BMC Genomics
BioMed Central
Open Access
Research article
In silico characterization of the family of PARP-like
poly(ADP-ribosyl)transferases (pARTs)
Helge Otto1, Pedro A Reche2,3, Fernando Bazan2,4, Katharina Dittmar5,
Friedrich Haag1 and Friedrich Koch-Nolte*1
Address: 1Institute of Immunology, University Hospital Hamburg-Eppendorf, Martinistr. 52, 20246 Hamburg, Germany., 2DNAX Research
Institute, Palo Alto, CA 94304, USA., 3Dana-Farber Cancer Institute, Harvard University, Boston, MA 02115, USA., 4Depts. of Molecular Biology
and Protein Engineering, Genentech, SF, CA 94080, USA. and 5Department of Integrative Biology, Brigham Young University, Provo, UT 84602,
USA.
Email: Helge Otto - [email protected]; Pedro A Reche - [email protected]; Fernando Bazan - [email protected];
Katharina Dittmar - [email protected]; Friedrich Haag - [email protected]; Friedrich Koch-Nolte* - [email protected]
* Corresponding author
Published: 04 October 2005
BMC Genomics 2005, 6:139
doi:10.1186/1471-2164-6-139
Received: 13 May 2005
Accepted: 04 October 2005
This article is available from: http://www.biomedcentral.com/1471-2164/6/139
© 2005 Otto et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
Background: ADP-ribosylation is an enzyme-catalyzed posttranslational protein modification in which
mono(ADP-ribosyl)transferases (mARTs) and poly(ADP-ribosyl)transferases (pARTs) transfer the ADPribose moiety from NAD onto specific amino acid side chains and/or ADP-ribose units on target proteins.
Results: Using a combination of database search tools we identified the genes encoding recognizable
pART domains in the public genome databases. In humans, the pART family encompasses 17 members.
For 16 of these genes, an orthologue exists also in the mouse, rat, and pufferfish. Based on the degree of
amino acid sequence similarity in the catalytic domain, conserved intron positions, and fused protein
domains, pARTs can be divided into five major subgroups. All six members of groups 1 and 2 contain the
H-Y-E trias of amino acid residues found also in the active sites of Diphtheria toxin and Pseudomonas
exotoxin A, while the eleven members of groups 3 – 5 carry variations of this motif. The pART catalytic
domain is found associated in Lego-like fashion with a variety of domains, including nucleic acid-binding,
protein-protein interaction, and ubiquitylation domains. Some of these domain associations appear to be
very ancient since they are observed also in insects, fungi, amoebae, and plants. The recently completed
genome of the pufferfish T. nigroviridis contains recognizable orthologues for all pARTs except for pART7.
The nearly completed albeit still fragmentary chicken genome contains recognizable orthologues for
twelve pARTs. Simpler eucaryotes generally contain fewer pARTs: two in the fly D. melanogaster, three
each in the mosquito A. gambiae, the nematode C. elegans, and the ascomycete microfungus G. zeae, six in
the amoeba E. histolytica, nine in the slime mold D. discoideum, and ten in the cress plant A. thaliana.
GenBank contains two pART homologues from the large double stranded DNA viruses Chilo iridescent
virus and Bacteriophage Aeh1 and only a single entry (from V. cholerae) showing recognizable homology
to the pART-like catalytic domains of Diphtheria toxin and Pseudomonas exotoxin A.
Conclusion: The pART family, which encompasses 17 members in the human and 16 members in the
mouse, can be divided into five subgroups on the basis of sequence similarity, phylogeny, conserved intron
positions, and patterns of genetically fused protein domains.
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Background
ADP-ribosylation is a posttranslational protein modification in which the ADP-ribose moiety is transferred from
NAD onto specific amino acid side chains of target proteins [1-4]. ADP-ribosylation was originally discovered as
the pathogenic principle of Diphtheria toxin, a multidomain secreted protein which inactivates elongation factor
2 by ADP-ribosylation after translocation into eucaryotic
cells [5]. Subsequently, numerous other bacterial toxins
were shown to ADP-ribosylate target proteins in host cells.
Moreover, endogenous toxin-like ADP-ribosylating
enzyme activities were detected in eucaryotic cells. Several
of these enzymes were purified to homogeneity,
sequenced, expressed as recombinant proteins, and
crystallized.
Sequence and structural analyses revealed the existence of
two distinct families of toxin-related ADP-ribosyltransferases in mammals [6,7]: The RT6 family of GPIanchored and secretory mono-(ADP-ribosyl)transferases
(mARTs) catalyzes mono-ADP-ribosylation of cell surface
and secretory proteins [8]. The PARP family of nuclear and
cytoplasmic poly(ADP-ribosyl)transferases (pARTs) catalyzes poly-ADP-ribosylation of nuclear and cytosolic proteins [9-12]. While mARTs have been implicated to
mediate signalling functions of extracellular NAD, pARTs
have been shown to play important roles in DNA repair
and maintenance of genome integrity [8,9,12].
In this paper we use the term pART (poly ADP-ribosyltransferase) rather than the more established term PARP
(poly-ADP-ribosyl-polymerase) for various reasons.
Firstly, to emphasize the structural and functional similarities of the poly- and mono-ADP-rib syltransferase subfamilies. Secondly, with respect to the biochemical
classficiation of enzymes the term transferase is more
appropriate than polymerase: ADP-riboslytransferases
belong to the family of glycosyltransferases; the term
polymerase is more commonly used for template-dependent DNA or RNA synthesizing enyzmes. Thirdly, use of the
term PARP would have confounded comparison of our
results with those of the recent review by Ame et al. [11],
who used the term PARP and a numbering system without
regard to structural similarities among gene family
members.
The 3D-structures of rat ART.2 (PDB accession number
1og3), chicken PARP-1 (1a26, 3pax), mouse PARP-2
(1gs0), and numerous ADP-ribosylating toxins uncovered
a common NAD binding fold with a conserved core of five
β strands arranged in two abutting β sheets [13-19]. These
two β sheets form the upper and lower jaws of a Pacmanlike active site crevice (Figure 1). Remarkably, only a single amino acid residue, the catalytic glutamic acid residue
at the front edge of the fifth conserved β-strand, is strictly
http://www.biomedcentral.com/1471-2164/6/139
conserved in all known 3D structures of enzymatically
active mARTs and pARTs. In a seminal study, Collier and
co-workers pinpointed the corresponding glutamic acid
residue in PARP-1 (before its 3D structure was solved) on
the basis of barely detectable sequence similarity to Diphtheria toxin [20,21]. More recently, the 3D structures of
anthrax lethal factor, VIP2, and iota toxin have been discovered to harbour ART-domains that lack a corresponding glutamic acid residue and may represent inactivated
enzymes [16,22,23].
Comparative structure and amino acid sequence analyses
revealed that PARP-1 and PARP-2 share additional secondary structure and conserved amino acids with Diphtheria toxin and Pseudomonas exotoxin A, which
evidently are not conserved in other mARTs (Fig. 1) [6,7].
These additional elements include a sixth β strand, an
alpha helix between β strands 2 and 3, and a trias of
amino acids, the so-called H-Y-E motif, encompassing a
histidine resdiue in β strand 1, a tyrosine residue in β
strand 3 and the catalytic glutamic acid residue at the front
edge of β strand 5. These features, highlighted in the 3D
structures of PARP-1 and Diphtheria toxin in Figure 1,
clearly distinguish the structures of PARP-1, PARP-2, and
DT/ETA from those of a second major ART subfamily that
includes rat ART2 and the Bacillus cereus VIP2 toxin. Distinguishing features of the ART2/VIP2 subfamliy include a
seventh β strand that displaces β strand 6, three conserved
alpha helices preceding β strand 1, and an R-S-E trias of
amino acid residues in place of the H-Y-E motif of PARP1 and DT. Interestingly, the recently reported 3D-structure
of a prototype member of the family of tRNA:NAD 2'
phosphotransferases (TpT) [24] revealed a striking resemblance to the structures of the PARP-1/DT subfamily
rather than to those of the ART2/VIP subfamily, including
the sixth β strand, the alpha helix between β strands 2 and
3, and a variant H-Y-E motif (H-H-V). These enzymes catalyze removal of a splice junction 2' phosphate from
ligated tRNA. This reaction resembles the reaction catalyzed by ARTs but yields ADP-ribose 1"-2" cyclic phosphate rather than ADP-ribosylated proteins [25].
The remarkable degree of plasticity of ART amino acid
sequences poses a challenging problem for genome data
base mining [7] and even the most sensitive database
search programs fail to connect all known members of the
ART gene family. Notwithstanding, the results of such in
silico analyses can provide important insight into the
structural and phylogenetic relationship of ART subfamilies. We have previously demonstrated that the
known members of the mART gene family in the human
and mouse could be faithfully connected with many
known bacterial ADP-ribosylating toxins, but not with
pARTs or Diphtheria toxin [26,27]. These analyses also
pointed out the presence of mART-encoding genes in the
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4
4
S
Y
1
1 H
N
R
N
3
3
7
6
C
E
2
E
2
loop
5
loop
5
C
6
PARP +
ART2 +
C
3
1
2
loop
loop
N
N
C
DT +
VIP2 +
3
1
2
N
C
loop
loop
N
C
TpT
N
loop
C
Schematic
Figure 1 illustration of the distinguishing structural features of the PARP-1/DT vs. the ART2/VIP2 subfamilies of ADP-ribosyltransferases
Schematic illustration of the distinguishing structural features of the PARP-1/DT vs. the ART2/VIP2 subfamilies of ADP-ribosyltransferases. Two abutting sheets of anti-parallel β strands form the upper and lower jaws of a
Pacman-like NAD-binding crevice in all known structures of ADP-ribosyltransferases. The distinguishing structural features of
the PARP/DT and ART2/VIP2 subfamilies are depicted schematically on top and are highlighted in the structures of chicken
PARP-1 (3pax), diphtheria toxin (DT) (1tox), an archael tRNA:NAD 2'-phosphotransferase (TpT) (1wfx), rat ART2 (1og3) and
B. cereus VIP2 toxin (1qs2) below. The structures are depicted from the "front view" with a full view of the ligands bound in the
active site crevice. The ligands NAD and 3MB are colored cyan and are depicted as stick models. The central four β-strands
(from top to bottom: β 5, β 2, β 1, β 3, colored orange) are conserved in all mARTs and pARTs. The β strands at the edges of
the respective sheets (β 4 and β 6, colored pink) show greater structural variation than the central β strands. The H-Y-E motif
residues are depicted in red and their side chains are shown as sticks. The glutamic acid residue at the front edge of β 5 is the
critical catalytic residue in both diphtheria toxin and PARP-1 – a corresponding glutamic acid residue is observed also in the 3D
structures of rat ART2 and numerous bacterial mARTs. Diphtheria toxin (1tox), pseudomonas exotoxin A (1aer), PARP-1
(3pax), and PARP-2 (1gs0) share the following structural features which are not conserved in either rat ART2 (1og3) or most
other bacterial mARTs: the orientation of β 6, the alpha helix between β 2 and β 3 (colored yellow) and the conserved histidine and tyrosine amino acid residues in β 1 and β 3. The loop between β 4 and β 5 (colored magenta) is thought to play a role
in the recognition of target proteins and ADP-ribose polymers. Distinguishing features of ART2, VIP2, iota toxin (1gir), and the
C3 exoenzymes (1g24, 1ojz) include three conserved alpha helices upstream of β strand 1, a seventh β strand that displaces β
strand 6 and an R-S-E- motif instead of the H-Y-E motif of PARP-1 and DT. (Note that the depicted ART2 structure carries a
site directed mutation of the catalytic glutamic acid residue E189I). The recently determined 3D structure of the tRNA:NAD
2'-phosphotransferase (1wfx) bears striking resemblance to that of DT and PARP-1 and carries an H-H-V variant of the H-Y-E
motif. Note that the structure of the diphtheria toxin catalytic domain shown here in complex with NAD is truncated C-terminally at the proteolytic cleavage site that separates this domain from the translocation domain. The PARP-1 catalytic domain
shown here is truncated N-terminally at the position of the phase 0 intron that separates this domain from a neighboring helical domain. The TpT catalytic domain is truncated N-terminally at the point of fusion to a winged-helix domain.
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BMC Genomics 2005, 6:139
genomes of many but not all other model organisms. Of
note, no mART-encoding genes could be detected in
plants, fungi, or archaea. Here we provide an in depth
analysis of the pART gene family.
Results and discussion
Identification of human and mouse pART family members
in the EST database
The human and mouse pART gene family members were
identified using a combination of data base search tools.
The human and mouse EST databases as well as the nonredundant GenBank database (nr) were screened with
tBLASTn using as queries the amino acid sequences of the
catalytic domains of the known and newly identified
pART family members. Whenever possible, the full coding
sequence of the catalytic domain and of the adjacent
regions was assembled using the sequences of published
cDNAs and overlapping ESTs. Screening of the EST and nr
databases was initiated in 1997 and was repeated in regular intervals. The coding sequences were extended when
suitable new sequences became available. When the
sequences of the human, mouse and rat genomes were
published in 2000, 2001, and 2004, respectively, the EST
database searches were complemented with corresponding tBLASTn and BLASTn searches of the genome
sequences [28-30]. Thereby, 17 pART family members
were identified in the human. These genes were designated pART1-pART17. Numbering reflects the degree of
amino acid sequence similarity to PARP-1 (= pART1) and
the degree of similarity within each of the pART subgroups. An orthologue for each of these genes was
detected in the mouse and in the rat, with the sole exception of pART7.
A complete list of human pART family members, including the common names and aliases of known genes, is
presented in Figure 2. Based on the degree of amino acid
sequence similarities, conserved intron positions, and
fused protein domains, the mammalian pART family can
be divided into five major subgroups. Group 1 (pART1pART4) contains PARP and its closest relatives, PARP-2,
PARP-3 and VPARP. Group 2 (pART5, pART6) contains
tankyrase 1 and tankyrase 2. Group 3 (pART7-pART10)
contains four proteins including the recently described BAggressive Lymphoma Protein (BAL = pART9) [31] and a
myc-interacting protein with PARP activity (PARP-10)
[32]. Group 4 (pART11-pART14) contains four proteins
including the recently described Zinc-finger Antiviral Protein (ZAP = pART13) [33] and TCDD-inducible PARP
(TiPARP) [34]. Group 5 (pART15-pART17) contains three
proteins of unknown function.
The steady growth in the number of matching ESTs
obtained for each of the human pART gene family members over the past 6 years is illustrated in additional file 1
http://www.biomedcentral.com/1471-2164/6/139
("Representation of pART gene transcripts in the database
of expressed sequence tags"). By October 2004, each
human pART except pART7 was represented by more than
100 ESTs. Interestingly, each pART except pART7 is represented by more ESTs than poly (ADP-ribose) glycohydrolase (PARG), the single known enzyme capable of
removing poly-ADP-ribose from pART target proteins.
The large number of ESTs corresponds to a large variety of
tissues found to contain pART ESTs and presumably
reflects an ubiquitous pattern of gene expression, i.e. akin
to that of the house keeping enzymes hypoxanthine-guanine phosphoribosyltransferase (HPRT) and glyceraldehyde-3-phosphate
dehydrogenase
(GAPD).
For
comparison, the members of the mART gene family
(ART1-ART5), which exhibit highly restricted patterns of
expression, are each represented by much fewer ESTs than
the pARTs. As of January 2005, the mammalian gene collection http://mgc.nci.nih.gov contains annotated fulllength cDNA sequences for 10 of the 17 human pARTs
and for 12 of 16 mouse pARTs (Fig. 2).
Chromosomal localizations and exon/intron structures of
the human and mouse pART gene family members
The results of tBLASTn and BLASTn searches of the
human, mouse, and rat genome sequences yielded the
chromosomal localization and the exon/intron structure
of each pART gene family member. The chromosomal
localizations of the pART genes are represented schematically in Figure 2. All human and mouse pART orthologues
lie in regions of conserved synteny. There are three conserved pART gene clusters containing two related paralogues (pARTs 8 and 9; pARTs 12 and 13; pARTs 15 and
17). However, the two most closely related pairs of pARTs
(pARTs 5 and 6; pARTs 16 and 17) each are located on different chromosomes. All other pARTs are distributed as
single copy genes on different autosomes. In the human
genome, the cluster containing pARTs 8 and 9 also contains pART7. Additional file 2 illustrates the local chromosomal environment of this pART gene cluster on human
chromosome 3q and the syntenic region on mouse chromosome 16B3. The local order of genes is similar in the
human and mouse. However, the region corresponding to
pART7 is missing in the mouse. The corresponding region
is also missing in the rat genome (not shown).
The total number of exons in each pART gene is depicted
in Figure 2 and the exon structure of the catalytic domain
is illustrated schematically for the human pARTs in Figure
3. All intron positions within the coding region are fully
conserved in human and mouse orthologues. With the
sole exception of pART4 (VPARP), the catalytic domain is
encoded by the 3' terminal exons. Remarkably, in all
pART genes, with the exception of pART4 (VPARP) and
pART14 (TiPARP), the exons encoding the catalytic
domain are separated from the rest of the respective
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gene
symbol
aliases
1
PARP1
PARP
2
PARP2
3
PARP3
4
5
pART
Hs protein
accession #
chrom. localization
# of exons
amino acids
MGC accession #
Hs
Mm
Hs
Mm
Hs
Mm
Hs
Mm
P09874
1q41-q42
1 H5
23
23
1014
1014
BC037545
BC012041
PARP-2
CAB41505
14q11.2-q12
14 C1
16
16
583
559
na
BC062150
PARP-3
AAM95460
3p21.1-22.2
9 F1
11
11
540
528
(BC014260)
BC014870
PARP4
vaultPARP
AAD47250
13q11
14 C1
34
> 28
TNKS
Tankyrase
AAC79841
8p23.1
8 A4
27
Tankyrase2
1724 > 1446
na
na
27
1327
1320
na
BC057370
na
6
TNKS2
NP_079511
10q23.3
19 C2
27
28
1166
1337
na
7
PARP15
NP_689828
3q21.1
---
8
---
444
---
na
---
8
PARP14
AAN08627
3q21.1
16 B3
12
12
1518
1535
na
(BC021340)
BC003281
9
PARP9
BAL
NP_113646
3q13.3-q21
16 B3
11
11
854
830
(BC039580)
10
PARP10
PARP-10
BAB55067
8q24.3
15 D3
11
11
1025
960
na
na
11
PARP11
AAF91391
12p13.3
6 F3
8
9
331
331
BC017569
BC040269
12
ZC3HDC1
NP_073587
7q34
6 B1
12
12
701
711
BC081541
na
13
ZC3HAV1
ZAP
NP_064504
7q34
6 B1
13
> 11
902
996
(BC025308)
(BC029090)
TiPARP
14
TIPARP
NP_056323
3q25.31
3 E1
6
6
657
657
BC050350
BC068173
15
PARP16
AAH31074
15q22.2
9C
6
7
322
322
BC006389
BC055447
16
PARP8
NP_078891
5q11.2
13 D2.3
26
26
854
852
(BC075801)
(BC021315)
17
PARP6
CAB59261
15q22.23
9C
22
22
630
630
(BC026955)
BC062096
Hs
3
9
8
1
1
2
4
5
16
7
14
3
11
6
13
12
4
5
6
7
8
16
17
18
19
20
10
9
10
11
12
21
22
X
Y
2
15
17
13
14
15
Mm
13
12
14
5
17
15
11
1
1
2
3
4
5
2
4
3
6
7
8
13
16
14
17
18
19
10
X
Y
11
8
9
10
12
9
15
6
16
Figure 2
Chromosomal
localizations and exon compositions of the human and mouse pART family members
Chromosomal localizations and exon compositions of the human and mouse pART family members. A) pART
family members are sorted by subgroup on the basis of similarities in amino acid sequence, intron positions and associated protein domains. Color-coding of subgroups is as follows: 1 = red, 2 = pink, 3 = orange, 4 = green, 5 = grey. This color-coding is
used in subsequent figures. Official gene designations, common aliases and accession numbers are shown. Exon compositions
and lengths of open reading frames are given for the longest known or predicted gene transcripts. Available full length cDNAs
from the Mammalian Gene Collection (MGC) are indicated with their respective accession numbers. MGC cDNAs which
apparently do not contain the full open reading frame are indicated in parentheses. Hs = Homo sapiens, Mm = Mus musculus. B)
Chromosomal localizations of pART genes were determined by tBLASTn searches of the respective genome sequences using
the amino acid sequences of the catalytic domains of individual pARTs. Members of the five pART family subgroups are colorcoded as in A).
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A
799
931
0
0
99
1
153
18
19
275
0
135
2
155
361
246
0
0
2
208
0
7
99
13
14
0
427
115
174
4
9
0
161
2
10
2
125
178
1
2
160
24
H
0
1931
0
1
6
106
23
2350
2
25
H
27
11
5
6
0
26
1206
927
0
2
76
63
12
2
0
2104
157
13
1
5446
125
14
0
15
132
16
134
8
0
10
0
383
131
10
140
200bp
utr
CDS
catalytic
domain
0
9
290
14
12
279
4
5
intron
4850
134
10
conserved exon/intron-boundaries
9
0
380
15
2
3
11
Y
16
H
Y
2122
1342
1
2
142
2
118
6
5
Y
Y
255 1513
0
I
Y
4
2815
0
1124
882
0
1
1
6951
2
49
113
73
70
74
70
85
103
19
20
21
22
23
24
25
26
H
I
252
752
0
2
I
Y
704
6545
399
171
1
0
1
0
343
2
49
110
73
70
80
64
85
15
16
17
18
19
20
21
H
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
448
172
H
17
B
0
207
1139
347
10
H
V
6
T
Y
1
2
3241
0
11
76
175
13
Y
H
138
10
Q
260
12
L
Y
1
1
131
981
0 161 2
1462
175
2
L
4166
9
I
5597
11
7398
1
H
591
12
Y
Y
321
11
E
Y
91
Y
175
326
11
8
7423
1
152
10
H
13
290
7
H
235
I
1311
27
616
6
8
Y
E
0 175 1
7
11
184
131
9
1109
170
317
7
H
781
157
Y
2800
1
156
1597
E
187
24
7684
73 0 131 2 152 1
87
26
Y
E
12
81
6387
157
25
1736
75
16
10
96
2
187
415
1419 6376
11
23
3680
1
H
E
1396
138
5
441
106
117
15
9
4362
0
75
23
H Y
1592
901
82
22
Y
8
2
E
100
226
87
1
21
Y
12
150
62
20
H
3
2
H
128
11
782 1015
128
Y
103
22
I
E
26
53
52
17
86
34
34
34
34
34
34
64
64
57
169
47
37
36
phase 0-Intron
phase 1-Intron
phase 2-Intron
Figure
Schematic
3 diagram of the exon/intron structures of the regions encoding the catalytic domain of pART family members
Schematic diagram of the exon/intron structures of the regions encoding the catalytic domain of pART family
members. A) Exon/intron structures were determined by BLASTn searches of the human genome sequence with individual
pART cDNA sequences. Only the exons corresponding to the catalytic domain of PARP-1 are shown. The coding region is
marked in red, the 3' untranslated region (utr) is marked in white, and a blue bar marks the region corresponding to the catalytic domain. Exons are represented as boxes with the width of each box reflecting the size of the respective exon (the 3' utr
is not drawn to scale). Exon numbers are given with exon 1 corresponding to the exon encoding the presumptive initiation
methionine. In all cases except pART4 (VPARP) the catalytic domain is encoded by the 3' terminal exons. Exon sizes (or size of
coding region in case of the 3' exons) in basepairs are indicated on top of the boxes. Introns are depicted as triangles and are
not drawn to scale. Intron sizes in base pairs are indicated on top of the triangles. The position of each intron with respect to
the reading frame is indicated in the triangles (0 = between codons, +1 = between codon positions 1 and 2, +2 = between
codon positions 2 and 3). Conserved exon boundaries are marked by colored arrows. Codons corresponding to the H-Y-E
motif in the NAD binding crevice of DT and PARP-1 (see Fig. 1) are marked by yellow circles. B) The catalytic domain as delineated in this paper is indicated by the dashed rectangle. For each pART the cDNA coding region within the catalytic domain is
marked by a straight line, regions extending beyond this domain in the 5' direction (and in the 3' driection in case of pART4)
are marked by dashed lines. The positions of the codons corresponding to the H, Y, E residues in the NAD-binding crevice are
indicated by vertical lines. Intron phases are indicated by circles (phase 0), boxes (phase 1), and triangles (phase 2). Numbers
indicate the distance in codons between the conserved histidine in β 1 and the next upstream phase 0 intron. Color-coding of
conserved introns corresponds to that shown in A). Nonconserved introns are indicated in blue (filled) icons.
Page 6 of 23
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BMC Genomics 2005, 6:139
coding exons by a phase 0 intron shortly upstream of the
codon for the first residue of the conserved H-Y-E catalytic
site motif, the conserved histidine in β 1 (Fig. 3). For most
pARTs, the amino acid sequences encoded by exons
upstream of this phase 0 intron do not show any detectable similarities, except for members of a particular subgroup. We used the position of this phase 0 intron in
pART1 to delineate the N-terminal border of the catalytic
domain (e.g., see the green labeled end of the PARP-1model in Figure 1 and the dashed rectangle in Figure 3B).
The exon/intron structures of the pART catalytic domains
reveal a number of intriguing features (Fig. 3). The region
encoding the catalytic domain is disrupted by a remarkable variety of introns with the number of introns varying
from one in subgroup 3 and in pART14 to six in pARTs 16
and 17. The catalytic domain of pART1 (PARP-1) and
those of its closest relatives in subgroup 1 are disrupted by
three (pARTs 3 and 4) or four (pARTs 1 and 2) introns.
Strikingly, not one of these 14 intron positions is conserved. The catalytic domains of the two closely related
tankyrases in subgroup 2 each are interrupted by three
conserved introns. In subgroup 3, the catalytic domains of
pARTs 7–10 each contain a single conserved intron. The
pARTs of subgroup 4 (pARTs 11–14) share a single conserved intron in their catalytic domains, pARTs 11–13
share a second conserved intron in the catalytic domain,
which is missing in pART14. The pARTs of subgroup 5
(pARTs 15–17) share two conserved introns in their catalytic domains, pARTs 16 and 17 share four additional conserved introns in the catalytic domain, which are missing
in pART15.
Conserved structural features revealed by multiple amino
acid sequence alignments and secondary structure
predictions
PSI-BLAST is a powerful, position sensitive iterative program designed to detect distantly related proteins in the
protein database [35]. Initial matches in the first iteration
correspond to those detected by classic BLASTp searches
and typically reveal proteins with an amino acid sequence
identity to the query sequence of > 30%. PSI-BLAST then
derives a position specific scoring matrix from the aligned
protein sequences obtained in the first iteration, which is
then used for the subsequent search of the protein database. This process is repeated in an iterative fashion until
no further matches are detected and the search 'converges'. We performed PSI-BLAST searches of the protein
database using as query the amino acid sequences of the
catalytic domain of each member of the pART gene family. Figure 4 schematically illustrates the tiling paths of
PSI-BLAST searches obtained with the stringent default
threshold setting (0.005 for the expect value) for a representative member of pART family subgroups 1, 3, 4 and 5.
Typically, the other members of the same subgroup were
http://www.biomedcentral.com/1471-2164/6/139
detected in the first iteration and obtained the highest
scores. The pARTs of other subgroups were usually
detected within two additional iterations, except in case of
pART15. Here, five iterations were required to detect all
pART family members.
The amino acid sequence alignments generated by PSIBLAST typically contained the highest degree of sequence
similarity in secondary structure motifs corresponding to
the NAD-binding cores in the known 3D structures of
chicken PARP-1 (1a26) and mouse PARP-2 (1gs0).
Separate multiple amino acid sequence alignments were
generated with T-Coffee for each of the pART subgroups
using the orthologous sequences from human and mouse
[36]. PSIPRED was used to predict secondary structure
units and GenTHREADER was used to predict the optimal
alignment of pART amino acid sequences with the 3D
structures of chicken PARP-1 and mouse PARP-2 [37]. In
all cases, predictions and alignments yielded consistent
results with respect to the sole alpha helix and five of the
six β-strands of the PARP-1 catalytic domain (see additional files 3, 4, 5, 6, 7: "Multiple amino acid sequence
alignments, secondary structure predictions and threading results for pART subgroups 1–5"). The small β strand
(β 4) at the upper edge of the active site crevice was
aligned and predicted congruently only for subgroups 1–
4, and could not be predicted with confidence for the
most distant relatives of PARP-1 (pARTs 15–17). Regions
corresponding to connecting loops showed significant
sequence identities only for members of a particular pART
subgroup. Most likely, these regions fold similarly only in
closely related pART family members.
A striking result of the alignment analyses is that the H-YE catalytic site motif is fully conserved only in subgroups
1 and 2 (pARTs 1–6). All other pARTs show deviations
from this motif. The histidine in β 1 is conserved in 9 of
the 11 members of subgroup 3–5, the tyrosine in β 3 is
conserved in all family members, yet the presumptive catalytic glutamic acid at the N-terminal end of β 6 is
exchanged in each of the pARTs 7–17.
Moreover, the amino acid sequence of the loop immediately upstream of β 5 and the active site glutamic acid residue deviates markedly from those of PARP-1 and PARP-2
in most other family members except for the tankyrases
(pARTs 5 and 6). A growing body of evidence indicates
that this region influences the target specificity of pARTs
and mARTs [38-40]. In the 3D structure of PARP-1 with
carba-NAD (3pax), the ligand was found to interact with
this loop outside of the active site crevice, and it was proposed that this may reflect the binding of the ADP-ribose
polymer in the target protein [14].
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input
Hs.pART1
Hs.pART9
Hs.pART12
Hs.pART15
Ci.pART
iteration 1
=
traditional
Blastp
searches
Ag.pARTa
Dm.pARTa
Hs.pART2
Ce.pARTa
Hs.pART3
Ce.pARTb
Hs.pART4
Ce.pARTc
Hs.pART5
Ci.pART
Ag.pARTb
Dm.pARTb
Hs.pART8
Hs.pART10
Hs.pART7
Hs.pART13
Hs.pART14
Hs.pART12
Hs.pART11
Hs.pART6
Hs.pART5
Hs.pART11
Hs.pART14
Hs.pART13
Hs.pART7
Hs.pART8
Hs.pART10
Ag.pARTb
Hs.pART16
Hs.pART6
Dm.pARTb
Hs.pART9
Hs.pART5
Ag.pARTc
Hs.pART17
Ag.pARTa
Hs.pART1
Hs.pART2
Dm.pARTa
iteration 2
Hs.pART6
Hs.pART14
Hs.pART13
Ba.pART
Hs.pART16
Hs.pART7
Hs.pART11
Ag.pARTc
Hs.pART17
Hs.pART12
Ag.pARTb
Dm.pARTb
Hs.pART3
Hs.pART1
Dm.pARTa
Ce.pARTb
Hs.pART4
Ce.pARTc
Ag.pARTa
Hs.pART3
Dm.pARTa
Ce.pARTc
Hs.pART1
Hs.pART4
Ce.pARTb
Ag.pARTa
Hs.pART2
Ce.pARTa
Ci.pART
Hs.pART16
Hs.pART2
Ag.pARTa
Ce.pARTa
Hs.pART3
Ce.pARTa
Hs.pART4
Ce.pARTb
Ce.pARTc
Dm.pARTb
Hs.pART5
Ag.pARTb
Hs.pART6
Hs.pART14
Hs.pART16
Hs.pART7
Ba.pART
iteration 3
Hs.pART8
Hs.pART10
Hs.pART15
Hs.pART9
Hs.pART2
Ce.pARTa
Hs.pART15
Ci.pART
Ag.pARTc
Hs.pART16
Hs.pART17
Ba.pART
Hs.pART17
Hs.pART15
Ag.pARTc
Dm.pARTa
Hs.pART1
Hs.pART3
Ce.pARTc
Ce.pARTb
Hs.pART4
Hs.pART5
Ag.pARTb
Dm.pARTb
Hs.pART8
Hs.pART10
Hs.pART12
Hs.pART11
Hs.pART13
Ag.pARTc
Hs.pART17
Hs.pART15
iteration 4
Hs.pART6
Hs.pART14
Hs.pART7
Hs.pART13
Hs.pART11
Ci.pART
Hs.pART12
Ba.pART
Hs.pART9
iteration 5
Hs.pART8
Hs.pART10
Hs.pART9
iteration 6
iteration 7
iteration 8
converged
Ba.pART
converged
converged
converged
converged
Figuremembers
Representative
family
4
tiling paths of PSI-BLAST searches initiated with the catalytic domain amino acid sequences of selected pART
Representative tiling paths of PSI-BLAST searches initiated with the catalytic domain amino acid sequences of
selected pART family members. PSI-BLAST searches were initiated with the catalytic domain amino acid sequences of the
pARTs indicated on top as query sequences with the default threshold setting for the expect value of 0.005. Matching
sequences from selected model organisms are indicated at the iteration in which they first appeared above threshold. pART
subgroups are color coded as in Figure 2. Accession numbers of the indicated pARTs are listed in Figures 2 and 9. Species of
origin is color-coded in the two letter abbreviation of the organism as follows: Homo sapiens (Hs) red, Drosophila melanogaster
(Dm) and Anopheles gambiae (Ag) purple, Caenorrhabditis elegans (Ce) blue, Chilo iridescent virus (Ci) and Bacteriophage Aeh
(Ba) brown.
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BMC Genomics 2005, 6:139
The results of the secondary structure prediction and
threading analyses were used to refine a multiple amino
acid sequence alignment of the catalytic domains of all
human pART family members. The resulting alignment is
shown in Figure 5. The conserved secondary structure
units corresponding to the catalytic NAD binding core
(the six beta strands and one alpha helix marked in Figure
1) are indicated schematically below the alignment. The
corresponding amino acid residues are highlighted in the
alignment. Intron positions are projected onto the amino
acid sequence in Figure 5. The positions of conserved
introns are marked by colored arrows below the alignment. Note that the alignment diverges most strongly
both in length and in sequence in the loops immediately
downstream and upstream of β 3.
Figure 6A shows a condensed version of the alignment in
which the diverging intervening loops are indicated only
by the number of amino acid residues. These 66 amino
acid residues can be superimposed well in the 3D structures of PARP-1, PARP-2, DT, and ETA. The respective
amino acid sequences of DT, ETA and the putative Chilo
iridescent virus pART are also shown for these regions. Figure 6B shows the calculated amino acid sequence identities of the pART family members in this region. The
percentage amino acid sequence identity in the aligned
core region is higher among members of a particular subgroup than between members of different subgroups,
lending support to the subgroup assignments. For each
pART, the next most closely related paralogue is a member
of the same subgroup. Note that two pairs of pART paralogues show very close sequence similarity: pARTs 5 and 6
(94% identity in the aligned core region) and pARTs 16
and 17 (86% identity). This close similarity is reflected
also in the conserved exon intron structures of the respective pART pairs (see Fig. 3).
Comparison of mouse and human pART orthologues
shows that seven of such pairs exhibit 100% sequence
identity in the aligned core region (pARTs 1, 5, 6, 11, 14,
16, and 17) and six show > 90% identity (pARTs 2, 3, 4,
10, 12, and 15). The mouse and human orthologues of
pARTs 8, 9, 13 show the least degrees of sequence identity
in this region (82%, 82%, and 70%, respectively) (Fig.
6B).
Phylogenetic analysis of the amino acid sequences of the
catalytic cores of pARTs resulted in three very similar trees
when using Maximum Parsimony (PAUP), Maximum
Likelihood (PhyML), and Bayesian Markov Chain Monte
Carlo (MrBayes) optimization criteria (Figure 7). All
topologies showed moderate to high support values for
the recovered relationships. All trees recovered five basic
clades corresponding to the subgroups 1–5. The results
indicate that pARTs of subgroups 1 and 2 are more closely
http://www.biomedcentral.com/1471-2164/6/139
related (sistergroups) to one another than to members of
the other subgroups. A similar relationship is seen for
pARTs of subgroups 3 and 4. Note that the putative Chilo
iridescent virus pART clusters with the mammalian pARTs
of subgroup 1, suggesting that this large double stranded
DNA virus may have acquired its pART by horizontal gene
transfer.
The pART catalytic domain has become genetically fused
to a wide spectrum of protein domains
With the exception of closely related members within a
subgroup, the amino acid sequence similarity between
pART family members breaks off upstream of β 1. Interestingly, loss of sequence similarity correlates well with the
presence of a phase 0 intron upstream of β 1. All pART
family members except pART4 and pART14 contain such
a phase 0 intron 26–64 codons upstream of the conserved
histidine in β 1 (Fig. 3B).
Using the sequences flanking the catalytic domain of each
pART family member as queries, we performed further
PSI-BLAST analyses and searches of the Conserved
Domain Database [41]. The results, summarized in Figure
8, reveal that each of the 17 human pARTs with the possible exception of pART15 is a multi-domain protein. Strikingly, the pART catalytic domain is associated – in a Lego
like fashion – with a broad spectrum of known protein
domains. In all family members except pART4 the catalytic domain represents the C-terminal domain.
A number of associated domains occur in two or more
human pART family members. Note that domain sharing
generally is restricted to members of a particular pART
subgroup. For example, all members of subgroup 1 contain a helical domain preceding the catalytic domain,
whereas this domain is missing in members of other pART
subgroups. The two members of subgroup 2 share SAM
and ankyrin-repeat domains. Three of four pARTs in subgroup 3 share A1pp domains [42], all members of subgroup 4 share WWE domains, and two members of
subgroup 5 contain a second, truncated pART domain,
reminiscent of the duplicated inactive ART domain found
in the VIP2 and iota mART toxins [16,23].
Several pARTs carry recognizable zinc-fingers containing
putative RNA-, DNA-, or ubiquitin-binding domains
(pART1, pART2, pART10, pART12, pART13). This indicates that the genetic fusion of a pART catalytic domain
with zinc-fingers has occurred repeatedly in evolution.
Representation of pARTs in other model organisms
We also used PSI-BLAST to screen the protein database for
recognizable pART family members in other organisms
using as queries the amino acid sequences of catalytic
domains of each of the 17 human pARTs (Figure 9). The
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798 T D I K V V D R D S E E
pART1
366 C A L R P L D H E S Y E
pART2
330 C Q L Q L L D S G A P E
pART3
379 C K I E H V E Q N T E E
pART4
pART5 1112 P E D K E Y Q S V E E E
959 P D D K E F Q S V E E E
pART6
253 D M N H Q L F C M V Q L
pART7
pART8 1327 D M K Q Q N F C V V E L
633 Q D E M K E N I I F L K
pART9
pART10 815 P W N N L E R L A E N T
pART11 126 T Q V P Y Q L I P L H N
pART12 493 P D P G F Q K I T L S S
pART13 723 S S K K Y K L S E I H H
pART14 457 P S Q D F I Q V P V S A
89 L S S K V L T I H S A G
pART15
pART16 644 I S S N R S H I V K L P
pART17 421 I S S N R S H I V K L P
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5
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6
Figure 5amino acid sequence alignment of the catalytic cores of the human pART family
Multiple
Multiple amino acid sequence alignment of the catalytic cores of the human pART family. The multiple sequence
alignment was generated with T-Coffee and manually adjusted using the results of the PSI-BLAST, PSIPRED, and GenTHREADER analyses. Numbers at the sequence ends indicate the number of additional residues upstream and downstream of
the alignment shown. Residues corresponding to the H Y E motif in the NAD binding crevice of diphtheria toxin are in red and
marked by asterisks. The conserved β sheets and alpha helix are shaded in green and yellow. Conserved intron positions are
marked in the multiple alignment using the same color-coding as in Figure 3. Conserved intron positions are indicated also
above the alignment with arrows. Non-conserved intron positions are marked in blue in the alignment.
Page 10 of 23
(page number not for citation purposes)
BMC Genomics 2005, 6:139
A
13
1tox
1aer 457
3pax 851
49
ci
h 1 854
m 1 854
h 2 420
1gs0 396
h 3 383
m 3 371
h 4 430
m 4 542
h 5 1176
m 5 1169
h 6 1023
m 6 1194
h 7 317
m7
h 8
m8
h 9
m9
h 10
m 10
h 11
m 11
h 12
m 12
h 13
m 13
h 14
m 14
h 15
m 15
h 16
m 16
h 17
m 17
1391
1408
697
668
879
828
189
189
556
566
779
870
524
524
144
144
689
687
465
465
http://www.biomedcentral.com/1471-2164/6/139
1
*
MENFSSYHGTKP
GYVFVGYHGTFL
HNRQLLWHGSRT
KKTRLLIHGTRC
HNRRLLWHGSRT
HNRRLLWHGSRT
HNRMLLWHGSRM
PNRMLLWHGSRL
GNRKLLWHGTNM
GNRRLLWHGTNV
GNVRPLLHGSPV
GNVRLLFHGSPV
HNERMLFHGSPF
HNERMLFHGSPF
ANERMLFHGSPF
ANERMLFHGSPF
NNERLLFHGTDA
24
23
25
26
25
25
25
25
21
21
30
30
20
20
20
20
23
MNEKQLFHGTDA
RNEKHLFHGTEA
PVSHRLFQQVPY
SGSQRLFQQVPH
PVEQVLYHGTTA
PVEQVLYHGTSE
INEQMLFHGTSS
INEQMLFHGTSS
VDERQLFHGTSA
VDERQLFHGTSA
EEGKLLFYATSR
KTEMFLFHAVGR
INERHLFHGTSQ
INERHLFHGTSQ
RDLIYAFHGSRL
RDLIYAFHGSRL
FGSTFAFHGSHI
FGSTFAFHGSHI
YGSTFAFHGSHI
YGSTFAFHGSHI
23
23
23
23
23
23
23
23
23
23
23
23
23
23
21
21
26
26
26
26
1
B
tox
aer
ci
g01
h01
m01
h02
m02
h03
m03
h04
m04
h05
m05
h06
m06
h07
h08
m08
h09
m09
h10
m10
h11
m11
h12
m12
h13
m13
h14
m14
h15
m15
h16
m16
h17
m17
tox
***
30
15
18
18
18
18
17
20
20
15
14
15
15
15
15
17
21
17
09
07
14
12
14
14
15
15
9
11
11
11
17
17
11
11
12
12
ddt
aer
30
***
18
17
17
17
20
21
20
20
18
17
17
17
17
17
15
14
15
06
05
20
20
15
15
17
17
12
7
12
12
15
15
9
9
9
9
aer
2
2
*
WKGFYSTDNKYDAAGYS
WRGFYIAGDPALAYGYA
GKGIYFADMVSKSANYC
GEGNYFSEHVQKSLNYT
GKGIYFADMVSKSANYC
GKGIYFADMVSKSANYC
GKGIYFADMSSKSANYC
GKGIYFADMSSKSANYC
GKGIYFASENSKSAGYV
GKGIYFASENSKSAGYV
GSGIYFSDSLSTSIKYS
GSGIYFSDSLSTSIKYA
GAGIYFAENSSKSNQYV
GAGIYFAENSSKSNQYV
GAGIYFAENSSKSNQYV
GAGIYFAENSSKSNQYV
GKGTYFAVDASYSAKDT
GKGTYFAVNANYSANDT
GKGTYFAVKASYSACDT
GAGIYFTKNLKNLAEKA
GAGIYFTKSLKNLADKV
GKGVYFAKRASLSVQDR
GQGVYFAKRASLSVLDR
GKGTYFARDAAYSSRFC
GKGTYFARDAAYSSRFC
GKGSYFARDAAYSHHYS
GKGSYFARDAAYSHHYS
GKGIYFAKDAIYSHKNC
GKGNYFTKEAMYSHKSC
GQGSYFAKKASYSHNFS
GQGSYFAKKASYSHNFS
GEGTYLTSDLSLALIYS
GEGTYLTSDLSLALIYS
GSGIYLSPMSSISFGYS
GSGIYLSPMSSISFGYS
GKGIYLSPISSISFGYS
GKGIYLSPISSISFGYS
2
2
10
12
7
4
7
7
7
7
9
9
7
7
20
20
20
20
8
AGGVVKVTYPGL
NGALLRVYVPRS
IGLILLGEVALG
DQILLIYEVHVG
IGLILLGEVALG
IGLILLGEVALG
TGLLLLSEVALG
TGLLLLSEVALG
VGYMFLGEVALG
VGYMFLGEVALG
TRLLLICDVALG
SRLLVVCDVALG
HRQMLFCRVTLG
HRQMLFCRVTLG
HRQLLFCRVTLG
HRQLLFCRVTLG
RKHMYVVRVLTG
3
45
32
18
8
18
18
19
19
19
19
19
19
18
18
18
18
24
VVLSL
LDAIT
HSVKG
YNGDR
HSVKG
HSVKG
HSTKG
HSTKG
DSVIA
DSVIA
DSVHG
DSVHG
HSVIG
HSVIG
HSVTG
HSVTG
DSVTN
7
7
35
26
35
35
38
38
40
40
12
12
8
8
8
8
4
5
6
*
SVEYINNWEQKAALSVELEINF
RLETILGWPLAERTVVIPSIPT
YNEYIVYDV--AQVNLKYLLKL
NSEIISYNE--DQSKIKYIIHI
YNEYIVYDI--AQVNLKYLLKL
YNEYIVYDI--AQVNLKYLLKL
YNEYIVYNP--NQVRMRYLLKV
YNEFIVYSP--NQVRMRYLLKI
QSEYLIYQE--SQCRLRYLLEV
QSEYLIYKE--SQCRLRYLLEI
DDEFVVYKT--NQVKMKYIIKF
DDEFVVYKT--NQVKMKYIVKF
YAEYVIYRG--EQAYPEYLITY
YAEYVIYRG--EQAYPEYLITY
LAEYVIYRG--EQAYPEYLITY
LAEYVIYRG--EQAYPEYLITY
PKLFVVFFD--NQAYPEYLITF
368
40
9
2
9
9
8
8
2
2
1160
>672
19
19
11
11
2
8
8
8
8
8
8
25
25
5
5
5
5
6
6
23
23
35
35
35
35
RKHVYYVRVLTG
RKYMYYVRVLTG
LIYVFEAEVLTG
LIYVFEAEVLTG
HKAVFVARVLTG
YKAVFVAQVLTG
YKSMFLARVLIG
YKSMFLARVLIG
THTMFLARVLVG
SHMMFLARVLVG
NVVMFVAQVLVG
GTVMFVARVLVG
VHFMFLAKVLTG
VHFMFLAKVLTG
IDHPDVKCQTKK
IDHPDVKCQIKK
LQSRNLKCIALC
LQSRNLKCIALC
LQSRNLNCIALC
LQSRNLNCIALC
24
24
23
23
24
23
23
23
23
23
16
16
25
25
6
6
6
6
6
6
DTVTD
DTVTD
DSVVD
DSVVD
DSAVD
DSAVD
DSCVD
DSCVD
DSCVN
DSCVN
DSCVD
DSCVD
DSCVD
DSCVD
DRRRA
DRSRA
DLHKH
DLHKH
DLQKH
DLQKH
4
4
4
4
4
4
4
4
4
4
4
4
4
4
11
11
0
0
0
0
PSLFVAFYD--YQAYPEYLITF
PSIFVVFYD--NQTYPEYLITF
PETFVIFSG--MQAIPQYLWTC
PETIVVFNG--MQAMPLYLWTC
PSIFVIFHD--TQALPTHLITC
PRIFVIFHD--TQALPTHLITC
PKIFVVFDA--NQIYPEYLIDF
PKIFVVFDA--NQIYPEYLIDF
PSIFVIFEK--HQVYPEYVIQY
PTIFVVFEK--HQVYPEYLIQY
PSVFVIFQK--DQVYPQYVIEY
PSVFVIFRK--EQIYPEYVIEY
PQIFVIFND--DQSYPYFVIQY
PQIFVIFND--DQSYPYFVIQY
PKYFVVTNN--QLLRVKYLLVY
PKYFVVTNN--QLLRVKYLLVY
GEIWVVPNT--DHVCTRFFFVY
GEIWVVPNT--DHVCTRFFFVY
GNIWVCPVS--DHVCTRFFFVY
GNIWVCPVS--DHVCTRFFFVY
2
2
33
38
21
8
1
1
24
24
9
12
9
9
51
51
32
32
32
32
3
4
4
5
6
ci g01 h01 h02 h03 h04 h05 h06 h07 h08 h09 h10 h11 h12 h13 h14 h15 h16 h17
15 18 18 18 20 15 15 15 17 21
9
14 14 15
9
11 17 11 12
18 17 17 20 20 18 17 17 15 14
6
20 15 17 12 12 15
9
9
*** 36 38 36 32 36 30 32 26 26 15 21 21 27 24 27 18 17 15
36 *** 97 79 56 47 47 44 33 29 23 26 33 27 26 26 23 26 26
38 97 *** 79 56 49 49 46 35 29 23 24 32 29 26 27 23 26 26
38 97 100 79 56 49 49 46 35 29 23 24 32 29 26 27 23 26 26
36 79 79 *** 58 50 47 46 33 27 21 24 32 29 26 27 23 30 29
36 76 76 92 53 52 44 44 35 29 26 27 33 30 27 27 24 29 29
32 56 56 58 *** 36 44 41 36 33 29 32 33 35 36 33 21 23 24
35 56 58 55 95 41 46 42 38 32 29 32 33 36 33 35 21 23 24
36 47 49 50 36 *** 46 47 38 29 26 26 32 33 27 30 24 30 26
32 46 47 47 36 91 44 46 39 29 29 26 32 30 29 29 26 30 26
30 47 49 47 44 46 *** 94 46 41 35 38 39 41 32 38 21 24 23
30 47 49 48 44 46 100 94 46 41 35 38 39 41 32 38 21 24 23
32 44 46 46 41 47 94 *** 46 42 35 38 38 39 30 36 21 24 23
32 44 46 46 41 47 94 100 46 42 35 38 38 39 30 36 21 23 23
26 33 35 33 36 38 46 46 *** 79 36 55 62 55 47 50 29 17 17
26 29 29 27 33 29 41 42 79 *** 39 55 55 50 42 47 21 14 15
24 30 30 30 38 33 41 41 79 82 36 55 61 52 44 53 24 18 18
15 23 23 21 29 26 35 35 36 39 *** 46 35 33 39 36 18 15 14
18 26 24 24 29 27 33 33 36 36 82 41 36 30 35 35 20 20 15
21 26 24 24 32 26 38 38 55 55 46 *** 52 50 46 52 20 15 17
21 24 23 23 30 26 33 33 50 50 46 91 52 47 47 55 20 15 15
21 33 32 32 33 32 39 38 62 55 35 52 *** 64 53 59 24 20 20
21 33 32 32 33 32 39 38 62 55 35 52 100 64 53 59 24 20 20
27 27 29 29 35 33 41 39 55 50 33 50 64 *** 62 67 24 23 24
26 29 30 29 32 33 39 38 56 49 32 47 65 94 59 65 26 24 24
24 26 26 26 36 27 32 30 47 42 39 46 53 62 *** 56 18 17 18
24 21 21 23 30 26 38 36 47 44 39 47 55 61 70 53 20 15 17
27 26 27 27 33 30 38 36 50 47 36 52 59 67 56 *** 21 26 24
27 26 27 27 33 30 38 36 50 47 36 52 59 67 56 100 21 26 24
18 23 23 23 21 24 21 21 29 21 18 20 24 24 18 21 *** 30 26
18 23 23 23 21 24 20 20 29 21 18 20 24 24 18 21 97 30 26
17 26 26 30 23 30 24 24 17 14 15 15 20 23 17 26 30 *** 86
17 26 26 30 23 30 23 23 17 14 15 15 20 23 17 26 30 100 86
15 26 26 29 24 26 23 23 17 15 14 17 20 24 18 24 26 86 ***
15 26 26 29 24 26 23 23 17 15 14 17 20 24 18 24 26 86 100
ci g01 h01 h02 h03 h04 h05 h06 h07 h08 h09 h10 h11 h12 h13 h14 h15 h16 h17
ddt
aer
ci
g01
h01
m01
h02
m02
h03
m03
h04
m04
h05
m05
h06
m06
h07
h08
m08
h09
m09
h10
m10
h11
m11
h12
m12
h13
m13
h14
m14
h15
m15
h16
m16
h17
m17
Figure 6 based amino acid sequence alignment of the catalytic cores of the pART gene family
Structure
Structure based amino acid sequence alignment of the catalytic cores of the pART gene family. A) The alignment
is restricted to those regions corresponding to the conserved secondary structure units of PARP-1 and DT as highlighted in
Figure 1. The H Y E motif is marked by asterisks and is highlighted in red. Black numbers indicate amino acid residues from the
N- and C-terminal ends of the protein and within the loops connecting the structure units shown. For proteins with known 3D
structures the pdb accession number is given and the residues corresponding to respective secondary structure units are
underlined. 1tox = diphtheria toxin; 1aer = pseudomonas exotoxin A, 3pax = chicken PARP-1 (pART1), 1gs0 = mouse PARP-2
(pART2). Human and mouse pARTs are indicated by colored numbers. The sequence of the putative pART from Chilo iridescent virus is also shown for comparison (ci). B) Pairwise percentage sequence identities were calculated for the 66 amino acid
residues shown in A), which correspond to the conserved core secondary structure units in Figure 1.
Page 11 of 23
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BMC Genomics 2005, 6:139
http://www.biomedcentral.com/1471-2164/6/139
1tox
1aer
100
Hs15
Mm15
99
87
Hs16
100
Mm16
100
Hs17
Mm17
100 Hs05
99
Ms05
100
100
Hs06
Mm06
75
Ci
99
70
Hs04
Ms04
95
61
Hs03
Mm03
75
3PAX
100
96
69
Hs01
91
90
Mm01
Hs02
Mm02
Hs09
100
100
Ms09
100
91
Mm10
Hs07
100
Hs08
99
99
Mm08
100
Hs11
Mm11
98
0.05
Hs10
100
Hs13
Mm13
89
97
Hs12
Mm12
83
100
Hs14
Mm14
Figure 7 of the evolutionary relationship of the pART family
Phylogram
Phylogram of the evolutionary relationship of the pART family. Evolutionary relationships of the amino acid
sequences in the catalytic core of the pARTs shown in Figure 6 are illustrated as a maximum a posteriori phylogram (MAP) of
Bayesian Markov Chain Monte Carlo analysis (pP = 0.92). Posterior probabilities were converted into percentages and are
shown above the branches. Members of the five pART family subgroups are color-coded as in Figure 2: subgroup 1 = red, 2 =
pink, 3 = orange, 4 = green, 5 = grey. Hs = Homo sapiens, Mm = Mus musculus.
Page 12 of 23
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BMC Genomics 2005, 6:139
1 DBD
1
BRCT
Dm.pARTa
DBD
BRCT
WGR
http://www.biomedcentral.com/1471-2164/6/139
1
DBD
Ce.pARTa
WGR
WGR
icons
1 DBD
BRCT
WGR
At.pARTa
1
1 DBD
1
WGR
HYE
HYE
1014
994
HYE
WGR
1 Cen
2
WGR
3
1 BRCT
1
WGR
Ce.pARTb
1 BRCT
HYE
637
WGR
Ba.pART
1
Gz.pARTa
HYE
538
4
1
HYE
vWFA
VIT
MVPI
HYE
WGR
Ce.pARTc
11 HYI
HYE
HYE
1
SAM
SAM
1 pRBD
1 WWE
WWE WWE
12
Dm.pARTb
WWE WWE
1 RF
1181
13
1
1 A1pp
1
HYL
1518
1 A1pp A1pp
na
RRM
RNA-recognition
motif
70
pfam00076
85
pfam05406
135
pfam02877
SAM
IBR
YYV
1
Dd.pARTg
A1pp
15
QYT
HYI
HYY
1
Ag.pARTc
16
UI UI
HYI
Gz.pARTc
1
854
10
HYI
1
1025
HYH
17
1077
cd00166
75
pfam00533
Appr-1" processing
135
smart00506
His-Pro-Ser
region
180
na
ankyrin repeats
20 x 30
cd00204
VIT
vault protein
inter alpha trysin
130
smart00609
vWFA
von Willebrand
factor type A
160
cd00198
MVPI
major vault protein
interacting
160
na
WWE
WWE-domain
trp/trp/glu
75
pfam02825
UI
ubiquitin interaction
motif
18
na
RF
C3HC4 type zinc
finger (RING finger)
45
pfam00097
UB
U-box
75
pfam04564
IBR
in between RING
fingers
65
pfam01485
HYI
630
65
HPS
HYY
259
1604
854
1
sterile alpha
motif
breast cancer suppressor
protein C-terminal
A1pp
HYL
HYI
322
9
1 RRM
pfam00642
55
BRCT
657
1 UB
pfam00645
PARP regulatory
358
14
HYL
2 x 90
PARP-type
zinc finger
SAF/Acinus/PIAS
SAP-domain
centriolelocalization
WWE
8
cd00195
Cen
Eh.pARTf
WWE WWE
444
A1pp A1pp A1pp
140
pfam02037
568
902
7
Ubiquitin-conjugating
enzyme catalytic UBCc
4 x 30
VHE
HYI
701
1 pRBD
HYE
HYE
na
2 x 35
2276
1327
1
120
protein-interaction domains
WGR-domain
WGR
tryp/gly/arg
At.pARTe
331
5
truncated
pART catalytic
CCCH-type
zinc finger
1 WWE
SAM
pfam00644
pRBD
DBD
752
1
HPS
180
nucleic acid binding domains
DBD
1211
1724
1
pART catalytic
HYE
HYE
540
accession #
At.pARTb
HYE
583
181
983
1 DBD
size
HYE
HYE
945
designation
catalytic domains
Ci.pART
Figure 8 diagram of the domain structures of human pARTs and pARTs from distantly related organisms
Schematic
Schematic diagram of the domain structures of human pARTs and pARTs from distantly related organisms.
Recognizable protein domains in the pART family are represented by the icons defined on the right. The domain structures of
human pARTs (on the left, numbered Pacman icons) and related pARTs from other species are illustrated schematically. Potential DNA binding domains are boxed in red, potential ubiquitylation motifs are boxed in green. Members of the five pART family subgroups are grouped within colored boxes using the color-coding as in Figure 2: subgroup 1 = red, 2 = pink, 3 = orange,
4 = green, 5 = grey. Amino acids corresponding to the HYE catalytic site motif of DT and PARP-1 are shown in the mouths of
the Pacman icons. Black numbers indicate protein lengths in number of amino acids. Species of origin is color-coded in the two
letter abbreviation of the organisms as in Figures 4 and 9: Drosophila melanogaster (Dm) and Anopheles gambiae (Ag) purple,
Caenorrhabditis elegans (Ce), Dictyostelium discoideum (Dd), Entamaoeba histolytica (Eh), and Gibberella zeae (Gz) blue, Arabidopsis
thaliana (At) green, Chilo iridescent virus (Ci) and Bacteriophage Aeh (Ba) brown. Protein database accession numbers for the
illustrated pARTs are listed in Figures 4 and 9. On the right, the approximate size of each domain is indicated in number of
amino acid residues. The accession numbers of the respective domain families in the pfam, cd, and smart databases are indicated. In case of zinc finger (zf) containing domains, the number of recognizable zinc fingers is indicated by colored bars within
the icon.
order in which PSI-BLAST picked up putative pART
sequences from the database in successive iterations was
similar for different members of a particular pART sub-
group but differed markedly for members of different subgroups (see additional file 8: "Representative tiling paths
of PSI-BLAST searches initiated with the catalytic domain
Page 13 of 23
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BMC Genomics 2005, 6:139
pART
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
protein
PARP1
PARP2
PARP3
PARP4
TNKS
TNKS2
PARP15
PARP14
PARP9
PARP10
PARP11
ZC3HDC1
ZC3HAV1
TIPARP
PARP16
PARP8
PARP6
pART
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
protein
PARP1
PARP2
PARP3
PARP4
TNKS
TNKS2
PARP15
PARP14
PARP9
PARP10
PARP11
ZC3HDC1
ZC3HAV1
TIPARP
PARP16
PARP8
PARP6
aliases
PARP
vaultPARP
Tankyrase
Tankyrase 2
BAL
ZAP
TiPARP
nematode
AAM27195 a
Q09525 b
AAC04454_c
http://www.biomedcentral.com/1471-2164/6/139
human
P09874
Q9UGN5
AAM95460
AAD47250
AAC79841
NP_079511
NP_689828
AAN08627
NP_113646
BAB55067
AAF91391
NP_073587
NP_064504
NP_056323
AAH31074
NP_078891
CAB59261
mouse
NP_031441
NP_033762
NP_663594
XP_283217
AAH57370
XP_129246
--XP_488522
NP_084529
AAH24074
NP_852067
NP_766481
BAB32047
NP_849223
NP_803411
AAH21881
XP_134863
chicken
NP_990594
slime mold
CAD59237_a
CAD58666_c
CAD59238_d
CAD59240_e
CAD59239_b
AA051129 f
fungi
amoeba
weed
viruses
bacteria
EAA75569_a
EAL47198_a
EAL50270_b
NP_850165_a
CAA88288_b
BAB09119_c
AAB94432
AAQ17796
1AERA
760286A
AAW80252
EAL43406_c
EAL50270_d
EAL49071_e
EAL45174 f
NP_849739_d
AAC36170_e
XP_417150
NP_989671
NP_989672
XP_422113
XP_422116
XP_416489
XP_416333
XP_423977
XP_422828
XP_413903
XP_424786
fish
CAG09179
CAF92030
CAG06805
CAG08214
CAG12585
CAG04910
fly
P35875 a
mosquito
XP 312938 a
AAF56487_b
XP_321116_b
CAF98988
CAG12587
CAG05989
CAG01913
CAF98285
CAF96305
CAF96664
CAG05566
CAG05573
CAF95416
XP 308419 c
AAS38928_g
EAA73885_c
Figure in9 distantly related species
pARTs
pARTs in distantly related species. pART relatives were identified by PSI-BLAST searches as in Figure 4. Matching
sequences from other organisms were sorted by group on the basis of sequence similarity and associated domains. Accession
numbers are given for pARTs from Homo sapiens (human), Mus musculus (mouse), Gallus gallus (chicken), Tetraodon nigroviridis
(puffer fish), Drosophila melanogaster (fruit fly), Anopheles gambiae (malaria mosquito), Caenorhabditis elegans (nematode), Dictyostelium discoideum (slime mold), Gibberella zeae (ear root microfungus), Entamaoeba histolytica (amoeba), Arabidopsis thaliana
(cress plant), Chilo iridescent virus and Bacteriophage Aeh1 (viruses), Pseudomonas aeruginosa, Corynebacterium diphtheriae and
Vibrio cholerae (bacteria). Lower case letters in black indicate the pART designations used in Figure 8.
amino acid sequences of selected pART family members"). In many instances, PSI-BLAST detected pART
sequences from distantly related organisms in earlier iterations than the human pART paralogues from other
subgroups.
ated domains, pARTs from other vertebrates including
fish and chicken, generally can be assigned to a particular
human pART orthologue. In contrast, pARTs of lower
eucaryotes can be assigned to a subgroup but not to a particular vertebrate pART.
Figure 9 summarizes the matches of pART-related proteins
found in model organisms with completed genome
sequences. On the basis of amino acid sequence similarity, conserved intron positions and/or conserved associ-
pART homologues were found in many model organisms
from the animal, plant, fungi, and protist kingdoms. The
recently completed genome of the pufferfish T. nigroviridis
contains recognizable orthologues for all pARTs except for
Page 14 of 23
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BMC Genomics 2005, 6:139
pART7 [43]. The nearly completed albeit still fragmentary
chicken genome contains recognizable orthologues for all
pARTs except for pARTs 2, 3, 7, 10, and 17 [44]. Simpler
eucaryotes generally contain fewer pARTs (two in the fruit
fly D. melanogaster, three each in the malaria mosquito A.
gambiae, the nematode C. elegans, and the ascomycete G.
zeae; six in the amoeba E. histolytica, nine in the slime
mold D. discoideum, and ten in the cress plant A. thaliana).
Remarkably, the yeast S. cerevisae and the archaea lack
detectable pARTs. Only two matches were found in the
viral proteome: these derive from two double stranded
DNA viruses: the insect virus Chilo iridescent virus and
the bacteriophage Aeh1. Although PSI-BLAST initially
failed to connect the pART family with Diphtheria toxin
and Pseudomonoas exotoxin A, these toxins were readily
connected with the eucaryotic pARTs when using as query
a chimera, e.g. of Diphtheria toxin and Chilo iridescent
virus pART in which the sequences of three of the conserved structure units highlighted in Figures 1 and 6A were
interchanged. These searches uncovered a DT/ETA-like
putative ADP-ribosyltransferase in V. cholerae, but no
other proteins in the microbial proteome in GenBank.
Of note, none of the known R-S-E motif bacterial or vertebrate mARTs were ever connected by PSI-BLAST with the
DT/ETA/pART group. In several cases, however, we
observed intriguing matches just slightly below threshold
(in the region surrounding the conserved H in β 1) to
members of the family of RNA:NAD 2' phosphotransferases. These enzymes catalyze a reaction during tRNA
splicing that is similar to the reaction catalyzed by ARTs,
but in which ADP-ribose is transferred to the 2'-phosphate
in immature tRNA rather than to an amino acid residue in
a protein [25]. The 3D-structure of a prototype member of
this gene family, indeed, reveals a structure closely resembling that of PARP-1 and Diptheria toxin (see Fig. 1), providing strong support for the relevance of the matches
detected by PSI-BLAST.
For the pART homologues shown in Figure 9 we also analyzed the sequences flanking the pART catalytic domain
for associated conserved domains. The results reveal that
many pARTs, even from very distantly related organisms,
share domain associations found in human and mouse
pARTs. Some of these are illustrated in Figure 8. For example, the association of regulatory, BRCT, and DNA
binding domains observed in pART1 (PARP-1) is found
also in similar proteins encoded by fruit fly, nematode,
microfungi and cress plant genomes. Tankyrase-like association with ankyrin repeats is found in pARTs from the
fruit fly and nematode. The association of a pART catalytic
domain with an A1pp domain, as seen in human pART
subgroup 3, is found also in a pART from the slime mold
Dictyostelium discoideum. The combination with a WWE
http://www.biomedcentral.com/1471-2164/6/139
domain, as seen in human pART subgroup 4, is found also
in putative pARTs from cress plant. A domain
corresponding to the unknown upstream region of the
smallest human pART (pART15) is observed also in a
pART from the malaria mosquito Anopheles gambiae, and
a duplicated truncated pART catalytic domain as in pARTs
16 and 17 is observed also in a pART from the microfungus Gibberella zeae. These results indicate that many of the
domain combinations observed in human and mouse
pARTs represent evolutionary ancient inventions.
Some pARTs of distantly related proteins are associated
with domains not found in any of the human pARTs. A
striking example is that of G. zeae pARTc, which most
closely resembles human pARTs 16 and 17, but is associated with a second potential catalytic, ubiquitin ligase
domain (Fig. 8). A similar pART is found also in the
related microfungus Aspergillus nidulans [GenBank:
EAA66581]. These microfungal pARTs are the only
examples found so far, in addition to vertebrate pART4,
where a distinct domain(s) is genetically fused to the Cterminal end of the pART catalytic domain. The large
domain(s) associated with the putative pART from bacteriophage Aeh1 does not bear any resemblance to pARTassociated domains in vertebrates but shows distant similarity to viral coat proteins. The only organism containing
an isolated pART domain reminiscent of the isolated ART
domain found in verbetrate mARTs [27] is the Chilo iridescent insect virus. This "naked" viral pART catalytic
domain contains the H-Y-E motif of PARP-1 and DT. It
will be interesting to determine whether this protein
exhibits the predicted pART activity.
A striking example of domain shuffling is observed in one
of the three C. elegans pARTs: like the human tankyrases
(pARTs 5 and 6), Ce.pARTc contains ankyrin repeats, but
also harbors the regulatory and WGR domains typical of
human group 1 pARTs instead of the SAM domain found
in human pARTs 5 and 6 (Fig. 8). A similar variation of
domains as in Ce.pARTc is found also in one of the ten
pARTs of D. discoideum (Dd.pARTb).
Finally, we addressed the question whether the striking
differences in exon/intron compositions of the closest
PARP-1-homologues in groups 1 and 2 might be reflected
in similar differences in pART orthologues of distantly
related species. To this end we determined the exon/
intron structures of distant pART orthologues by BLASTn
searches of the respective genome databases using cDNA
sequences as queries; and compared the results with those
obtained for human pART genes. The results are illustrated schematically in Figure 10, with conserved intron
positions highlighted. As in case of most other genes, the
pART genes of 'lower' animals, protists, and plants in general contain fewer and shorter introns than the human
Page 15 of 23
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BMC Genomics 2005, 6:139
A
http://www.biomedcentral.com/1471-2164/6/139
Hs.pART1
5430
0
281
1
9899 1574
1
166
0
116
2
1654 2213 645
2
215
3
100
4
0
117
5
0
6
2320
1827
903
0
1
1
177
148
7
141
8
280 298 1838
1
243
9
1
69
10
2
133
11
2753 3708 2112
0
196
12
0
129
13
0
84
14
590
0
123
15
1427 799
0
129
16
0
99
17
931
18
1
1
0
1122 1737
1
2
1
62
1
35198 64273 737 28862 1429 557
225
1
1
96
3
2
1
37
2
76
4
0
2
95 67
5
6
0
1373
1462
1
0
187
7
122
8
9
95 10153
2
92
79
10
0
11
23
E
Y
561
804
181
275
155
361
246
1
2
0
0
2
0
0
0
51
97
76
103
163
139
64
135
128
100
99
125
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
0
80
12
2243
1420
2
0
146
13
H
166
14
220
292
12537
3396
1
0
0
110
16
15
189
697
18
17
1
238
19
8889
83
0
20
1510 1471
121
1
0
98
21
22
901
441
0
1
75
106
23
Dm.pARTa
0
1
190
36000
>20000
2
334
534
2
3
4
3680
187
24
2394
1
688
1
H
60
1
1881
649
0
720
1
1094
0
123
578
3
4
2
158
64
0
1653
2
205
5407
0
1551
0
333
5
1390
0
Ce.pARTb
1
1335
88 1
554
1
91
1
50
1
199
1712
3
2
77
0
4
119
0
114
2295
5
6
46
0
H
488
2
245
49
0
343
7
51
0
432
8
At.pARTa
294
1
50
80
2
2
217
97
0
3
187
111
1
282
4
149
2
5
196
99
0
6
162
72
0
7
61
82
1
64
8
92
2
84
0
484
9
10
83
2
113
77
161
11
12
13
93
0
183
14
212
1
77
0
268
2
81
1
146
3
92
0
181
1
82
236
4
5
196
0
214
1
130
56
6
7
93
0
117 76
2
1
74
47
50
8
9
10
107
0
63
11
93
0
83
138
16
17
109
0
81
111
12
13
GR
ca.200bp
Zn finger
utr
CDR
SAP
Intron
Ankyrin
SAM
91
0
E
129
0
183
Y
103
1
WGR
pART rd
19
121 111
0
2
86
0
76
143
74
106
14
15
16
17
H
Y
198
18
E
543
1
Y
E
pART cd
H
B
273
E
H
BRCT
229
0
18
Ci.pART
conserved exon-intron-boundaries
domains
Y
15
H
204
11
109
GR
At.pARTb
E
136 110
1
0
83
0
326
5
59
0
H
103
1
47
2
Y
645
GR
749
0
E
125
10
9
79
10
Y
4
GR
49
0
46
2
254
9
3
2
7
E
2145
0
49
0
698
135
6
8
H
78
60
0
157
Y
216
7
GR
44
1
68
2
5
237
6
E
Y
293
4
556
Ce.pARTc
27
6
3
65
0
232
E
Y
H
93
0
26
1344
5
2
Ce.pARTa
6387
157
2
570
64
1
783
2
25
GR
Dm.pARTb
16
E
6500
2
142
1
260
Y
H
55
2
71
1
1300
117
195
6095 4169
172
737
22
GR
23546
2
115
21
53
Hs.pART5
678
424
0
2
20
H
267 1398 3612
1244
782 1015 427
128
19
GR
Hs.pART2
0
153
Y
E
Dm.pARTa
Hs.pART1
Ce.pARTa
Dm.pARTb
Hs.pART5
At.pARTa
Hs.pART2
Ce.pARTb
At.pARTb
phase 0 intron
Ankyrin
phase 1 intron
WGR
pART rd
phase 2 intron
pART cd
Schematic
Figure 10diagram of the exon/intron structures of pART family members of distantly related organisms
Schematic diagram of the exon/intron structures of pART family members of distantly related organisms. A)
Exon/intron structures were determined by BLASTn searches of the genome browsers using the pART cDNA sequences. The
positions of codons corresponding to the H Y E motif in the NAD-binding crevice of diphtheria toxin are marked by yellow
circles. The position of the conserved glycine and arginine pair of residues within the WGR domain is marked in blue. Coding
regions for catalytic and other domains are indicated by colored bars. Conserved introns are marked by colored arrows. B)
The diagram contains only those introns that are conserved in at least two distantly related species. Color-coding of the
introns corresponds to that shown in A). The position of codons encoding/corresponding to the H, Y, E residues in the NAD
binding crevice are indicated by vertical lines. The position of each intron with respect to the codon is indicated by circles
(phase 0 introns), boxes (phase 1 introns), and triangles (phase 2 introns). Coding regions for catalytic and other selected
domains are indicated by colored lines as in A).
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BMC Genomics 2005, 6:139
homologues. However, some of the introns found in
human pART genes are found also in homologues of
distantly related organisms. For example, all six introns
observed in D. melanogaster pARTb are found in corresponding positions also in human pART5 (tankyrase 1);
yet human pART5 contains 14 additional introns not
found in the fruit fly pART. The other pART of the fruit fly
shares two of its five introns with human pART1 (PARP1). The three pARTs of the nematode C. elegans show a different, only partially overlapping set of conserved introns:
Ce.pARTa shares seven of its nine introns with human
pART1, Ce.pARTb shares three of its four introns with
human pART2, whereas Ce.pARTc does not seem to share
any of its introns with pART5, despite the similar domain
organization on the protein level (see Fig. 8). The pARTs
from the model plant Arabidopsis thaliana contain a fairly
high number of introns, however only very few intron
positions correspond to ones found also in human pARTs.
For example, At.pARTa which is most closely related to
human PARP-1 in terms of amino acid sequence similarity and organization of conserved protein domains, evidently does not share any of its 18 introns with human
pART1. Strikingly, however, the introns found in the catalytic domain of this pART exhibit conserved positions
with two different human pARTs: two of the four intron
positions in the catalytic domain of At.pARTa are found in
corresponding positions in human pART5 (tankyrase),
another intron is found at a corresponding position in
human pART2 (Fig. 10), whereas the fourth intron is not
found in any human pART. At.pARTb which is most
closely related to human pART2 in terms amino acid
sequence similarity and domain organization, shares one
of its 17 introns with human pART2. Note further, that in
only two cases (Chilo iridescent virus pART and pARTa of
the fruit fly), the pART catalytic domain lacks introns, i.e.
is encoded by a single exon as in case of the vertebrate
mARTs [27].
Discussion
The results of our study illustrate the great power and utility of the public genome databases and database search
programs. Moreover, they provide important novel
insights into the molecular structure and evolution of the
pART gene family.
Our results differ in some details from those of a recent
report by Ame and coworkers [11]. These discrepancies
can be explained by errors in the draft sequence of the
human genome available at the time of the previous
report. For example, the database entry AK023746 given
by Ame et al. for PARP-5c evidently represents a truncated
cDNA for pART6 (alias tankyrase 2 or PARP-5b). This
entry contains two point mutations and a 65 bp deletion
in the 3' utr vs. the cDNA and genomic sequences of
pART6. Blast analyses of the high quality sequence of the
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human genome and of the EST database with the
AK023746 sequence provide no evidence for a distinct
copy of this gene in the human genome. We conclude that
the PARP-5c gene identified by Ame et al. represents an
allelic variant or cloning/sequencing error rather than a
genuine pART gene family member; i.e. that the total
number of human pART genes is 17 rather than 18 suggested in the previous report. Large discrepancies exist
also in the number of amino acids assigned in the two
reports for pART7/PARP-15 (444 vs. 989) and for
pART16/PARP-8 (854 vs. 501). The earlier database
entries for PARP-8 (XM_018395) and PARP-15
(XM_093336) have hence been removed as a result of
standard genome annotation processing because these
entries evidently contained frameshift mutations and/or
fused cDNA sequences that led to erroneous amino acid
assignments. Similarly, the small differences in assignments for five other PARPs/pARTs can be accounted for by
differences in the draft vs. high quality sequence of the
human genome (Ame et al./our study): pART2/PARP2
(583/570), pART3/PARP3 (540/533), pART10/PARP10
(1020/1025), and pART14/PARP7 (657/680).
We assigned the 17 human pARTs into five distinct subgroups (Fig. 2). This assignment is supported by several
independent lines of evidence: Firstly, members of a particular subgroup show higher amino acid sequence
identities to one another than to members of other subgroups (Fig. 6). This is reflected in the tiling paths of PSIBlast searches, where members of the same subgroup were
detected in the first iteration, whereas members of other
subgroups generally were detected in later iterations (Fig.
4). Secondly, members of a particular subgroup typically
share one or more associated domains not found in members of other subgroups (Fig. 8); pARTs 8, 10 and 15 pose
exceptions to this rule. Thirdly, members of a particular
subgroup typically share one or more intron positions not
found in members of other subgroups (Fig. 3); pARTs 1–
4 pose notable exceptions to this rule. Fourthly, when
genes of two or more pARTs are physically linked in a cluster on the same chromosome, they belong to the same
subgroup – possibly reflecting regional duplications (Fig.
2). Finally, results of all phylogenetic analysis converged
in topologies with clearly distinct clades for each of the
subgroups (Fig. 7). Members of subgroups 1 and 2 evidently are more closely related to one another than to
other subgroups (Figs. 6 and 7). Similarly, members of
subgroups 3 and 4 are sister-groups to one another, indicating a close relationship.
Members of the pART family are found fused to a striking
variety of associated domains (Fig. 8). It is not farfetched
to hypothesize that the associated domains direct the
respective pARTs to subcellular structures and/or target
proteins. Genetic fusion of group 1 and group 2 pARTs
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BMC Genomics 2005, 6:139
with DNA-binding domains is in line with their established roles in DNA-repair, chromosome remodeling, and
mitotic spindle formation [9,11,12]. Moreover, the SAM
and ankyrin domains of pARTs 5 and 6 have been shown
to mediate interactions with target proteins in telomereassociated protein complexes [45]. Similarly, the C-terminal domain of pART4 evidently plays a role in targeting
pART4 to the major vault particles [46]. A flurry of
domains implicated in the ubiquitination pathway point
to a possible connection between ubiqutitination and
ADP-ribosylation. Indeed, it has recently been reported
that ADP-ribosylation of TRF1 by tankyrase (pART5)
results in the release of the protein from telomers and its
subsequent ubiquitination [47]. Strikingly, pARTs from
the microfungi G. zea and A. nidulans provide examples
for the genetic fusion of two enzyme domains catalyzing
these post-translational protein modifications into a single polypeptide.
So far, only a single example of a 'naked' pART catalytic
domain akin to the isolated catalytic domain of the vertebrate ecto-ARTs 1–5 [27] was recovered from the public
database. This putative pART from Chilo iridescent virus
clusters with the mammalian pARTs of subgroup 1 (Fig.
7), suggesting that this large double stranded DNA virus
[48] may have acquired its pART by horizontal gene
transfer.
The definition of the pART catalytic domain proposed in
this paper is somewhat smaller than that commonly used
in the field [11]. We used the position of the common
phase 0 intron upstream of the first conserved β sheet to
set the N-terminal end of the catalytic domain (e.g. see
Figs. 1 and 3B). The pARTs of subgroup 1 are extended Nterminally of this position by an alpha helical domain
(Fig. 8) which is often included as part of the PARP-1 catalytic domain. However, since other pART family members lack this region, we propose to omit it from the
proper pART catalytic domain. Moreover, this N-terminal
delineation of the catalytic domain corresponds well to
the N-terminus of the 'naked' pART of Chilo iridescent
virus as well as to those of Diphtheria toxin and Pseudomonas exotoxin A after proteolytic processing of the
signal sequence or translocation domain (Fig. 1).
With the exception of pART4, the group 1 pARTs are
extended upstream of this helical region by another
domain named after its conserved motif of tryptophane
(W) – glycine (G) – arginine (R) residues. This WGR
domain is found also in poly-A-polymerases, its function
is unknown. Many group 1 pARTs from distantly related
organisms, e.g. plants, insects, nematodes, and microfungi, also contain these two domains. Interestingly, in
Drosophila melanogaster pARTa these three domains (WGR,
helical, catalytic) are encoded by a single, large exon (Fig.
http://www.biomedcentral.com/1471-2164/6/139
10). Human pARTs 5–17 lack the WGR and helical
domains. However, pART5/6 (tankyrase)-like pARTs from
C. elegans (Ce.pARTc) and D. discoideum (Dd.pARTb) contain the WGR and helical domains whereas a SAM
domain is found at this position in human pARTs 5 and 6
(Fig. 8).
A puzzling finding is the lack of conservation of the classic
H-Y-E motif found in the catalytic cores of PARP-1, PARP2, Diphtheria toxin and Pseudomonas Exotoxin A (Fig. 1).
This motif is conserved only in members of subgroups 1
and 2. All other human pARTs carry notable variations
from this motif. In particular, all other pARTs carry a
replacement of the glutamic acid residue in β 5, i.e. the
residue that was shown to be critical for the catalytic activities of DT, PARP-1 and many other pARTs and mARTs
[6,7,20,21]. In six cases, this glutamic acid is replaced by
an isoleucine residue, in two cases by leucine, and in one
case each by threonine, valine, or tyrosine. Enzyme
activity has been reported recently for two of the six pARTs
that carry an H-Y-I motif instead of the H-Y-E motif
(pARTs 10 and 14) [32,34]. Thus, it is not unlikely that the
four other pARTs carrying the H-Y-I motif turn out to be
active enzymes (pARTs 11, 12, 16, and 17). Mouse pART8
also carries an H-Y-I motif, whereas its human orthologue, like pART7, carries an H-Y-L variant motif. H-Y-I
and H-Y-L variant motifs are also found in pARTs from the
slime mold (Dd.pARTg) and amoeba (Eh.pARTf) (Fig. 8).
Human pART15 carries an H-Y-Y variant motif, which is
conserved in its orthologues from mouse and the malaria
mosquito (Fig. 8). It will be interesting to determine
whether and how site directed mutagenesis of the H-Y-E
motif in pARTs 1–6 to the variant motifs of pARTs 7–17 –
and vice versa – affects their enzyme activities. Moreover,
it remains to be determined whether the most striking variation of the H-Y-E motif – to Q-Y-T in human and mouse
pART9 is compatible with enzyme activity.
The results of our PSI-BLAST and PSIPRED analyses (Figs.
4, 5, 9 and additional files 3, 4, 5, 6, 7, 8) support the conclusions that the pART gene family described here and the
mART gene family described in our previous study [27]
constitute two distinct ART subfamilies, and further, that
the family of tRNA:NAD 2'-phosphotransferases [24,25]
constitutes a branch that is more closely related to the
pART subfamily than to the mART subfamily. Our results
illuminate the power and limits of PSI-BLAST searches:
PSI-BLAST readily connected members of the pART subfamily in many different species, while DT, ETA and TpTs
were found at or below the threshold. In contrast PSIBLAST searches never connected pART family members
with members of the mART subfamily or vice versa. The
results of PSI-BLAST searches, thus, are in accord with
insights gained from the known 3D structures of representative ADP-ribosyltransferases (Fig. 1), i.e. that certain
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BMC Genomics 2005, 6:139
conserved structural features clearly distinguish these two
subfamilies. Is it possible that some of the pART gene
family members described here actually possess monoADP-ribosyltransferase rather than poly-ADP-ribosyltransferase activity? Given the structural similarity to DT/
ETA this is a possibility. Moreover, it cannot be excluded
that some family members may have lost enzyme activity
and have acquired a new function. In any case, the respective proteins clearly are more closely related to the pART
than to the mART gene family, in line with the nomenclature proposed here. Have all ARTs encoded in the human
genome been identified? A number of ADP-ribosylation
reactions have been described in mammalian cells that
cannot yet be accounted for by the ARTs identified in this
study or our previous study, e.g. mono-ADP-ribosylation
of actin, rho, glutamate dehydrogenase, and of the alpha
and beta subunits of heterotrimeric G proteins [3,4,8].
Given the fact that the pART subfamily described here and
the mART subfamily described in our previous study [27]
could not be interconnected by PSI-BLAST, it reamins an
intriguing possibility that other ART subfamilies in the
human genome still await to be identified.
Conclusion
The family of proteins containing a PARP-like catalytic
domain consists of 17 members in the human and 16 in
the mouse, rat, and pufferfish. The vertebrate pART family
can be divided into five subgroups on the basis of
sequence similarity, phylogenetic relationships, conserved intron positions, and patterns of genetically fused
protein domains. The four members of group 1 and the
two members of group 2 each contain a conserved trias of
residues (H-Y-E motif) also observed in Diphtheria toxin
and Pseudomonas exotoxin A. The eleven other pART proteins carry variants of this motif (six H-Y-I, two H-Y-L, and
one each Q-Y-T, Y-Y-V, H-Y-Y). All human pARTs are
multi-domain proteins in which the pART catalytic
domain is associated in a Lego-like fashion with other
putative protein-protein interaction, DNA binding and
ubiquitination domains. In all but one case (pART4) the
catalytic domain represents the C-terminal end of the
multi-domain protein. Most of the domain associations
observed in human pARTs appear to be very ancient
inventions since they can be found also in insects, plants,
microfungi, and amoeba.
Methods
Database searches
Protein databases were searched using PSI-BLAST [35].
Genome databases were searched using BLASTn and
tBLASTn [49]. Tissue distributions of pART-ESTs were analyzed using Electronic Northern calculations at the GeneCard website [50].
http://www.biomedcentral.com/1471-2164/6/139
Structure and sequence analyses
Amino acid sequence alignments were performed with TCoffee [36]. Secondary structure predictions were
performed with PSIPRED [37]. Threading of amino acid
sequences onto known 3D structures in PDB were performed with GenTHREADER [37]. Sequence analyses
were performed using DNA-Star software, 3D-images
were prepared with PyMol [51] software.
Phylogenetic analyses
Phylogenetic analyses were applied to the 36 catalytic core
amino acid sequences using the dataset in Figure 6. Phylogenetic analyses were performed on the computational
cluster of the College of Biology and Agriculture at
Brigham Young University by using maximum parsimony
and Bayesian Markov chain Monte Carlo approaches
The
topologies
were
http://babeast.byu.edu.
reconstructed using equally weighted maximum parsimony (MP) analysis as implemented in PAUP* 4.0b10
[52], maximum likelihood (ML) with simultaneous
adjustment of topology, and branch length as implemented in PhyML [53], as well as Bayesian methods coupled with Markov Chain Monte Carlo inference (BMCMC,
MrBayes) [54]. The best fit likelihood model for amino
acid evolution was determined based on the lowest
Akaike Information Criterion (AIC) or Bayesian Information Criterion (BIC) score as implemented in ProtTest
1.2.6 [53,55,56].
The MP analysis was run using 5000 random addition
replicates and tree bisection-reconnection branch swapping. Nonparametric bootstrap values were calculated for
MP and ML analyses (10.000/100 bootstrap replicates,
100/1 heuristic random addition replicates) to assess confidence in the resulting relationships. ML analysis was run
implementing the RtREV+I+G+F model of amino acid
evolution (AIC= 4907.73; -lnL= 2800). The a priori information obtained by ProtTest 1.2.6 was incorporated into
the BMCMC analysis. Bayesian phylogeny estimation was
achieved using random starting trees, run for 3 × 106 generations, with a sample frequency of 1000, and ten chains
(nine heated, temperature= 0.2). Analyses were repeated
three times to check for likelihood and parameter mixing
and congruence. Likelihood scores were plotted against
generation time to determine stationery levels. Sample
points before reaching stationery were discarded as "burnin". Repeated analyses were compared for convergence on
the same posterior probability distributions [57]. The
maximum a posteriori tree (MAP) is presented in this
paper, showing to percentage converted posterior probabilities (pP%).
Abbreviations used
ART = ADP-Ribosyltransferase, BLAST = basic local alignment search tool, 3MB = 3-methoxybenzamide, NAD =
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BMC Genomics 2005, 6:139
nicotinamide adenine dinucleotide, PDB = protein
database.
Authors' contributions
This study was initiated in the summer of 1997 while FKN
was a visiting scientist in FB's lab at DNAX. Initial database searches were performed by FKN and FB, later
searches by HO, PAR, and FKN. KD performed the phylogenetic analyses. FKN supervised the study with essential
contributions by FH. HO prepared the figures and FKN
wrote the paper. The results represent the partial fulfillment of the requirements for the graduate thesis of HO.
Additional material
Additional File 1
Representation of pART gene transcripts in the database of expressed
sequence tags The public EST database was screened for ESTs encoding
pARTs using tBLASTn and the amino acid sequences of the catalytic
domain of known pART family members as queries at the dates indicated
on top. Accession numbers of the corresponding Unigene clusters are indicated. Blank fields indicate lack of detectable ESTs encoding the respective
pART catalytic domain. Tissue distribution analyses were performed for
each cluster by "electronic Northern" analyses. For each family member,
the two tissues with the highest numbers of ESTs are indicated. Tissue
abbreviations: BMR bone marrow, BRN brain, HRT heart, MSL muscle,
PNC pancreas, PST prostate, KDN kidney, LNG lung, LVR liver, LYN
lymph node, SPC spinal chord, SPL spleen, TMS thymus, UTR uterus
Click here for file
[http://www.biomedcentral.com/content/supplementary/14712164-6-139-S1.pdf]
Additional File 2
Schematic illustration of the local human and mouse chromosomal
environments of the pART subgroup 3 gene cluster The figure schematically illustrates the local chromosomal environment of the syntenic cluster
of pART genes and neighboring genes on human chromosome 3q (top)
and mouse chromosome 16B3 (bottom). The order and orientation of all
genes in the depicted cluster is conserved. Known transcripts in GenBank
are indicated schematically with their respective accession number. Exons
are indicated by boxes. The direction of transcription is marked by arrows.
Grey vertical bars correspond to a scale of 10.000 base pairs. The figure
was modified from the respective online UCSC human and mouse genome
browsers http://genome.ucsc.edu.
Click here for file
[http://www.biomedcentral.com/content/supplementary/14712164-6-139-S2.pdf]
http://www.biomedcentral.com/1471-2164/6/139
Additional File 3
Multiple amino acid sequence alignments, secondary structure predictions, and threading results for pART subgroup 1 A multiple sequence
alignment was generated for the catalytic domains of pARTs 1–4 with TCoffee. Each residue in the sequence is reported as a single letter code. Secondary structure units in the 3D structures of chicken PARP-1 (1a26)
and mouse PARP-2 (1GS0) are indicated on top of the alignment. Positions with identical residues in all sequences are marked by asterisks, similarities are marked with colons and periods below the alignment. Residues
corresponding to the H Y E motif in the NAD binding crevice of diphtheria
toxin are marked in red. Intron positions are projected onto the multiple
alignment and are marked in grey (phase 0), blue (phase 1), and yellow
(phase 2). Secondary structure predictions were generated for human
pART1 with PSIPRED and are indicated in blue below the alignment
(pr1); the confidence of the prediction is indicated in orange (highest confidence = 9). Secondary structure units are abbreviated as follows: H =
helix; B = residue in isolated beta bridge; E = extended beta strand; G =
310 helix; I = pi helix; T = hydrogen bonded turn; S = bend.
Click here for file
[http://www.biomedcentral.com/content/supplementary/14712164-6-139-S3.pdf]
Additional File 4
Multiple amino acid sequence alignments, secondary structure predictions, and threading results for pART subgroup 2 A multiple sequence
alignment was generated for the catalytic domains of pARTs 5 and 6 with
T-Coffee. Residues, identities, intron positions, and secondary structure
units are marked as in additional file 3. Indicated secondary structure predictions were generated for human pART5 (pr5) with PSIPRED.
Click here for file
[http://www.biomedcentral.com/content/supplementary/14712164-6-139-S4.pdf]
Additional File 5
Multiple amino acid sequence alignments, secondary structure predictions, and threading results for pART subgroup 3 A multiple sequence
alignment was generated for the catalytic domains of pARTs 7–10 with TCoffee. Residues, identities, intron positions, and secondary structure
units are marked as in additional file 3. Indicated secondary structure predictions were generated for human pART7 (pr7) with PSIPRED.
Click here for file
[http://www.biomedcentral.com/content/supplementary/14712164-6-139-S5.pdf]
Additional File 6
Multiple amino acid sequence alignments, secondary structure predictions, and threading results for pART subgroup 4 A multiple sequence
alignment was generated for the catalytic domains of pARTs 11–14 with
T-Coffee. Residues, identities, intron positions, and secondary structure
units are marked as in additional file 3. Indicated secondary structure predictions were generated for human pART11 (pr11) with PSIPRED.
Click here for file
[http://www.biomedcentral.com/content/supplementary/14712164-6-139-S6.pdf]
Page 20 of 23
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BMC Genomics 2005, 6:139
http://www.biomedcentral.com/1471-2164/6/139
8.
Additional File 7
Multiple amino acid sequence alignments, secondary structure predictions, and threading results for pART subgroup 5 A multiple sequence
alignment was generated for the catalytic domains of pARTs 15–17 with
T-Coffee. Residues, identities, intron positions, and secondary structure
units are marked as in additional file 3. Indicated secondary structure predictions were generated for human pART15 (pr15) and for human
pART16 (pr16) with PSIPRED.
Click here for file
[http://www.biomedcentral.com/content/supplementary/14712164-6-139-S7.pdf]
13.
Additional File 8
14.
Representative tiling paths of PSI-BLAST searches initiated with the
catalytic domain amino acid sequences of selected pART family members PSI-BLAST searches were initiated with the query sequences indicated on top at a threshold setting for the expect value of 0.005 as in
Figure 4. pART subgroups are color coded as in Figure 2. Matching
sequences from the slime mold (D. discoideum, blue) and from a model
plant (A. thaliana, green) are indicated at the iteration in which they first
appeared above threshold. The respective pART homologues from these
species were arbitrarily numbered (pARTa-j) in the order in which they
were detected in the search that was initiated with human pART1 (PARP1). Protein data base accession numbers are listed in Figure 9. pARTs
indicated in black include short possibly truncated coding sequences of
pART homologues that could not be assigned to a particular subgroup with
certainty.
Click here for file
[http://www.biomedcentral.com/content/supplementary/14712164-6-139-S8.pdf]
9.
10.
11.
12.
15.
16.
17.
18.
19.
20.
Acknowledgements
This work was supported by grant No310/3 from the Deutsche Forschungsgemeinschaft to FKN. HO was a grantee of the Studienstiftung des
Deutschen Volkes. KD is funded by the NSF grants DEB-0120718 and DEB9983195. DNAX is fully funded by the Schering Corporation. We thank
Sahil Adriouch, Bernhard Fleischer, Stefan Kernstock, and Stefan Rothenburg (University Hospital Hamburg) for critical reading of the manuscript.
21.
22.
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