The ycaC-related protein from the amphioxus Branchiostoma belcheri (BbycaCR) interacts with creatine kinase Shengjuan Jiang1,2,*, Xuemei Sun1,* and Shicui Zhang1
1 Department of Marine Biology, Ocean University of China, Qingdao, China 2 College of Life Science, Anhui Science and Technology University, Fengyang, China
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 forming a homopolymer. Surprisingly, neither isochorismatase, ycaCR, 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.
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.
Abbreviations BbCK, Branchiostoma belcheri creatine kinase; Dig, digoxigenin; NCBI, National Center for Biotechnology Information.
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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) the genome. operon and surrounding regions of The ycaC gene product has been predicted to be a homo-octameric hydrolase with unknown substrate specificity, catalyzing isochorismatase-like reactions [6], Isochorismatase catalyzes the conversion of isochoris- mate, in the presence of H2O, 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 ((cid:2) 20%). For
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such bacteria from other
(NP_770892), indicating that ycaCRs,
rat
(NP_651869) fruit and fly 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, 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 targeting sequence BbycaCR had a mitochondrial MSGLVRRLGKVGVDT, suggesting that it was a mitochondrial protein.
therefore
although biochemical and functional characterization lacking. The ycaC genes have recently been is still as Bacillus isolated cereus (GenBank accession number: NP_832865), Salmonella typhimurium (NP_459926), Bradyrhizobium japonicum 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), (XP_001059344), dog (XP_850761), and frog (Q5PQ71), and invertebrates, including silkworm (NP_001040541). They all possess a ycaC-related domain, known as the isochorismatase domain, and called ycaC-related (ycaCR) genes. are the ycaCR product has not yet been However, 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
ycaCR (GeneID: 700714), rat
37944) and nematode 178169) both comprised three
A search of the recently completed draft assembly the Branchiostoma and automated annotation of 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), ycaCR monkey (GeneID: 684270) and dog ycaCR (GeneID: 609103) all comprised five coding exons, whereas fruit fly ycaCR ycaCR (GeneID: (GeneID: 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
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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–a sandwich 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 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
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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 His6-tag produced a single band of (cid:2) 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 forming a band of (cid:2) 26 kDa, expression vector, 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 (cid:2) 22 kDa, matching the molecular mass predicted by BbycaCR cDNA. These show that the rabbit antiserum prepared has conspicuous antigen- specific reactivity.
total protein of
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: the amphioxus homogenate. marker. Lane 1: 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.
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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 His6-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.
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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.
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 (cid:2) 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
further
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 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).
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.
Discussion
Expression of BbycaCR
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 approaches, submissions. Although computational
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. 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 (cid:2) 890 bp.
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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 section in situ hybridization was detected. Tissue revealed that BbycaCR transcript was abundant in the
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A
C
D
B
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 lm in (C), (D) and (E).
mainly localized in mitochondria, the in vivo sites of ATP production. However, whether BbycaCR usually functions as a polymer remains to be studied. these genes,
amidohydrolase
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including sophisticated sequence analysis, phylogenetic patterns, domain fusions, structural threading and gene neighborhoods, can be used to predict the likely the ultimate biochemical properties of 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 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 the recombinant BbycaCR. Surprisingly, none of isochorismatase, nicotinamidase and N-carbamoyl- sarcosine 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
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A
B
Both tissue section in situ hybridization and immu- nohistochemistry revealed that BbycaCR was expressed ubiquitously in B. belcheri, but the expression levels varied in different tissues, with most the abundant lev- els being found in the gill, hepatic caecum, hindgut, endostyle, and ovary. The ubiquitous expression pat- tern suggests that BbycaCR plays a conserved funda- mental cellular role in amphioxus, lending additional support to the proposal that BbycaCR is involved in energy transduction. The predominant expression in the gill, hepatic caecum, hindgut, endostyle and ovary may be in line with a rapid energy turnover in these tissues.
C
D
Experimental procedures
The gut cDNA library of adult amphioxus was constructed with the smart cDNA Library Construction Kit (Clontech, Palo Alto, CA, USA), as described by Liu et al. [14]. Dur- ing large-scale sequencing of the gut cDNA library with the ABI PRISM 377XL DNA sequencer, more than 5000 clones were analyzed for coding probability with the dnatools program. Comparison against the GenBank protein database was performed using the blast network server at the NCBI. Multiple protein sequences were aligned using the megalign program by the clustal w method in the dnastar software package. The phylogenetic tree was constructed by the neighbor-joining method within the phylip 3.6 c software package, using 1000 bootstrap replicates.
(A, C) Fig. 7. Localization of BbycaCR by immnohistochemistry. Micrographs show the strong presence of BbycaCR in the hepatic caecum, gill, hindgut, endostyle and ovary in amphioxus. (B, D) Control sections. hc, hepatic caecum; hg, hindgut; g, gill; o, ovary; t, testis. Scale bars represent 100 lm.
Cloning and sequence analysis of cDNA
Expression and purification of recombinant protein
known subfamilies of the isochorismatase superfam- ily. Together, that BbycaCR these findings suggest may represent a novel member of the isochorisma- tase superfamily.
The complete coding region of BbycaCR was amplified by PCR with the upstream primer 5¢-CCGGAATTCATGTCG GGACTAGTGCG-3¢ (EcoRI site underlined) and the down- stream primer 5¢-AAGGAAAAAAGCGGCCGCCAAGTA GCACGTTAGGAC-3¢ (NotI site underlined). The reaction was carried out under the following conditions: initial dena- turation at 94 (cid:2)C for 5 min, followed by 30 cycles of denatur- ation at 94 (cid:2)C for 30 s, annealing for 30 s at 53 (cid:2)C, and extension at 72 (cid:2)C for 1 min. The PCR product was digested with EcoRI and NotI, and subcloned into the pET-28a expression vector (Novagen, Madison, WI, USA) previously cut with the same restriction enzymes. The identity of the insert was verified by sequencing, and the plasmid was desig- nated pET28a–BbycaCR.
Cells of E. coli BL21 were transformed with the plasmid in LB broth pET28a–BbycaCR, and cultured overnight containing kanamycin (30 lgÆmL)1). The culture was diluted 1 : 100 with LB broth and subjected to further
(amphioxus) invertebrate to is clear that
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A homology search over the recently completed draft assembly and automated annotation of the B. floridae genome shows that the coding region of BbycaCR is highly conserved within species. It is of interest to note that the ycaCR exon–intron structures were not conserved throughout animal evolution. The exons and introns of ycaCR genes numbers of increased during evolution of the gene from proto- stome invertebrates (nematode and fruit fly) to deuter- ostome vertebrates the amphioxus ycaCR (mammals). It genomic organization appears at a stage of evolution of protostome invertebrates to vertebrates, agreeing with its established phylogeny.
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incubation at 37 (cid:2)C for 3 h. The expression of BbycaCR was induced by addition of isopropyl-thio-b-d-galactoside to the culture at a final concentration of 0.1 mm. After incubation at 19 (cid:2)C for 12 h, bacterial cells were harvested by centrifugation, resuspended in 50 mm NaCl ⁄ Pi (pH 8.0) containing 0.3 m NaCl and 10 mm imidazole (binding buf- fer), and sonicated on ice. The recombinant protein was purified as described by Fan et al. [15]. Protein concentra- tions were determined by the method of Bradford [16], using BSA as a standard.
Tissue section in situ hybridization
Sexually mature B. belcheri organisms were cut into three to four pieces and fixed in freshly prepared 4% paraformal- dehyde in 100 mm NaCl ⁄ Pi (pH 7.4) at 4 (cid:2)C for 8 h. The samples were dehydrated in an ethanol gradient, embedded in paraffin, and sectioned at 7 lm. The sections were mounted on poly(l-lysine)-coated slides, dried at 42 (cid:2)C for 36 h, and deparaffinized in xylene for 20 min (two changes for 10 min each); this was followed by immersion in abso- lute ethanol for 10 min (two changes for 5 min each). They were then rehydrated, and finally equilibrated in double-dis- tilled water containing 0.1% diethylpyrocarbonate. Tissue section in situ hybridization histochemistry was carried out as described by Xue et al. [21].
Preparation of polyclonal antibody
The purified recombinant BbycaCR protein was used for raising antibody in two rabbits. Approximately 400 lg of the purified BbycaCR was emulsified with Freund’s com- plete adjuvant and injected subcutaneously at multiple sites of the rabbits. Two booster injections of 100 lg of antigen mixed with Freund’s incomplete adjuvant were administered subcutaneously at intervals of 2 weeks. Eight days after the final booster, the blood was collected from the rabbits by carotid puncture, and serum was prepared. The antisera were aliquoted and stored at )70 (cid:2)C. The antibody titer in the sera was determined by dot blot assay.
Western blotting
the
enzyme
activities of
Two amphioxus organisms were homogenized in 1 mL of 50 mm Tris ⁄ HCl (pH 7.2) with 50 mm NaCl on ice and cen- trifuged at 15 000 g for 20 min at 4 (cid:2)C. The supernatant was pooled and subjected to 12% SDS ⁄ PAGE. The cell lysates of isopropyl-thio-b-d-galactoside-induced E. coli BL21, control cell lysates and purified protein were also run on the same gel. The gel was washed for 15 min in 20 mm NaCl ⁄ Pi (pH 7.4) containing 0.1% Tween-20, and proteins on the gel were blotted on nitrocellulose membrane (Hybond, Amersham Pharmacia, Piscataway, NJ, USA). Blotted membrane was incubated in 20 mm NaCl ⁄ Pi (pH 7.4) containing 3% defat- ted milk powder at 30 (cid:2)C for 1.5 h, and then in the rabbit antisera diluted 1 : 800 with the same buffer for 2 h at 30 (cid:2)C. After being washed in 20 mm NaCl ⁄ Pi (pH 7.4), the mem- brane was incubated in the peroxidase-conjugated goat anti- rabbit serum diluted 1 : 1000 at 30 (cid:2)C for 2 h. Bands were visualized using 4-dimethylaminobenzene and 0.03% H2O2.
Enzymatic activity assays
As BbycaCR protein belongs to the isochorismatase super- family and has structural similarities to some other enzymes of the superfamily, including isochorismatase, nicotinami- dase and N-carbamoylsarcosine amidohydrolase [4,6], all three recombinant protein expressed in E. coli were tested. Isochorismatase activity was assayed as described by Rusnak et al. [1], by using chorismate acid as the alternative substrate [17], and nicoti- namidase activity was measured as described by Anderson et al. [18], by using nicotinamide as the substrate. For N-carbamoylsarcosine amidohydrolase activity assay, the substrate N-carbamoylsarcosine was chemically synthesized with sarcosine and potassium cyanate by the method of Nyc & Mitchell [19], and the enzyme activity was detected as described by Zajc et al. [20].
Amphioxus organisms were cut into four pieces and fixed in freshly prepared 4% paraformaldehyde (w ⁄ v) in 100 mm NaCl ⁄ Pi (pH 7.4) at 4 (cid:2)C for 12 h. After dehydration, the samples were embedded in paraffin and sectioned at 7 lm. The sections were mounted on slides and dried at 42 (cid:2)C for 36 h, and the immunohistochemical staining was performed as described by Liang et al. [22].
Immunohistochemistry
Northern blotting
Total RNA was extracted with Trizol (Gibco, Grand Island, NY, USA) from adult B. belcheri ground in liquid nitrogen. Aliquots of 3 lg of RNAs were each electropho- resed and blotted onto a Nylon membrane (Roche, Mann- heim, Germany). The digoxigenin (Dig)-labeled BbycaCR riboprobes of about 890 bp were synthesized in vitro from linearized plasmid DNA, following the Dig-UTP supplier’s instructions (Roche). Northern blot analysis was carried out as described previously [15].
The His-tag pull-down experiments were performed by a method modified from Boutell et al. [23] and Arifuzzaman et al. [24]. Cells of E. coli BL21 with the plasmid pET28a– BbycaCR were induced, harvested, and sonicated on ice as above. After centrifugation, the supernatant containing the recombinant protein was mixed with 50 lL of MagExtractor His-tag particles (TOYOBO, Osaka, Japan) at 4 (cid:2)C with
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His-tag pull-down assay
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Cruz, CA, USA) at 4 (cid:2)C for 5 h. A total of 20 lL of protein A ⁄ G PLUS-Agarose (Santa Cruz Biotechnology) was added to the reaction mixture and incubated at 4 (cid:2)C overnight. After centrifugation at 1000 g at 4 (cid:2)C for 5 min, the agaroses were washed four times with NaCl ⁄ Pi (pH 7.8), and the precipitated proteins were eluted from the agaroses with 30 lL of SDS ⁄ PAGE loading buffer and separated by 12% SDS ⁄ PAGE [26,27]. The immunoblotting analysis was incubations carried out as above. Two parallel reactions, of the monoclonal antibody with BbCK alone or with BbycaCR alone followed by interaction with protein A ⁄ G PLUS-Agarose, were the negative controls, and incubation of the monoclonal antibody with the homogenates of B. belcheri followed by interaction with protein A ⁄ G PLUS- Agarose was the positive control.
end-over-end agitation for 12 h. The particles were washed sequentially with binding buffer, wash buffer I (50 mm NaCl ⁄ Pi, pH 8.0, containing 0.3 m NaCl and 20 mm imidaz- ole) and wash buffer II (50 mm NaCl ⁄ Pi, pH 8.0, containing 0.3 m NaCl and 40 mm imidazole) three times each. Mean- while, 1 g of the gut, hepatic caecum or whole amphioxus was homogenized in 10 mL of homogenate buffer consisting of 50 mm Hepes (pH 7.4) with 250 mm glucose, 1 mm EDTA and 1 mm dithiothreitol (added freshly) on ice, and centri- fuged at 10 000 g at 4 (cid:2)C for 15 min, and the supernatants (3 mgÆmL)1 proteins) were pooled. Ten per cent of deoxy- cholic acid in H2O was added to the supernatants at a final concentration of 0.4%, and the supernatants were sonicated on ice to release the proteins in the cell organelles. The washed particles were incubated with 1 mL of the superna- tants at 4 (cid:2)C overnight with vigorous shaking. The particles were washed three times with 800 lL of homogenate buffer and three times with 800 lL of binding buffer. The controls consisted of the particles incubated with both BbycaCR recombinant protein and the homogenate buffer, and the particles incubated with pET28a-expressed soluble protein and the tissue homogenates. The interacted complexes were eluted with 50–100 lL of elution buffer (50 mm NaCl ⁄ Pi, pH 8.0, containing 0.3 m NaCl and 250 mm imidazole) and separated by 9% SDS ⁄ PAGE.
Preparation of homogenate, cytosolic and mitochondrial fractions and subcellular distribution assay
All steps were performed on ice. The homogenate, cytosolic and mitochondrial fractions were prepared by the method of Meffert et al. [28]. Approximately 0.5 g of the gut was homogenized in 4.5 mL of buffer A (250 mm sucrose, 5 mm Hepes, 0.5 mm EGTA, pH 7.4). The homogenate was sub- jected to centrifugation at 800 g for 5 min. The pellet was discarded, and the supernatant was centrifuged for 4 min at 5100 g (B). The supernatant of B was further clarified by centrifugation for 12 min at 12 300 g, yielding the cytosolic fraction. The pellet of B was resuspended in 1 mL of buf- fer A, and centrifuged for 10 min at 12 300 g (C). After resuspension of the C pellet in 1 mL of buffer A and centri- fugation for a further 10 min at 12 300 g, the sediment was resuspended in 0.4 mL of buffer A, yielding the mitochon- drial fraction. The different fractions were aliquoted and immediately frozen in liquid nitrogen and stored at )70 (cid:2)C until used. Protein concentrations were determined by the method of Bradford, using BSA as a standard. The same amounts of both cytosolic and mitochondrial fractions were analyzed by 12% SDS ⁄ PAGE and immunoblotting.
MS
Acknowledgements
To identify the pulled-down proteins, the Coomassie blue- stained bands were cut out of the gel, and the gel pieces were subjected to trypsin digestion and further processed as described by Gevaert & Vandekerckhove [25]. Peptide mass fingerprinting (PMF) analysis was performed on a Voyager DE Pro MALDI ⁄ TOF mass spectrometer (ABI, Framing- ham, MA, USA). Spectra were obtained by accumulation of 200 consecutive shots with the same laser level. The data obtained by MS were then submitted to the NCBI database for protein identification using mascot explorer software (http://www.matrixscience.com/; version: 9 April 2007) with ± 0.2 Da peptide mass tolerance. Protein scores > 79 were considered to be significant (P < 0.05). The mass calibra- tion was done externally on the target using a sequazyme mass STDs Kit (ABI).
Reverse pull-down assay This work was supported by the Ministry of Science and Technology (MOST) of China (2008AA09Z411, 2006CB101805).
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