT, suggesting that it was a
mitochondrial protein.
A search of the recently completed draft assembly
and automated annotation of the Branchiostoma
floridae genome was also carried out. It revealed
the presence of a Florida amphioxus ycaCR cDNA
and its genomic DNA sequence (estExt_fgenesh2;
Brafl1 ⁄scaffold_22; http://genome.jgi-psf.org/cgi-bin/
dispTranscript?db=Brafl1&id=113855&useCoords=1).
Sequence comparison demonstrated that BbycaCR
shared 93% identity with the deduced protein
encoded by the Florida amphioxus gene at the
amino acid level. Analysis of the genomic structure
showed that Florida amphioxus ycaCR consisted of
four exons and three introns. The four coding exons
of 138, 216, 139 and 110 bp, respectively, were inter-
spaced by three introns of 585, 407 and 649 bp,
which all begin with GT and end with AG,
sequences that are thought to be necessary for cor-
rect RNA splicing of various other eukaryotic genes.
It was notable that human ycaCR (GeneID: 79763),
monkey ycaCR (GeneID: 700714), rat ycaCR
(GeneID: 684270) and dog ycaCR (GeneID: 609103)
all comprised five coding exons, whereas fruit fly
ycaCR (GeneID: 37944) and nematode ycaCR
(GeneID: 178169) both comprised three coding
exons. Moreover, the first two coding exons in all
the species mentioned above encoded the respective
homologous protein domains. Exon 3 and the first
half of exon 4 in the vertebrates were combined in a
single third exon in BbycaCR, and the rest of exon 4
and exon 5 in the vertebrates were combined in a
single fourth exon in BbycaCR. Exon 3 and exon 4
in BbycaCR were combined in a single third exon in
protostome invertebrates (data not shown).
Biochemical properties of recombinant BbycaCR
An expression vector including the entire ORF of
BbycaCR and an additional 5¢-tag of pET28a was
constructed and successfully transformed into E. coli,
and this resulted in the original N-terminal Met in the
ycaCR interacts with creatine kinase S. Jiang et al.
4598 FEBS Journal 275 (2008) 4597–4605 ª2008 The Authors Journal compilation ª2008 FEBS
recombinant protein being replaced by Met-Gly-Ser-
Ser-(His)
6
-Ser-Ser-Gly-Leu-Val-Pro-Arg-Gly-Ser-His-Met-
Ala-Ser-Met-Thr-Gly-Gly-Gln-Gln-Met-Gly-Arg-Gly-
Ser-Glu-Phe-Met, which increased the size of the
recombinant protein by approximately 4 kDa. The
recombinant protein was purified by affinity chroma-
tography on a Ni–nitrilotriacetic acid resin column,
and the purified BbycaCR with His
6
-tag produced a
single band of 26 kDa on SDS ⁄PAGE gel after
Coomassie blue staining, coinciding with its theoretical
size (Fig. 1).
Rabbit antiserum against the purified recombinant
BbycaCR with a titer of 1 : 800 was obtained. Western
blotting analysis demonstrated that the antiserum
reacted with the supernatant of the cell lysate of iso-
propyl-thio-b-d-galactoside-induced E. coli BL21 with
expression vector, forming a band of 26 kDa,
whereas it was not reactive to the supernatant of the
lysate of the same E. coli cells before induction by
isopropyl-thio-b-d-galactoside. The antiserum also
reacted with whole amphioxus homogenates, forming a
single band of 22 kDa, matching the molecular mass
predicted by BbycaCR cDNA. These show that the
rabbit antiserum prepared has conspicuous antigen-
specific reactivity.
To test whether BbycaCR had any isochorismatase,
nicotinamidase or N-carbamoylsarcosine amidohydro-
lase activity, different amounts of recombinant Bby-
caCR protein were assayed for the activities of all the
three enzymes. None of the three enzyme activities
were detected under the conditions tested, although the
protein possessed the putative catalytic triad Asp19,
Arg(Lys)84 and Cys118.
We then used the pull-down technique to identify
the proteins that might interact with the recombinant
protein [9,10]. It was found that two proteins (A and
B in lane 2 of Fig. 2) from amphioxus tissue homogen-
ates bound to the His
6
-tagged BbycaCR. The mole-
cular masses of proteins A and B were approximately
42 and 22 kDa, respectively. Thirteen peptide frag-
ments of protein A were measured by MALDI-
TOF MS, accounting for 35% of peptide sequence
coverage. The measured peptides were matched against
the creatine kinase from B. belcheri, and the mowse
score obtained was 120, which was 1.5-fold greater
than the threshold for identification. The matched pep-
tide fragments were located at residues 8–20, 21–27,
21–31, 133–142, 147–166, 210–217, 218–230, 237–245,
287–298, 303–311, 315–336, 316–336 and 367–374 of
the creatine kinase protein, respectively. Similarly, 15
peptide fragments of protein B were measured by
MALDI-TOF MS, accounting for 61% of peptide
sequence coverage. The measured peptides were
matched against the native ycaCR encoded by ycaCR
of B. belcheri, and the mowse score obtained was 128,
which was 1.6-fold greater than the threshold for iden-
tification. The matched peptide fragments were located
at residues 1–7, 27–32, 33–43, 47–63, 47–63, 64–79,
64–80, 120–141, 142–152, 142–152, 142–152, 147–154,
155–178, 155–178 and 179–184 of the native ycaCR,
respectively. The control experiments showed that
none of the proteins bound to the MagExtractor
His-tag particles in the absence of BbycaCR (lane 3 in
Fig. 2). These suggested that BbycaCR was able to
form a polymer and was able to bind to creatine
kinase.
Fig. 1. SDS ⁄PAGE of BbycaCR. Lane M: marker. Lane 1: extracts
from E. coli BL21 containing pET28a–BbycaCR before induction.
Lane 2: extracts from E. coli BL21 containing pET28a–BbycaCR
after isopropyl-thio-b-D-galactoside induction. Lane 3: recombinant
BbycaCR purified on an Ni–nitrilotriacetic acid resin column. The
arrow indicates the location and size of recombinant BbycaCR.
Fig. 2. SDS ⁄PAGE (9%) analysis of the pull-down assay. Lane M:
marker. Lane 1: total protein of the amphioxus homogenate.
Lane 2: BbycaCR protein and the interaction proteins. Lane 3: the
control particles incubated with the pET28a-expressed soluble pro-
tein and the tissue homogenates. Lane 4: the control particles incu-
bated with both BbycaCR recombinant protein and the homogenate
buffer. Lane 5: extracts from E. coli BL21 containing pET28a–Bby-
caCR after induction. A and B indicate the pulled-down proteins
(fi). The triangle indicates the BbycaCR recombinant protein.
S. Jiang et al. ycaCR interacts with creatine kinase
FEBS Journal 275 (2008) 4597–4605 ª2008 The Authors Journal compilation ª2008 FEBS 4599
To verify that BbycaCR can bind to creatine kinase,
the reverse pull-down assay in vitro was carried out.
B. belcheri creatine kinase (BbCK) was expressed in
E. coli strain BL21 and purified. It had a molecular
mass of 61 kDa. Both purified BbycaCR and BbCK
were incubated together with mouse monoclonal anti-
body against human creatine kinase, and then with
protein A ⁄G PLUS-Agarose. After stringent washing,
the complexes were analyzed by western blotting. As
expected, BbycaCR was detected in the complexes as
well as in the positive control (lanes A, B and C in
Fig. 3). In contrast, no positive signals were observed
in the negative controls (lanes D and E in Fig. 3).
These showed that BbycaCR was pulled down by
forming the BbycaCR–BbCK–antibody–agarose com-
plex, confirming that BbycaCR was able to interact
with BbCK specifically.
Subcellular localization of BbycaCR
In order to examine the subcellular localization of
BbycaCR, immunoblotting of the cytosolic and mito-
chondrial fractions from the gut with rabbit anti-Bby-
caCR serum was performed. The results showed that
BbycaCR was mainly present in the mitochondrial
fraction (Fig. 4), at a level that was about six-fold
higher than that in the cytosolic fraction, agreeing with
the presence of a mitochondrial targeting sequence in
the protein.
Expression of BbycaCR
Northern blotting was conducted to assess the presence
and size of BbycaCR transcript. As shown in Fig. 5,
an approximately 890 bp band of BbycaCR transcript
was detected. Tissue section in situ hybridization
revealed that BbycaCR transcript was abundant in the
gill, hepatic caecum, hindgut, endostyle and ovary, and
was present at a lower level in the epidermis, epipha-
ryngeal groove, testis, muscle, neural tube and noto-
chord (Fig. 6). This was further supported by
immunohistochemical staining using rabbit antiserum
against the purified recombinant BbycaCR, which
demonstrated that BbycaCR was predominantly local-
ized in the gill, hepatic caecum, hindgut, endostyle and
ovary (Fig. 7).
Discussion
The progress in gene ⁄genome sequencing has led to an
accumulation of ycaCR genes with the isochorismatase
domain from a variety of animal species in GenBank
submissions. Although computational approaches,
Fig. 3. Western blotting of reverse pull-down proteins. Lanes A, B
and C show the presence of BbycaCR in the complexes eluted.
Lanes D and E are the two negative controls. Lane F is the positive
control. Arrows indicate the protein marker.
Fig. 4. Western blot analysis of the BbycaCR subcellular distribu-
tion. Lane 1: the purified recombinant protein. Lane 2: the extracts
from E. coli BL21 containing pET28a–BbycaCR before induction.
Lane 3: the mitochondrial fraction of the primitive gut. Lane 4: the
cytosolic fraction of the primitive gut. The arrow indicates the locali-
zation of the recombinant BbycaCR and the triangle shows the dis-
tribution of the native BbycaCR in different cell fractions of the
primitive gut.
Fig. 5. Northern blotting. Three microgram amounts of RNAs were
analyzed in 1.2% agarose formaldehyde-denaturing gel. The two
main bands are 28S and 18S RNA. The blot was hybridized with a
Dig-labeled BbycaCR RNA probe. The arrow indicates the molecular
size equivalent to 890 bp.
ycaCR interacts with creatine kinase S. Jiang et al.
4600 FEBS Journal 275 (2008) 4597–4605 ª2008 The Authors Journal compilation ª2008 FEBS
including sophisticated sequence analysis, phylogenetic
patterns, domain fusions, structural threading and gene
neighborhoods, can be used to predict the likely
biochemical properties of these genes, the ultimate
biochemical function of the proteins can only be estab-
lished via direct experimentation. The ycaCR gene
from B. belcheri,BbycaCR, coded for a protein that
interacts with creatine kinase, in addition to binding to
native BbycaCR, forming a homopolymer. Creatine
kinase catalyzes the reversible transfer of the phos-
phate group of phosphocreatine to ADP, yielding ATP
and creatine [11–13]. This indicates that BbycaCR may
be indirectly involved in energy transduction. This is
additionally supported by the fact that BbycaCR is
mainly localized in mitochondria, the in vivo sites of
ATP production. However, whether BbycaCR usually
functions as a polymer remains to be studied.
Previous structural studies have solidified the rela-
tionship of bacterial ycaC to a hydrolase, catalyzing
isochorismatase-like reactions [6]. We have therefore
tested the presence of any hydrolase activity for the
recombinant BbycaCR. Surprisingly, none of the
isochorismatase, nicotinamidase and N-carbamoyl-
sarcosine amidohydrolase activities was detected,
although BbycaCR had the catalytic triad Asp19,
Arg(Lys)84 and Cys118 found in ycaC protein. On
the other hand, the phylogenetic analysis revealed
that BbycaCR clustered separately from all the five
A
B
C
D
E
Fig. 6. Localization of BbycaCR transcripts
in different tissues of adult amphioxus. (A)
Low-magnification view of tissues of male
amphioxus. BbycaCR transcripts were
observed in the gill, hepatic caecum, epider-
mis, epipharyngeal groove, testis, muscle,
neural tube, and notochord. (B) Negative
control of male amphioxus (with antisense
RNA). (C) Enlargement of the box in (A). (D,
E) Positive signals in endostyle, ovary, and
hindgut. Arrows in the figure indicate the
positive signals. es, endostyle; eg, epipha-
ryngeal groove; g, gill; hc, hepatic caecum;
hg, hindgut; m, muscle; nc, notochord; nt,
neural tube; o, ovary; t, testis. Bars repre-
sent 200 lm in (A) and (B), and 100 lmin
(C), (D) and (E).
S. Jiang et al. ycaCR interacts with creatine kinase
FEBS Journal 275 (2008) 4597–4605 ª2008 The Authors Journal compilation ª2008 FEBS 4601