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Original Paper
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Acta Biochim Biophys
Sin 2006, 38: 549-555 |
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doi:10.1111/j.1745-7270.2006.00197.x |
Verification, characterization and tissue-specific expression of UreG, a urease accessory�
protein Gene, from the amphioxus Branchiostoma belcheri
Ji-Yu Xue, Shi-Cui Zhang*,
Nai-Guo Liu, and Zhen-Hui Liu
Department
of Marine Biology, Ocean University of China, Qingdao 266003, China
Received: March 12,
2006�������
Accepted: May 14,
2006
This work was
supported by the grants from the Ministry of Science and Technology of China
(2005AA626010) and the National Natural Science Foundation of China (30470203)
*Correspondence
author: Tel, 86-532-82032787; Fax, 86-532-82032787; E-mail, [email protected]
Abstract������� UreG genes have been found in bacteria, fungi and plants
but have not yet identified in animals, although a putative UreG-like gene has
been documented in sea urchin. In the course of a large-scale sequencing� of
amphioxus gut cDNA library, we have identified a cDNA with high similarity to
UreG genes. Both reverse transcription-polymerase chain reaction and nested
polymerase chain reaction, as well as in situ hybridization
histochemistry, verified that the cDNA represented an amphioxus UreG gene (AmphiUreG)
rather than a microbial contaminant of the cDNA library. This is further
supported by the presence of urease activity in amphioxus gut, gill and ovary. AmphiUreG
encodes a deduced protein of 200 amino acid residues including a highly
conserved P-loop, bearing approximately 46%-49%,
44%-48%, and 29%-37%
similarity to fungal, plant and bacterial UreG proteins, respectively. It shows
a tissue-specific expression pattern in amphioxus, and is especially abundant
in the digestive system. This is the first UreG gene identified in animal
species.
Key words���� ���amphioxus; Branchiostoma
belcheri; UreG; expression; urease
Urease (EC 3.5.1.5) is a
nickel-containing enzyme catalyzing the hydrolysis of urea to form ammonia and
carbon dioxide. Its activity has been found in bacteria, eukaryotic
microorganisms, plants and some invertebrates [1-3].
Biochemically, bacterial ureases are best characterized and their activation
requires the presence of several accessory proteins including UreD, UreE, UreF
and UreG [2]. The specific role of each of these proteins has not yet been
fully clarified, but the available data indicate that the activation process is
quite complex. Most of the information on this process has been obtained from
the analysis of bacterial in vitro activation systems. UreD binds to
urease and appears to induce a conformational change for the next steps in the
activation process [4]. UreF binds the UreD-urease complex and seems to
facilitate carbamoy�lation of the nickel-bridging lysine residue and to prevent
Ni2+ binding to the
noncarbamoylated urease [5]. UreG can form a quaternary complex with
UreDF-urease and appears to energize the enzyme activation process [6,7]. UreE
is required for maximal enzyme activity, and is thought to bind the
UreDFG-urease complex, acting as a nickel-binding protein involved in Ni2+
storage and possibly delivered to the active site of the enzyme [8-11].
In addition to its presence in
bacteria, UreG has been identified in plants [12,13], and the homologs of
bacterial UreD, UreE and UreF have recently been documented in Arabidopsis [14].
Sequence comparison of UreG proteins from both bacteria and plants shows that
they all contain a highly conserved P-loop motif usually consisting of
G-P-V-G-T-G-K-T and typically found in nucleotide-binding proteins [2,3].
Because crustacean and mollusk
have been shown to possess urease activity [15], one would expect the presence
of urease accessory proteins, including UreG, in invertebrates. Surprisingly,
to date, no UreG has been identified in animal species, although a putative
UreG-like gene (RefSeq accession No. xp_792077) has been found in sea urchin Strongylocentrotus
purpurtaus. In this report, we document for the first time the
identification of an amphioxus gene, AmphiUreG, which displays striking
similarity to bacterial, fungal and plant UreG genes, and describe its
expression pattern in adult amphioxus.
Materials and methods
Cloning and sequence analysis
of cDNA
Gut cDNA library of adult
amphioxus was constructed with the SMART cDNA library construction kit
(Clontech, Palo Alto, USA) as described previously [16,17]. In a large-scale
sequencing of amphioxus gut cDNA library with ABI PRISM 377XL DNA sequencer
(Invitrogen, Carlsbad, USA), more than 5000 clones were analyzed for coding
probability with the DNATools program (Rehm BH, Munster, Germany) [18].
Comparison against the GenBank protein database was carried out using the BLAST
network server (http://www.ncbi.nih.gov/BLAST)
[19]. Multiple protein sequences were aligned using the MegAlign program by the
CLUSTAL method in the DNASTAR software package (DNASTAR, Madison, USA) [20]. A
phylogenetic tree was constructed by the neighbor-joining method within the
PHYLIP 3.5c software package, supplied by Prof. J. FELSENSTEIN (Department of
Genomic Sciences, University of Washington, Seattle, USA), using 1000 bootstrap
replicates.
Reverse
transcription-polymerase chain reaction
Adult amphioxus Branchiostoma
belcheri collected during their breeding season were starved for 2 d in sterilized
filtered seawater before experiment to remove all food in the gut, and tissues
from ovary and digestive tract were dissected. Total RNAs were prepared with
Trizol (Sigma-Aldrich, St. Louis, USA) from whole organism, ovary and digestive
tract. For RT-PCR, the first strand cDNA was synthesized in 50 ml of avian myeloblastosis virus (AMV)/Tfl
1�reaction buffer containing 1 mg of total RNAs, 0.1 U/ml of AMV reverse transcriptase (Promega,
Madison, USA), 0.2 mM dNTP mix, 1 mM each
of antisense primer NF2 (5'-CACCAGTCAGGCACATC-3') and sense
primer NS2 (5'-ACACCCTCCTGTCTCCAC-3'), 0.1 U/ml Tfl DNA polymerase (Promega) and 1 mM
MgSO4.
The reaction was carried out at 48 �C for 45 min. After AMV reverse
transcriptase inactivation and RNA/cDNA/primer denaturation at 94 �C for 2 min,
the second-strand cDNA synthesis and PCR amplification were carried out in 30
cycles using the following parameters: denaturation at 94 �C for 30 s,
annealing at 61 �C for 1 min, and elongation at 68 �C for 2 min. The reaction
was continued for a final extension at 68 �C for 7 min and terminated at 4 �C.
Normalization was carried out by amplification of cytoskeletal b-actin mRNA using an antisense primer (5'-GCTGGGCTGTTGAAGGTC-3') and a
sense primer (5'-CTCCGGTATGTGCAAGGC-3'),
under the same conditions as described above. The RT-PCR product from ovary
RNAs was purified and ligated into pGEMT-Easy Vector (Promega), transformed
into Escherichia coli JM109 cells, and sequenced with the ABI PRISM
377XL DNA sequencer.
Nested PCR
The digestive tracts of adult
amphioxus B. belcheri were removed, and the genomic DNAs were isolated
from the gut-free amphioxus according to Ma et al. [21]. The nested PCR
primers used were external primers NF1 (5'-ATGGCATCTACTGATCAAGT-3')
and NS1 (5'-CCTGATAATAGCTTTCATTT-3'), that match the 20
nucleotides of both 5' and 3' ends of AmphiUreG, and
internal (nested) primers NF2 (5'-CCACCAGTCAGGCACATC-3') and NS2
(5'-ACACCCTCCTGTCTCCAC-3'), that were both designed by PRIMER
5.0. The reaction for the first step of nested PCR was carried out in 25 ml of 1�PCR buffer (Mg2+ plus;
Takara, Takara, Japan) containing 1 mg
of genomic DNA, 0.2 mM dNTP mix, 0.8 mM each
NF1 and NS1 and 0.025 U/ml Taq DNA polymerase (Takara).
The reaction conditions were: 95 �C for 5 min; denaturating at 94 �C for 1 min,
annealing at 53.2 �C for 1 min, and elongating at 72 �C for 1 min, 25 cycles;
and final extension at 72 �C for 7 min. Aliquots of 5 ml of reaction mixtures were sampled and
run on an agarose gel to estimate the quantity of PCR products, then the
reaction mixtures were diluted to 1:1000, and used as the templates for the
second step of nested PCR. The second step of nested PCR was carried out in 25 ml of 1�PCR buffer (Mg2+ plus)
with 1 ml of diluted products from the
first step of nested PCR, 0.2 mM each dNTP, 0.8 mM
both NF2 and NS2, and 0.025 U/ml Taq DNA polymerase.The
reaction parameters were: 95 �C for 5 min; denaturating at 94 �C for 1 min, 30
cycles; annealing at 61 �C for 1 min and elongating at 72 �C for 1 min; and final
extension at 72 �C for 7 min. A single PCR amplification product of
approximately 900 bp was purified and ligated into pGEMT-Easy Vector,
transformed into E. coli JM109 cells, and sequenced with ABI PRISM 377XL
DNA sequencer.
In situ hybridization histochemistry
All reagents used for in
situ hybridization histochemistry were prepared with 0.1% diethyl
pyrocarbonate in double-distilled H2O to rid the working solutions
of RNases. Digoxigenin (Dig)-labeled AmphiUreG riboprobes of
approximately 700 bp were synthesized in vitro from linearized plasmid
DNA following the Dig-UTP supplier's instructions (Roche, Basel, Switzerland).
Sexually mature amphioxus were
cut into two or three pieces and fixed in freshly prepared 4% paraformaldehyde
in 100 mM phosphate-buffered saline (PBS; pH 7.4) at 4 �C for 8 h. After
dehydration, they were embedded in paraffin, sectioned at 6 mm, mounted on poly-L-lysine coated
slides, and dried at 42 �C for 36 h. The sections were dewaxed in xylene for
two times (10 min each time), followed by immersion in 100% ethanol for two
times (5 min each time). After rehydration, they were brought to
double-distilled H2O with 0.1% diethyl pyrocarbonate,
digested with 6 mg/ml proteinase K in 100 mM
Tris-HCl buffer (pH 8.0) containing 50 mM EDTA at 37 �C for 30 min, post-fixed
in 4% paraformaldehyde in 10 mM PBS (pH 7.4) at room temperature for 30 min,
acetylated in freshly prepared 100 mM triethanolamine-HCl (pH 8.0) with 0.25%
acetic anhydrite at room temperature for 10 min, and dehydrated with graded
ethanol. They were then pre-hybridized in a hybridization buffer containing 50%
(v/v) deionized formamide, 100 mg/ml
heparin, 5�standard saline citrate, 0.1% Tween-20, 5 mM EDTA, 1�Denhardt's solution
and 1 mg/ml total yeast RNA at 55 �C for 3 h, and hybridized in the same
hybridization buffer with 1 mg/ml Dig-labeled AmphiUreG
riboprobes at 55 �C for 12-16 h in a humidified chamber.
Subsequently, the sections were subjected to RNase A (Promega) digestion buffer
(20 mg/ml in 2�standard saline
citrate) at 37 �C for 30 min, washed in 100 mM Tris-HCl (pH 7.4) with 150 mM
NaCl for three times (15 min each time), pre-incubated in 1% blocking reagent
(Roche) in 100 mM Tris-HCl (pH 7.4) with 150 mM NaCl for 1 h at room
temperature, and incubated with anti-Dig alkaline phosphatase conjugated
antibody (Roche) diluted 1:1000 in 1% blocking reagent in 100 mM Tris-HCl (pH
7.4) with 150 mM NaCl for 2 h at room temperature. The sections� were washed in
100 mM Tris-HCl (pH 7.4) containing 100 mM NaCl and 50 mM MgCl2 for
three times (5 min each time), then incubated with a coloring solution
consisting of 4.5 mg/ml NBT and 3.5 mg/ml BCIP in 100 mM Tris-HCl (pH 8.0)
with 100 mM NaCl and 50 mM MgCl2 (Boehringer-Ingelheim, Mannheim,
Germany) for 2-24 h in the dark. The color
reaction was stopped in PBS for 10 min. After rinsing in distilled water, the
sections were dehydrated, mounted in Canada balsam, and photographed under a
BX51 Olympus microscope (Olympus, Tokyo, Japan).
Urease activity assay
Urease activity was measured
according to the instructions of the test kit (Sanqiang, Sanming, China) at
room temperature. Briefly, gut, gill and ovary were dissected out of B.
belcheri, which had been cultured for 2 d in sterilized filtered seawater
to remove all food in the gut, rinsed in 50 mM Tris-HCl buffer (pH 7.2)
containing 50 mM NaCl, and cut into small pieces (2-3
mm3).
The pieces were placed in the reaction solution, and the enzyme activity was
demonstrated by the color change in the reaction solution from yellow to pink
or red.
Results and discussion
The cDNA clone L239 (GenBank
accession No. AAT39417) obtained from the gut cDNA library of amphioxus B.
belcheri is 966 bp long. Its longest open reading frame consists of 603 bp
encoding a protein of 200 amino acid residues with a predicted molecular weight
of approximately 22.41 kDa and an isoelectric point of 6.6. The 5'-untranslated
region is 284 bp long with a typical oligopyrimidine motif and an in-frame stop
codon (TAG), and the 3'-untranslated region is 78 bp long with a
polyadenylation tail (Fig. 1). Therefore, the clone L239 encodes a
full-length sequence protein.
The initial BLASTP search
revealed that the protein encoded by clone L239 shared 70% (119/169) and 62%
(95/151) identity with the UreG-like protein of sea urchin S. purpuratus
and the UreG of fungus Cryptococcus neoformans (Genbank accession No.
AAW41177), respectively. It was thus further compared with other members of the
UreG and UreG-like family. Fig. 2 shows an alignment of the amino acid
sequence of the protein with that of known UreG/UreG-like proteins from 15
species including sea urchin, fungi, plants and bacteria. The proteins encoded
by clone L239 were approximately 66%, 46%-49%,
44%-48%, and 29%-37%
similar to sea urchin, fungal, plant and bacterial UreG/UreG-like proteins,
respectively. Moreover, they all possessed a highly conserved P-loop.
Therefore, the clone L239 encodes a UreG-like protein.
To verify that the UreG-like
gene is from amphioxus itself, we carried out RT-PCR and nested PCR analyses.
RT-PCR, based on RNAs from digestive tract, ovary and whole organism, all
formed a single amplification band of the expected size (324 bp) (Fig. 3),
and sequencing of the RT-PCR product from ovary RNAs showed that it exactly
matched the expected sequence (data not shown). This suggests that the clone
L239 did not represent a bacterial� or fungal contaminant. Nested PCR also
yielded a single amplification product of approximately 900 bp [Fig. 4(A)].
Sequencing of the nested PCR product produced� a resulting sequence of 701 bp,
which included two introns, 1 and 2, with intron 2 being complete [Fig. 4(B)].
The predicted mRNA sequence of nested PCR product� well agrees with that
deduced from clone L239 [Fig. 4(C)]. It is of particular interest to
note that intron 2 begins with GT and ends with an AG dinucleotide, sequences�
thought to be necessary for correct RNA splicing� of various other eukaryotic
genes [22]. The identification� of the introns and consensus splice acceptor�
site in the nested PCR product indicates that the clone L239 represents an
expressed eukaryotic gene rather than a transcribed pseudogene. In addition, in
situ hybridization histochemistry revealed a tissue-specific expression
pattern of the clone L239 in amphioxus (see below). All of these results show
that the clone L239 stands for an amphioxus homolog of UreG gene, therefore
designated AmphiUreG. This is also supported by a search of the recently
completed draft assembly and automated annotation of the Branchiostoma
floridae genome (http://shake.jgi-psf.org/Brafl1/Brafl1.home.html),
which revealed the presence of UreG, UreD and UreF (estExt_fgenesh2_pg.C_1500097;
estExt_fgenesh2_pg.C_1500095; fgenesh2_pg.scaffold_150000093). Moreover, in our
experiment, the instant color change in the reaction solution containing the
pieces of gut, gill and ovary demonstrated the presence of urease activity in
these tissues, providing additional evidence for the existence of UreG
in amphioxus. It is apparent that AmphiUreG is the first UreG gene fully
identified so far in an animal species.
The phylogenetic tree
constructed using the amino acid sequence of AmphiUreG and that of other
known UreG/UreG-like proteins from various species, including sea urchin,
fungi, plants and bacteria, showed that AmphiUreG and sea urchin
UreG-like protein clubbed together, forming a clade with eukaryotic UreG,
whereas all bacterial UreG proteins clustered together (Fig. 5).
Identification of other UreG-like genes from more animal species will shed
detailed light on the evolution of UreG.
In situ hybridization histochemistry demonstrated an abundant expression of AmphiUreG in the digestive system, including endostyle, hepatic caecum, foregut and hindgut, and in the gill. A faint expression of AmphiUreG was also detected in the ovarian oocytes, but not in the muscle, notochord, neural tube, nor testis (Fig. 6). It is clear that AmphiUreG is expressed in amphioxus in a tissue-specific manner, which is in contrast to the ubiquitous expression pattern of UerG in plants [23,16]. Wang et al. [25] has recently proven the presence of allantoicase activity, one of the urate-degrading enzymes, in amphioxus [26]. Here we show the existence of urease activity and a urease accessory protein gene, UreG, in amphioxus. It is therefore highly likely that AmphiUreG is similar to the microbial and plant UreG and is involved in the recycling of metabolically-derived urea.
References
1�� Polacco JC, Holland MA. Roles of urease in plant cells. Int Rev
Cytol 1993, 145: 65-103
2�� Mobley HL, Island MD, Hausinger RP. Molecular biology of microbial
ureases. Microbiol Rev 1995, 59: 451-480
3�� Sirko A, Brodzik R. Plant ureases: roles and regulation. Acta Biochim Pol 2000, 47: 1189-1195
4�� Park IS, Hausinger RP. Evidence for the presence of urease
apoprotein complexes containing UreD, UreF, and UreG in cells that are
competent for in vivo enzyme activation. J Bacteriol 1995, 177: 1947-1951
5�� Moncrief MB, Hausinger RP.
Purification and activation properties of UreD-UreF-urease apoprotein
complexes. J Bacteriol 1996, 178: 5417-5421
6�� Park IS, Hausinger RP. Evidence for the presence of urease apoprotein
complexes containing UreD, UreF, and UreG in cells that are competent for in
vivo enzyme activation. J Bacteriol 1995, 177: 1947-1951
7�� Soriano A, Hausinger RP. GTP-dependent activation of urease
apoprotein in complex with the UreD, UreF, and UreG accessory proteins. Proc
Natl Acad Sci USA 1999, 96: 11140-11144
8�� Lee MH, Pankratz HS, Wang S, Scott RA, Finnegan MG, Johnson MK,
Ippolito JA et al. Purification and characterization of Klebsiella
aerogenes UreE protein: a
nickel-binding protein that functions in urease metallocenter assembly. Protein
Sci 1993, 2: 1042-1052
9� Brayman TG, Hausinger RP. Purification, characterization, and
functional analysis of a truncated Klebsiella aerogenes UreE urease accessory
protein lacking the histidine-rich carboxyl terminus. J Bacteriol 1996, 178:
5410-5416
10� Soriano A, Colpas GJ, Hausinger RP. UreE stimulation of
GTP-dependent urease activation in the UreD-UreF-UreG-urease apoprotein
complex. Biochemistry� 2000, 39: 12435-12440
11� Zambelli B, Stola M, Musiani F, De Vriendt K, Samyn B, Devreese B,
Van Beeumen J et al. UreG, a chaperone in the urease assembly process,
is an intrinsically unstructured GTPase that specifically binds Zn2+. J Biol Chem 2005,
280: 4684-4695
12� Freyermuth SK, Bacanamwo M, Polacco JC. The soybean Eu3 gene
encodes� an Ni-binding protein necessary for urease activity. Plant J 2000, 21:
53-60
13� Witte CP, Isidore E, Tiller SA, Davies HV, Taylor MA. Functional
characterisation of urease accessory protein G (ureG) from potato. Plant Mol
Biol 2001, 45: 169-179
14� Witte CP, Tiller SA, Taylor MA, Davies HV. Leaf urea metabolism in
potato. Urease activity profile and patterns of recovery and distribution of
(15)N after foliar urea application in wild-type and urease-antisense
transgenics. Plant Physiol 2002, 128: 1129-1136
15� Takada Y, Noguchi T. Subcellular distribution, and physical and
immunological� properties of hepatic alanine: glyoxylate
aminotransferase isoenzymes in different mammalian species. Comp Biochem
Physiol B 1982, 72: 597-604
16� Liu Z, Zhang S, Yuan J, Sawant MS, Wei J, Xu A. Molecular cloning
and phylogenetic analysis of AmphiUbf80, a new member of ubiquitin
family from the amphioxus Branchiostoma belcheri tsingtauense. Curr Sci
2002, 83: 101-104
17� Li X, Zhang SC, Liu ZH, Li HY. Ribosomal Protein Genes S23 and L35
from Amphioxus Branchiostoma belcheri tsingtauense: Identification and
Copy Number. Acta Biochim Biophys Sin 2005, 37: 573-579
18� Rehm BH. Bioinformatic tools for DNA/protein sequence analysis,
functional� assignment of genes and protein classification. Appl Microbiol
Biotechnol 2001, 57: 579-592
19� Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W,
Lipman DJ. Gapped BLAST and PSI-BLAST: a
new generation of protein database search programs. Nucleic Acids Res 1997, 25:
3389-3402
20� Burland TG. DNASTAR�s Lasergene sequence analysis software. Methods
Mol Biol 2000, 132: 71-91
21� Ma L, Zhang S, Liu Z, Li H, Xia J. Characterization and copy number
of the S27 ribosomal protein gene from amphioxus Branchiostoma belcheri
tsingtauense. Genet Mol Biol 2005, 28: 839-842
22� Breathnach R, Benoist C, O�Hare K, Gannon F, Chambon P. Ovalbumin
gene: evidence for a leader
sequence in mRNA and DNA sequences at the exon-intron boundaries. Proc Natl
Acad Sci USA 1978, 75: 4853-4857
23� Witte CP, Rosso MG, Romeis T. Identification of three urease
accessory proteins that are required for urease activation in Arabidopsis.
Plant Physiol 2005, 139: 1155-1162
24� Polacco JC, Holland MA. Genetic control of plant ureases. In:
Setlow JK ed. Genetic Engineering. Vol 16. New York: Plenum Press 1994
25� Wang Y, Zhang S, Liu Z, Li H, Wang L. Identification and expression
of amphioxus h-microseminoprotein (MSP)-like gene encoding an ancient and
rapidly evolving protein in chordates. Comp Biochem Physiol Biochem Mol Biol
2005, 142: 251� 257
26� Fujiwara S, Noguchi T. Degradation of purines: Only
ureidoglycollate lyase out of four allantoin-degrading enzymes is present in
mammals. Biochem J 1995, 312: 315-318