Structure, studia, genomika

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Gene 275 (2001) 267–277
www.elsevier.com/locate/gene
Structure, chromosomal localization, and expression of the gene for mouse
ecto-mono(ADP-ribosyl)transferase ART5
Gustavo Glowacki
a
, Rickmer Braren
a
, Marina Cetkovic-Cvrlje
b
, Edward H. Leiter
b
,
Friedrich Haag
a
, Friedrich Koch-Nolte
a,
*
a
Institute for Immunology, University Hospital, Martinistrasse 52, 20246 Hamburg, Germany
b
The Jackson Laboratory, Bar Harbor, ME, USA
Received 7 May 2001; received in revised form 29 June 2001; accepted 11 July 2001
Received by A. Sippel
Abstract
Mono(ADP-ribosyl)transferases regulate the function of target proteins by attaching ADP-ribose to specific amino acid residues in their
target proteins. The purpose of this study was to determine the structure, chromosomal localization, and expression profile of the gene for
mouse ecto-ADP-ribosyltransferase ART5. Southern blot analyses indicate that Art5 is a single copy gene which maps to mouse chromosome
7 at offset 49.6 cM in close proximity to the Art1, Art2a and Art2b genes. Northern blot and RT-PCR analyses demonstrate prominent
expression of Art5 in testis, and lower levels in cardiac and skeletal muscle. Sequence analyses reveal that the Art5 gene encompasses six
exons spanning 8 kb of genomic DNA. The 5
0
end of the Art5 gene overlaps with that of the Art1 gene. A single long exon encodes the
predicted ART5 catalytic domain. Separate exons encode the N-terminal leader peptide and a hydrophilic C-terminal extension. Sequencing
of RT-PCR products and ESTs identified six splice variants. The deduced amino acid sequence of ART5 shows 87% sequence identity to its
orthologue from the human, and 37 and 32% identity to its murine paralogues ART1 and ART2. Unlike ART1 and ART2, ART5 lacks a
glycosylphosphatidylinositol-anchor signal sequence and is predicted to be a secretory enzyme. This prediction was confirmed by transfect-
ing an Art5 cDNA expression construct into Sf9 insect cells. The secreted epitope-tagged ART5 protein resembled rat ART2 in exhibiting
potent NAD-glycohydrolase activity. This study provides important experimental tools to further elucidate the function of ART5.
q
2001
Published by Elsevier Science B.V.
Keywords: ADP-ribosylation; NAD-glycohydrolase; Gene family; Gene structure; Splice variants; Toxin homologue
1. Introduction
of the target protein. ADP-ribosyltransferase activity of
bacterial toxins often is involved in the pathogenesis of
disease (Moss and Vaughan, 1990; Aktories, 1991). For
example, Diphtheria toxin ADP-ribosylates a diphthamide
residue in elongation factor 2, thereby shutting off host cell
protein synthesis (Honjo et al., 1968). Cholera and Pertussis
toxins interfere with signal transduction by ADP-ribosylat-
ing the alpha-subunit of heterotrimeric G proteins at specific
arginine or cysteine residues leading to uncoupling of
surface receptors from their downstream effector molecules,
thereby affecting adenylate cyclase activity and ion flux
(Moss and Vaughan, 1990). Other toxins ADP-ribosylate
arginine residues in actin (C2, iota, SpvB, VIP2) and ras
(exoS) or asparagine residues in rho (C3) (Rappuoli and
Montecucco, 1997; Han et al., 1999; Otto et al., 2000). In
photosynthetic bacteria, a mADPRT (DRAT) regulates
nitrogen fixation by ADP-ribosylating an arginine residue
of dinitrogenase reductase (Ludden, 1994).
Mounting
Mono(ADP-ribosyl)transferases (mADPRTs) are an
important class of enzymes with known regulatory functions
as bacterial toxins and metabolic regulators (Moss and
Vaughan, 1990; Aktories, 1991; Ludden, 1994). These
enzymes mediate the post-translational modification of
specific target proteins by transferring the ADP-ribose
moiety from NAD
1
to specific amino acid residues in
their target proteins. This usually inactivates the function
Abbreviations: Gapd, glyceraldehyde 3 phosphate dehydrogenase; GPI,
glycosylphosphatidylinositol; mADPRT, mono(ADP-ribosyl)transferase;
NAD
1
, nicotinamide adenine dinucleotide; PBS, phosphate-buffered
saline; PCR, polymerase chain reaction; RACE, rapid amplification of
cDNA ends; RT, reverse transcription; SDS-PAGE, sodium dodecyl sulfate
polyacrylamide gel electrophoresis; UTR, untranslated region
* Corresponding
author.
Tel.: 149-40-428033612;
fax: 149-40-
evidence
indicates
that
ADP-ribosyltrans-
428034243.
E-mail address: nolte@uke.uni-hamburg.de (F. Koch-Nolte).
ferases
play
important
regulatory
roles
also
in
higher
0378-1119/01/$ - see front matter
q
2001 Published by Elsevier Science B.V.
PII: S 0378-1119(01)00608-4
 268
G. Glowacki et al. / Gene 275 (2001) 267–277
animals (Haag and Koch-Nolte, 1997). In the mouse, for
example, cell surface mADPRTs have been implicated in
regulating myogenesis, long-term potentiation in hippocam-
pal neurons, and the activity of cytotoxic T cells (Zolk-
iewska and Moss, 1993; Schuman et al., 1994; Wang et
al., 1996). The first mammalian mADPRT (ART1) was
cloned from rabbit skeletal muscle (Zolkiewska et al.,
1992). Related genes, designated ART2–ART7, have been
cloned from mouse, human, and chicken (Koch-Nolte and
Haag, 1997). We have previously cloned and characterized
the mouse Art1 gene and mapped it to chromosome 7 at
offset 49.6 cM (Koch-Nolte et al., 1996a; Braren et al.,
1998). We now report the cloning, characterization, and
expression of the mouse Art5 gene and show that it maps
to the same region on mouse chromosome 7.
In the case of Art2, defects in gene structure and/or
expression have been found to coincide with susceptibility
for autoimmune disease in certain rat and mouse laboratory
strains (Greiner et al., 1987; Koch-Nolte et al., 1995).
Considering the presumptive regulatory role of mADPRTs,
it is conceivable that defects in other gene family members
may also be of clinical relevance. The description of the
mouse Art5 gene presented here provides the basis for
applying established transgene and knock-out technologies
to further analyze the function of the ART5 protein and to
determine the functional significance of Art5 gene defects.
with primers N11 and N41, purified and radiolabeled to high
specific activity (.10
8
cpm/mg). Hybridization and wash-
ing were performed essentially as described previously
(Koch-Nolte et al., 1995; Braren et al., 1998).
2.3. Isolation and sequencing of genomic and cDNA clones
A 129/SvJ mouse genomic P1 DNA library (Genome
Systems, St. Louis, MO) was screened by PCR with primers
N00 and N31. Purified P1 DNAs were subjected to restric-
tion mapping and suitable restriction fragments were
subcloned into pBluescript (Stratagene) for sequence
analyses. Mouse heart and testis ‘marathon’ cDNA
(BALB/c) were purchased from Clontech and subjected to
5
0
and 3
0
RACE reactions according to the manufacturer’s
protocol with nested primers N32 and N33 for 5
0
RACE and
N02 and N03 for 3
0
RACE. For defining the 5
0
cap site of
Art5 cDNA, testis CapSite cDNA
e
(Eurogentec) was
amplified with primers N32 and N07 according to the manu-
facturer’s instructions. PCR amplification products were
cloned into the pCR2.1 vector (Invitrogen). All sequences
were obtained by dideoxy sequence analysis with appropri-
ate vector- and Art5-specific primers using the big dye-
terminator sequencing kits (Applied Biosystems).
Sequences obtained from PCR products were confirmed
by sequencing clones obtained from two separate PCR reac-
tions. The nucleotide sequences described here have been
deposited
in
the
EMBL
database
(Accession
numbers:
2. Materials and methods
AJ295722, Y08028, and Y16835).
2.1. PCR primers and PCR reactions
2.4. Amino acid sequence alignment and structure
prediction analyses
Primers derived from the sequence of the mouse Art5 gene
were as follows: N02, ACT CTC TGG AGT TAT GAT CAG
ACC TG; N03, GCT GCA GCT CTC CAG AGC TGG ACC;
N00, AGG ATG ATT CTG GAG GAT CTG CTG ATG;
N33, GCG TGC AAG CTG AGG CAG CTG AG; N32,
CCA GGC CTC TTG TGC TGC TTC CCA; N99, CTG
CTT CCT GCA GCC GTT CAA AGC CC; N06, AGA
CAG ATT TGG CGA CTT AAC TAG C; N05, CTG ATC
TCA GGC CAG GAC TAG GC; N11, GAT ACC TTT GAT
GAT GCC TAT GTG GGC TG; N41, GAC AAC GCC TGG
ATA GGA GCC CCA AAA CA. PCR reactions were carried
out in 20 ml reaction volumes on purified plasmid or P1 DNA
(0.1–10 ng) or cDNA (Clontech) (10–50 ng) in 1 £ PCR
buffer (Perkin Elmer) with 2.5 units of Amplitaq-Gold poly-
merase (Perkin Elmer) for 25 cycles (20 s at 958C, 20 s at
558C, and 60 s at 728C). Samples were incubated for 8 min at
948C prior to the first PCR cycle.
Multiple sequence alignments, sequence distance and
phylogeny calculations were performed with the DNAstar
and Sequencher programs on a Macintosh computer. Signal
peptide cleavage sites were predicted with the Signal P
program (www.cbs.dtu.dk/services/SignalP/).
2.5. RT-PCR analyses
PCR analyses were carried out on cDNAs from different
mouse tissues normalized with respect to the transcript
levels for six house-keeping enzymes (MTC panel, Clon-
tech). cDNA fragments were amplified with primer pairs
N00 and N99, N06 and N99, and N05 and N99. PCR reac-
tions were performed in 20 ml reaction volumes with the
Advantage polymerase mix (Clontech), 0.2 mg template
cDNA, and 100 ng of primers derived from two separate
exons. The polymerase mix included the TaqStart antibody
(Clontech) for automatic hot start PCR. Cycling conditions
were 948C for 20 s, 708C for 30 s, and 728C for 180 s for
cycles 1–3; the annealing temperature was reduced to 658C
in cycles 4–6 and to 608C thereafter. Aliquots (6.5 ml) were
removed after the 26th, 31st, and 36th cycles and analyzed
by
2.2. Southern and Northern blot analyses
Genomic DNAs were prepared as described (Koch-Nolte
et al., 1995) or purchased from The Jackson Laboratory (Bar
Harbor, ME), restriction digested and subjected to Southern
blot analysis. Northern blots were purchased from Clontech.
A 545 bp fragment of mouse Art5 was generated by PCR
electrophoresis
on
1%
agarose
gels
and
ethidium
bromide staining.
G. Glowacki et al. / Gene 275 (2001) 267–277
269
2.6. Expression of recombinant ART5 protein and enzyme
assays
fic polymorphism with those of other loci previously
mapped in the BSS interspecific cross clearly showed link-
age of Art5 to markers on medial chromosome 7 (Fig. 2),
just distal to where Art2a and Art2b had been previously
mapped using the same backcross panel (Prochazka et al.,
1991; Koch-Nolte et al., 1996a). Art5 showed the identical
strain distribution pattern as Art1 (0/94 recombinants vs. 1/
94 recombinants with the Art2a/Art2b tandem pair) (Koch-
Nolte et al., 1996a). The latter show no recombinants with
the guanyl cyclase 2d (Gucyd2d) locus currently listed at
offset 48 cM and Art5 shows no recombinants with the
cholecystokinin b receptor locus (Cckbr) at offset 49.6
cM (map positions were retrieved in June 1999 from the
tics.jax.org).
Mouse ART1 and ART5 were produced as His6x–FLAG-
tagged recombinant proteins in Sf9 insect cells as described
previously for ART2.1 and ART2.2 (Koch-Nolte et al.,
1996b). The coding region of mouse Art5 was amplified by
PCR with primers BF6, ACT CGT TCT AGA ATG ATT
CTG GAG GAT CTG CTG ATG and BR6, AAG AGG
AGA TCT GGG TCC AGC TCT GGA GAG CTG; that of
mouse Art1 was amplified with BF9, GTC ACC TCT AGA
ATG AAG ATT CCT GCT ATG ATG TCT and BR9L, GCA
GGA GGA TCC AAT GGA GCC TGG GGC TGA GCT AC.
Amplification products were digested with XbaI and BglII
and cloned into a derivative of pVL1393 expression vector
(Pharmingen) containing a C-terminal chimeric His6x- and
FLAG-tag. Sf9 and Hi5 cells were transfected with purified
plasmid (3 mg/3 £ 10
6
cells), BaculoGold DNA (0.5 mg)
(Pharmingen), and Cellfectin (GIBCO/BRL). Cell superna-
tants were harvested 4 days after transfection and used for
two amplification rounds of infection. Sepharose-immobi-
lized FLAG-tag specific monoclonal antibody M2 was
purchased from Sigma. Soluble or immobilized proteins
were incubated in 50 ml 20 mM Tris (pH 8.0), 1 mM ADP-
ribose, 5 mM DTT, 0.5 mCi
32
P-labeled NAD
1
(Amersham)
and 10 mM NAD
1
(Sigma) for 60 min at 378C. Where indi-
cated, reactions also contained 2 mM agmatine (Sigma).
Proteins were analyzed by SDS-PAGE and Western blot
analyses; supernatants were analyzed by thin layer chroma-
tography as described previously (Haag et al., 1995; Koch-
Nolte et al., 1996b; Braren et al., 1998).
3.2. Structure of the Art5 gene
We confirmed the suspicion that Art5 and Art1 are in
3. Results and discussion
3.1. Southern blot analyses show that Art5 is a single copy
gene in close linkage with the Art1 and Art2 genes on mouse
chromosome 7
Considering that a single Art1 gene and two tandemly
linked, functional Art2 genes have been observed in the
mouse (Prochazka et al., 1991; Braren et al., 1998), it was
of interest to determine whether the Art5 gene might also
have been duplicated. To this end we performed Southern
blot analyses of restriction digested genomic mouse and rat
DNAs with an Art5-specific probe (Fig. 1). A single band
was obtained with all of the enzymes tested except for those
with a cognate site in the probe used (e.g. SacI: Fig. 1, lanes
1 and 6). These results indicate that the Art5 gene is a single
copy gene in the mouse and in the rat.
With TaqI we observed a restriction fragment length
polymorphism in genomic DNAs from C57BL/6J and
Mus spretus (SPRET/Ei) (1.7 vs. 2 kb). We used this
RFLP to map the gene in the C57BL/6J £ SPRET/Ei
(BSS) interspecific backcross panel (Rowe et al., 1994).
Comparison of the haplotype distribution of the Art5-speci-
Fig. 1. Southern blot analyses of the Art5 gene. (A) Genomic DNAs were
digested with SacI (lanes 1 and 6), HindIII (lanes 2 and 7), BamHI (lanes 3
and 8), EcoRI (lanes 4 and 9) and TaqI (lanes 5 and 10) and subjected to
Southern blot analysis with radiolabeled Art5-specific cDNA probe
N11xN41 (the localization of this probe in exon 4 is illustrated in Fig. 3).
DNAs were from a C57BL/6J mouse (lanes 1–5) and from the rat cell line
C58NT (lanes 6–10). (B) Genomic DNAs were digested with HindIII (lanes
1, 4, and 7), PstI (lanes 2, 5, and 8), and BamHI (lanes 3, 6, and 9) and
subjected to Southern blot analysis as in (A). DNAs were from a farmhouse
mouse caught in Northern Germany (lanes 1–3), from a C57BL/6J mouse
(lanes 4–6), and from inbred Mus spretus (lanes 7–9).
270
G. Glowacki et al. / Gene 275 (2001) 267–277
close physical proximity by molecular cloning and sequen-
cing of the Art5 gene. Primers N00 and N31 were derived
from the mouse Art5 cDNA sequence (Okazaki et al., 1996;
Haag and Koch-Nolte, 1997) and were used to isolate three
clones containing the Art5 gene from a 129/SvJ mouse
genomic P1 library by PCR screening. Given the close link-
age of Art5 with Art1 and the two Art2 genes, we also
probed these P1 clones with specific probes for each of
the other genes. The results revealed that each of the three
P1 clones does indeed contain Art5 and Art1 but not Art2a
or Art2b. Three overlapping restriction fragments contain-
ing Art5 and Art1 were subcloned and sequenced (Fig. 3A).
The results showed that the two genes are arranged in a head
to head fashion. The distance between the two polyadenyla-
tion sites is approximately 15 kb. The finding that the P1
genomic DNA clones containing the Art1–Art5 gene cluster
do not overlap with P1 clones containing the Art2 gene pair
indicates that these respective Art gene pairs are separated
by at least 40 kb.
Comparison of cDNA and genomic DNA sequences
showed that the Art5 gene is composed of six exons
(Figs. 3B and 4). Fig. 3B shows a comparison of the
Art5 exon/intron structure with those of the mouse Art1,
rat ART2b, and chicken ART7 genes (Davis and Shall,
1995; Haag et al., 1996; Braren et al., 1998). A common
feature of these genes is the presence of a long exon encod-
ing the predicted catalytic domain. The genes differ in the
5
0
and 3
0
regions. While the ATG initiation codon of
chicken ART7 is contained in the first exon, the 5
0
UTRs
of the mammalian ART genes are split into at least three
exons. Similarly, the C-terminal ends of the mammalian
ARTs are encoded by distinct exons whereas the C-term-
inal end of chicken ART7 is encoded by the same exon as
the catalytic domain (Fig. 3B). We note further that Art1
and Art5 both contain a small 3
0
exon (encoding seven and
ten amino acids, respectively) which is lacking in rat ART2
and chicken ART7 (Fig. 3B).
The finding that the Art1 and Art5 genes overlap at their
5
0
ends (Fig. 3A) suggests that their expression may be
regulated by a common promoter and/or regulatory
element(s). Indeed, Art1 and Art5 genes are coexpressed
in some tissues, e.g. heart and skeletal muscle (see Fig. 7).
Furthermore, the differently sized Art5 transcripts in testis
(1.6 kb) vs. skeletal muscle (1.35 kb) could reflect use of
alternative promoters, as has been described for rat ART2
(Kuhlenb
¨
umer et al., 1997). The results of our 5
0
RACE
and RT-PCR analyses (see Fig. 8) support this: exon 1 of
Art5 is preferentially used in skeletal muscle, while Art5
transcripts in testis evidently derive from a transcription
start site at the beginning of a long version of exon 2.
Note that the testis-specific transcription start site of Art5
lies outside of the Art1 gene, while the muscle-specific tran-
scription start site overlaps with the 5
0
end of the Art1 gene
(Fig. 3A). Further studies will be required to define the
common
and
gene-specific
regulatory
elements
in
the
Fig. 2. Chromosomal mapping of the Art5 gene. Map figure (top) from The
Jackson BSS backcross panel showing part of chromosome 7 with loci
linked to Art5. The map is depicted with the centromere toward the top.
A 3 cM scale bar is shown to the right of the figure. Loci mapping to the
same position are listed in alphabetical order. Raw data from The Jackson
cmdata. The known positions of human orthologues are depicted on the
left (top). Haplotype analyses are shown on the bottom. Each column
represents the chromosome identified in the backcross progeny that was
inherited from the (C57BL/6J £ SPRET/Ei) F1 parent. Solid boxes indicate
the presence of a C57BL/6J allele; open boxes indicate the presence of a
SPRET/Ei allele. Loci are listed in order with the most proximal at the top.
The number of offspring inheriting each type of chromosome is listed at the
bottom of each column. The percent recombination between adjacent loci is
given to the right of the figure, with the standard error (SE) for each percent
recombination.
Art1/Art5 gene pair.
3.3. Features of the predicted Art5 gene product
Fig. 4 shows the nucleotide and deduced amino acid
sequences of the Art5 coding region and the primers used
for PCR and sequence analyses. The sequence shown devi-
ates from the cDNA sequence published by Okazaki et al.
(1996) by two frame shift mutations and a dinucleotide
mutation. We obtained the same sequence as the 129/SvJ
mouse genomic sequence shown in all available EST
sequences and in cDNA from BALB/c mouse. Most of
the coding sequence for the predicted secretory protein
G. Glowacki et al. / Gene 275 (2001) 267–277
271
Fig. 3. Structure of the Art5 gene. (A) Map of the closely linked Art5 (right) and Art1 (left) genes. Restriction enzyme sites are marked by vertical bars (S, SacI;
E, EcoRI). Exons are boxed and coding regions are shaded. Fragments obtained by subcloning P1 DNA into plasmid vectors are shown on top with their
corresponding fragment length indicated in kilobases (kb). The PCR fragment obtained with primers N11 and N41 that was used as a probe for the Southern and
Northern blot analyses shown in Figs. 1 and 7 is indicated below exon 4 of Art5. Note that Art1 contains an exon (exon 1*) in addition to those reported by
Braren et al. (1998) (database Accession number: X95825). This exon is contained within the cDNA sequence obtained by Okazaki et al. (1996) (database
Accession number: U31510, residues 193–245) from a lymphoma cell line, but was contained neither in 5
0
RACE nor in PCR products obtained from skeletal
muscle cDNA (G.G. and R.B., unpublished data). (B) Schematic diagram of the exon/intron structure of the mouse Art5 gene compared to those of the mouse
Art1, rat ART2b, and chicken ART7 genes. Exons are boxed, coding regions are shaded and introns are shown as triangles. The exact lengths of exons and the
approximate lengths of introns are given in basepairs.
is contained within a single 730 bp long exon (exon 4 in
Fig. 3). Three separate exons encode the 5
0
UTR (exons
1–3), the presumptive N-terminal leader peptide is encoded
by the last 60 nt of exon 3, and the 3
0
UTR is encoded by
a single exon (exon 6). The predicted cleavage site for
the signal peptide (A23/V24) is encoded at the 5
0
contain two potential N-glycosylation sites, albeit at differ-
ent positions: N102 and N197 in mART5; N65 and N248 in
mART1. Both proteins also carry the characteristic R-S-
EXE active site motif of arginine-specific mADPRTs
(R174, S184, and E220XE222 in ART5) (Domenighini
and Rappuoli, 1996; Koch-Nolte et al., 1996b).
Fig. 6 shows a comparison of the deduced mouse ART5
amino acid sequence with that of its proposed orthologue
from the human hART5 (our own unpublished observation),
the mouse and human ART1 and the chicken ART6A and
ART7 paralogues. Note that mouse ART5 shows significant
sequence identity to human ART5 even in the N- and C-
terminal regions. ART5 is slightly more similar to ART1
(37% identity) than to ART6A or ART6B (31 and 33%
identity, respectively).
end of
exon 4.
Fig. 5 shows hydropathy profiles of ART5 and ART1.
Like ART1, ART5 also has a hydrophobic N-terminal signal
peptide, but lacks the C-terminal hydrophobic GPI-anchor
signal. In addition to four cysteine residues which are
conserved among the ART family, both proteins contain
two extra cysteine residues near the C-terminus: C254 and
C270 in ART5; C280 and C284 in ART1 (the latter pair is
also found in chicken ART7, see Fig. 6). Both proteins
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