From the Center for Biotechnology, the
§ Center for Structural Biology, Protein Analysis Unit,
Department of Biosciences at NOVUM, and the ¶ Department of
Medical Nutrition, Karolinska Institutet, Huddinge S-14157, Sweden, the
Department of Anatomy and Cell Biology, McGill University,
Montreal, Quebec H3A 2B2, Canada, the ** Department of
Anatomy and Cell Biology, Queen's University, Kingston, Ontario
K7L 3N6, Canada, and the
Department of
Biochemistry and Molecular Biology, University of Calgary,
Calgary, Alberta T2N 4N1, Canada
Received for publication, January 13, 2003, and in revised form, January 30, 2003
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ABSTRACT |
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We describe here the cloning and
characterization of a novel member of the thioredoxin family,
thioredoxin-like protein 2 (Txl-2). The Txl-2 open reading frame codes
for a protein of 330 amino acids consisting of two distinct domains: an
N-terminal domain typical of thioredoxins and a C-terminal domain
belonging to the nucleoside-diphosphate kinase family, separated
by a small interface domain. The Txl-2 gene spans ~28 kb, is
organized into 11 exons, and maps at locus 3q22.3-q23. A splicing
variant lacking exon 5 ( Thioredoxin (Trx)1 is a
small ubiquitous protein (12 kDa) that is conserved in all organisms
from lower prokaryotes to human and functions as a general
protein-disulfide reductase. The redox activity of thioredoxin
resides in the sequence of its conserved active site Cys-Gly-Pro-Cys
(CGPC), which undergoes reversible oxidation of the two cysteine
residues from a dithiol to a disulfide form (1). Thioredoxin is
maintained in its active reduced form by the flavoenzyme thioredoxin
reductase, a selenocysteine-containing protein that uses the reducing
power of NADPH (1). Several functions have been assigned to
thioredoxin, mostly dependent on its redox activity, including
regulation of transcription factor DNA binding activity, antioxidant
defense, modulation of apoptosis, and the immune response (2).
Moreover, abnormal thioredoxin expression has been correlated with a
number of pathological situations such as cancer and Alzheimer's and
Parkinson's diseases (3). The three-dimensional structure of
thioredoxin is conserved through evolution and consists of five central
stranded During recent years, the number of thioredoxin family members has
increased substantially. Based on protein sequence organization, two
distinct groups of thioredoxins can be distinguished. Group I includes
those proteins that exclusively encode a thioredoxin domain; Group II
is composed of fusion proteins of thioredoxin domains plus additional
domains. Among those belonging to Group I are Escherichia
coli Trx-1 (5), the three yeast thioredoxins (6, 7), and mammalian
Trx-1 and Trx-2 (8, 9). Examples of Group II thioredoxins are E. coli Trx-2 (10), Chlamydomonas DLC14 and DLC15 proteins
(11), mammalian Txl-1, and the spermatid-specific thioredoxins Sptrx-1
and Sptrx-2 (12-14). Until our discovery of Txl-2, Sptrx-2 was the
only mammalian member of the family where two different known protein
domains are present in the same polypeptide, as Sptrx-2 is a fusion
protein of an N-terminal thioredoxin domain followed by three
nucleoside-diphosphate (NDP) kinase domains. A similar domain structure
is also found in sea urchin axonemal protein IC1 (15).
NDP kinases (also known as nm23) constitute another well known family
of structurally and functionally conserved proteins identified across a
wide range of species from bacteria to human. NDP kinases catalyze the
transfer of We report here the characterization of a novel human fusion protein
composed of an N-terminal thioredoxin domain followed by an NDP kinase
domain, named Txl-2. Based on the considerations above, Txl-2 should be
classified as a member of the Group II of both thioredoxin and nm23
family of proteins. Txl-2 shares clear homology with Sptrx-2 with the
difference that the latter has three NDP kinase domains following the
thioredoxin domain. In accordance with nm23 nomenclature, Txl-2 should
also be denoted as nm23-H9.
cDNA Cloning of Human Txl-2 Gene--
The Basic Local
Alignment Search Tool (BLAST) (22) was used to perform a survey of
different data bases at the National Center for Biotechnology
Information (www.ncbi.nlm.nih.gov/) to identify new entries encoding
potential novel members of the thioredoxin family. Using the
thioredoxin domain of human Sptrx-2 as bait (14), we found the
expressed sequence tag entry AI341589 to encode a putative
thioredoxin-like sequence. Based on this sequence, the nested forward
primers F1 (5'-GGGCAGCAGGAAGAAGGATATTGC-3') and F2
(5'-CAGGTCAACATCAGCACACAACAGC-3') were used for 3'-RACE on a human
testis cDNA library (Clontech, Palo Alto, CA).
Based on the sequence obtained, the nested forward primers R1
(5'-CACCAGCCTAGATAGACATCAACAAC-3') and R2
(5'-CTCGATCCTCATCTTCTGGAAGAG-3') were used for 5'-RACE in the same
library. The resulting sequences were used to amplify by PCR the
full-length and Quantitative Real Time PCR--
Human cDNA multiple
tissue cDNA panels were purchased from Clontech,
oligonucleotide primers were purchased from Cybergene AB (Stockholm,
Sweden), and TaqMan probes and GAPDH kits were purchased from PE
Applied Biosystems (Warrington, UK). To detect total Txl-2 mRNA,
the forward primer was 5'-GGGCAGCAGGAAGAAGGAA-3' (nucleotides 30-48),
the reverse primer was 5'-TTAGTCCTTTGGAACTGAGCATTTC-3' (nucleotides
115-91), and the Txl-2 TaqMan probe was 5'-CCTGCAGGTCAACATCAGCACCCA-3' (nucleotides 54-77). To detect only full-length Txl-2 selectively, the
forward primer was 5'-TCGTCTTGATGTCCTCGAAAAGTAC-3' (nucleotides 234-258), the reverse primer was 5'-CCTCTAACCACAGCCACCAGTT-3' (nucleotides 323-302), and the Txl-2 long TaqMan probe was
5'-GAGCCAACCTTTCTGTTTTATGCAGGAGGAG-3' (nucleotides
189-212). All TaqMan probes were fluorescein labeled with the reporter
dye FAM and quencher dye TAMRA. All primer/probe sets were designed to
cross an exon-intron boundary to prevent detection of genomic DNA. 90 µl of Mastermix containing 400 nM primers, 400 nM TaqMan probe, and 1× TaqMan Universal Mastermix (PE
Applied Biosystems) was added to 1.8 µl of cDNA before aliquoting in triplicate to a 96-well microtiter plate. The cDNA was amplified using an ABI PRISM 7700 thermocycler (PE Applied Biosystems) under the
following conditions: 1 cycle at 50 °C for 2 min and 95 °C for 10 min followed by 40 cycles at 95 °C for 15 s and 60 °C for 1 min. The amounts of GAPDH and Txl-2 mRNA were calculated using the
standard curve method (separate tubes, following the instructions in
User Bulletin 2; PE Applied Biosystems). Fluorescence intensity was
measured during the PCR run. A graph was drawn with the threshold cycle
CT value versus the logarithm of the amount of
serially diluted control cDNA. Using this graph and the
CT value of GAPDH and transforming acidic coiled-coil
samples, the relative amount of TACC mRNA adjusted for GAPDH was
calculated. All real time PCR experiments were carried out in
triplicate and performed a minimum of three independent times with
similar results.
To determine the relative amounts of full-length and alternatively
spliced Expression and Purification of Human Txl-2--
The ORF encoding
human Txl-2 was cloned into the BamHI-EcoRI sites
of the pGEX-4T-1 expression vector (Amersham Biosciences) and used to
transform E. coli BL21(DE3). A single positive colony was
inoculated in 1 liter of LB medium plus ampicillin and grown at
37 °C until A600 = 0.5. The production of the
fusion protein was induced by the addition of 0.5 mM
isopropyl-1-thio- Mass Spectrometry Analysis--
Reduced Txl-2 was prepared by
incubation in the presence of 250 mM DTT on ice for 30 min,
and unreduced controls were treated under identical conditions but in
the absence of DTT. The samples were diluted 10 times with 0.1% (v/v)
trifluoroacetic acid and mixed with an equal volume of a saturated
solution of sinapinic acid in 33% (v/v) acetonitrile and 0.1% (v/v)
trifluoroacetic acid. An aliquot of 0.5 µl of this mixture was
crystallized on a microcrystalline layer that had been prepared with a
saturated solution of sinapinic acid (Fluka) in ethanol (23). The
spectra were acquired using a Reflex III mass spectrometer from Bruker (Germany) and calibrated using the high mass protein standard from
Aglient Technologies which contained 66,430.2-Da bovine serum albumin,
16,951.5-Da equine cardiac myoglobin, and 12,359.2-Da equine cardiac
cytochrome c. Data processing and evaluation were carried
out with the XMASS software from Bruker.
Antibody Production--
Purified GST-hTxl-2 was used to
immunize rabbits (Zeneca Research Biochemicals). After six
immunizations, serum from rabbits was purified by ammonium sulfate
precipitation. Affinity-purified antibodies were prepared using a
cyanogen bromide-activated Sepharose 4B column, onto which 0.5 mg of
recombinant Txl-2 fragment had been coupled using the procedure
recommended by the manufacturer (Amersham Biosciences). The specificity
of the antibodies was tested by Western blotting using recombinant and
in vitro translated human Txl-2 and Mouse/Rat Testis and Epididymis Sample Preparation,
Immunocytochemistry, and Electron Microscopy--
Adult male
Sprague-Dawley rats and CD mice were anesthetized, and testes and
epididymes were fixed by perfusion through the abdominal aorta and
heart, respectively, either with 0.5% glutaraldehyde and 4%
paraformaldehyde in 0.1 M phosphate buffer containing 50 mM lysine, pH 7.4, or with 4% paraformaldehyde (mice only)
or in Bouin's fixative (for light microscopy). Tissues destined for Lowicryl (SPI Supplies, West Chester, PA) embedding (for electron microscopy) were immersed in the respective fixatives for 2 h at
4 °C, washed three times in phosphate buffer, and incubated with
phosphate buffer containing 50 mM NH4Cl for
1 h at 4 °C. Tissues were subsequently washed in buffer,
dehydrated in graded methanol up to 90%, and infiltrated and embedded
in Lowicryl K4M. Thin sections were mounted on Formvar nickel-coated
grids for immunogold labeling. Bouin's fixed rat and human tissue were
washed extensively in 75% alcohol before being completely dehydrated in ethanol and embedded in paraffin. Paraformaldehyde-fixed tissue were
washed in phosphate buffer, dehydrated, and embedded in paraffin.
For light microscopic immunocytochemistry, 5-µm paraffin sections
were deparaffinized and hydrated through a graded series of ethanol
concentrations before immunoperoxidase localization with anti-Txl-2
antibody by standard procedures (24). For electron microscopic
immunocytochemistry ultrathin Lowicryl sections on Formvar
nickel-coated grids were immunogold labeled according to the procedure
of Oko et al. (25). Staging of the cycle of the seminiferous
epithelium and determining the steps of spermiogenesis were done
according to the classifications of Leblond and Clermont (26).
Mouse Lung Sample Preparation, Immunocytochemistry, and Electron
Microscopy--
Adult C57BL/6 mice were were killed by cervical
dislocation, the trachea was cannulated, and the cannula was tied
firmly in place. The anterior chest wall was removed and the lungs
dissected out. The lungs were infused via the tracheal cannula with 4%
paraformaldehyde, pH 7.4, at 20 cm H2O pressure and
maintained at this pressure for 5 min. The lungs were subsequently kept
in fixative overnight at 4 °C. After fixation the lungs were
dehydrated through a graded series of ethanol. Finally the lobes were
separated and placed into individual cassettes and embedded in
paraffin. The central portions of the blocks were sectioned at 5-µm
intervals, and the sections were mounted on glass slides,
deparaffinized, and hydrated before immunoperoxidase localization with
anti-Txl-2 antibody. To block unspecific binding of secondary
antibodies, sections were incubated in blocking solution (5% normal
goat serum). Primary antibody was added at a 1/20 dilution in blocking
solution, and in control experiments 0.03 mg/ml blocking peptide was
included. After several washes, the Vectastain-ABC kit (Vector
Laboratories, OH) was used for visualization. Sections were slightly
counterstained with Mayer's hematoxylin, dehydrated, and mounted.
Indirect immunogold labeling was used at the electron microscope level
to localize Txl-2 specifically. Small pieces (1 mm3) of
adult mouse lung were fixed in 4% paraformaldehyde in 0.1 M Sorensen's buffer, pH 7.4, for 4 h at 4 °C.
After washing in several changes of Sorensen's buffer at 4 °C, the
tissues were dehydrated in a graded series of methanol at progressively
lower temperatures to
Lowicryl thin sections were placed on Formvar nickel-coated grids and
incubated face-down on drops of blocking solution consisting of 1%
bovine serum albumin in 50 mM Tris, pH 7.6, with 100 mM sodium chloride. After 30 min, the grids were
transferred to drops of primary antibody diluted 1:10 in blocking
solution and left overnight in a humidity chamber at 4 °C. The
following day, the grids were washed face-down on wells containing 50 mM Tris, pH 7.6, with 100 mM sodium chloride
for 3 × 10 min. The grids were then incubated on blocking
solution for an additional 30 min prior to being transferred to drops
of secondary antibody (goat F(ab')2 anti-rabbit IgG
conjugated to 10 nm of colloidal gold; Ted Pella) diluted 1/30 in
blocking solution. After 1 h, the grids were washed as described
previously and then rinsed in distilled water. Immunolabeled sections
were counterstained with methanolic uranyl acetate followed by lead citrate.
Microtubule (MT) Binding Assay--
MTs were purified from rat
and mouse brain essentially as described previously (27), resulting in
MT preparations that contain MTs and MT-binding proteins including
mitogen-activated proteins and kinesins. In short, brain samples were
extracted in ice-cold BRB80 buffer containing protease inhibitors
pepstatin, leupeptin, and phenylmethylsulfonyl fluoride and centrifuged
at 55,000 rpm for 15 min at 4 °C. Supernatants containing
depolymerized MTs were removed and stored on ice. MT extracts were next
supplemented with 0.5 mM GTP, 15 units/ml hexokinase, and
20 mM D-glucose to deplete ATP. Next the extracts were
warmed to 30 °C and 5 µM paclitaxel was added.
After a 5-min incubation at 30 °C, 15 µM Taxol was added (20 µM total). 2 mM AMP-PNP was added
to stabilize kinesin heavy chains on MTs. The extracts were layered
onto sucrose cushions and spun at 40,000 rpm for 20 min at 22 °C.
The cushion was washed twice with BRB80 before being removed to avoid
contamination of the MT pellets. MT pellets were resuspended in 100 µl of BRB80 and depolymerized on ice for 20 min. 0.5 mM
GTP and 2 mM AMP-PNP were added to the MT preparations.
Extracts were warmed to 30 °C and polymerized and centrifuged as
above. MT pellets were subjected to another round of depolymerization
and polymerization as above. Final pellets of polymerized MTs were
resuspended in 10% glycerol and BRB80 and flash frozen in liquid nitrogen.
Pure polymerized MTs were made from purified tubulin (CytoSkeleton
Inc.) as follows: 10 µl of pure tubulin (10 mg/ml) was mixed in BRB80
buffer with 5 µl of glycerol and 3.3 µl of 0.15 mM
paclitaxel, and water was added to 50 µl. This reaction was incubated
at 33 °C for 15 min. The mix was loaded on a glycerol cushion of
equal volume and spun at 22,000 rpm for 30 min at room temperature. The
MT pellet was recovered and dissolved in 50 µl of BRB80 buffer and
10% glycerol.
To analyze binding of Txl-2 and cDNA Cloning, Sequence Analysis, Genomic Organization, and
Chromosomal Localization of the Human Txl-2 Gene--
By sequence
comparison with human Trx-1 (8), we found that GenBank expressed
sequence tag entry AI341589 (from pooled germ cell tumors) encoded a
putative novel human Trx-like sequence. Therefore, we designed specific
primers based on the AI341589 sequence and performed 5'- and 3'-RACE
PCR analysis using a human testis cDNA library to clone the
full-length cDNA sequence of this novel protein. The complete
sequence of the cDNA obtained consists of an ORF of 990 bp, a
5'-UTR of 27 bp, and a 3'-UTR of 432 bp upstream of the
poly(A)+ tail (Fig.
1A). In addition, the PCR also
rendered a smaller band, which corresponded to a putative splicing
variant (see below). Human Txl-2 ORF encodes a protein of 330 amino
acids with a calculated molecular mass of 36.9 kDa and a pI of 4.73. Analysis of the human Txl-2 sequence identified two distinct domains:
an N-terminal domain (comprising the first 105 residues) similar to
thioredoxins and a C-terminal domain composed of one NDP kinase domain
(Fig. 1B). The domains are separated by a small interface
domain. The Txl-2 protein domain organization resembles that of Sptrx-2
(14), with the exception that Sptrx-2 has three NDP kinase domains. Regarding the N-terminal thioredoxin domain, some of the structural amino acids that are conserved in previously characterized mammalian thioredoxins (including Sptrx-2) such as Asp-26, Trp-31, Pro-75, or
Gly-91 (numbers refer to those of human Trx-1) are also conserved in
Txl-2. However, other residues shown to be essential for catalysis, maintenance of three-dimensional structure, or protein-protein interactions are substituted, for instance Phe-11, Ala-29, Pro-40, Asp-58, or Lys-81 (4) (see Fig. 2B of Ref. 14).
Additionally, the Txl-2 C-terminal domain consists of one NDP kinase
domain. Following the nomenclature for NDP kinase protein family, Txl-2
should be considered the ninth member of this family and therefore
termed nm23-H9 (19). A protein alignment and phylogenetic analysis of
all human NDP kinase domains show that Txl-2 belongs to NDP kinase
Group II (Fig. 2, A and
B) (19). The protein alignment of the Txl-2 domain with all
of the previously identified thioredoxin proteins has been reported
elsewhere (14). The 3'-UTR of human Txl-2 mRNA contains a short
interspersed repetitive element of the Alu family spanning from
base 1156 to 1439 (Fig. 1A). This is not the first report of
the presence of such a repetition in a member of the NDP kinase family
as nm23-H6 mRNA also harbors an Alu sequence located in the 3'-UTR
of its mRNA (21). Interestingly, both proteins are testis-specific.
In x-ray crystallography and site-directed mutagenesis studies, nine
residues essential for catalysis and stability of nm23 proteins have
been identified (17, 19). Human Txl-2 has five of these nine conserved
residues regarded as crucial for enzymatic activity (Fig.
2A). Furthermore, the sequence of the active site (NAVH)
matches the consensus for this family of proteins (NXXH)
where X can be any residue (Fig. 1, A and
B).
A comparison of the protein sequence with PROSITE data base (28)
identified, along with the above mentioned thioredoxin and NDP kinase
domains, several potential phosphorylation sites and an
RCC1 (regulator of chromosome condensation-1) signature present between
residues 222 and 232 (Fig. 1A).
A homology search in the Human Genome Sequence Data Base
(www.ncbi.nlm.nih.gov/genome/guide/human/) identified the Txl-2 genomic region to be localized to chromosome 3q22.3-q23 (entry
NT_025664.5 Hs3_25820) (Fig. 3). Using
the Genomatix Software (www.genomatix.de/) we have determined that
the Txl-2 gene spans ~28 kb and is organized into 11 exons and 10 introns, all conforming to the GT/AG rule (see supplemental Table 1 in
on-line version of this work). Analysis of the splicing variant that
appeared during the cloning PCR indicated that it lacked exon 5.
Differential Expression Pattern of Txl-2 mRNA in Various Human
Tissues--
Initially, we used multiple tissue Northern blots to
determine the size and tissue distribution of human Txl-2 mRNAs
with either the ORF or the thioredoxin domain as probes. However, we were unable to detect any signal despite the use of low stringency conditions and extended time of exposure (data not shown). To improve
the sensitivity of our studies and to provide a means of
quantification, we therefore used real time PCR to determine Txl-2
mRNA levels in a variety of human adult tissues (Fig.
4A). Our results demonstrate
that total Txl-2 mRNA in adult tissues is very low.
However, highest levels are found in testis and lung, whereas lower levels are found in brain, thymus, spleen, prostate, kidney, and ovary. Txl-2 mRNA is virtually absent in colon, liver, and heart. We also determined the relative amounts of full-length Txl-2
versus alternatively spliced Expression and Enzymatic Activity of Human Txl-2
Protein--
Recombinant human Txl-2 migrated at 36 kDa in good
agreement with its theoretical size (Fig.
5A, inset). Members
of the NDP kinase family have been described as having an oligomeric
structure in their native conformations (19). To evaluate whether this was also the case for Txl-2, we performed gel filtration chromatography and found two peaks corresponding to the monomeric and the
dimeric conformation, indicating that in its native form, Txl-2 might be found in equilibrium between both forms (Fig. 5A).
Analysis of the fractions eluting from the column by SDS-PAGE ruled out the possibility of protein contamination or degradation as an explanation for the appearance of two peaks (data not shown). Next, we
used MALDI-TOF mass spectrometry to determine whether the dimeric
conformation is maintained by disulfide bonding. Fig. 5B
shows Txl-2 spectra in which two peaks can be identified, corresponding to the monomeric and dimeric forms. Incubation of the Txl-2 protein with DTT buffer resulted in a decrease of the dimeric peak, further demonstrating that disulfide bonds are responsible, at least in part,
for the dimeric conformation.
Enzymatic activity of thioredoxin is usually assayed by the capacity to
reduce the disulfide bonds of insulin using either DTT as artificial
reductant or NADPH and thioredoxin reductase as a more physiological
reducing system (1). We were unable to detect any enzymatic activity in
either enzymatic assay, using full-length Txl-2 or the
On the other hand, NDP kinases catalyze the transfer of a terminal
phosphate residue from NTPs to NDPs according to a ping-pong mechanism.
The first step of this reaction consists of the autophosphorylation of
the enzyme at a conserved histidine of the active site (29, 30). Txl-2
was unable to undergo autophosphorylation under the same experimental
conditions that allowed positive control yeast NDP kinase
autophosphorylation
(31).2
Cellular and Subcellular Localization of Txl-2 Protein--
As
determined by real time PCR, testis and lung were the tissues with the
highest amount of Txl-2 mRNA. We therefore selected these two
tissues to address the cellular and subcellular localization of Txl-2
protein. Thioredoxins and NDP kinases display a high degree of amino
acid identity between humans and rodents (www.ncbi.nlm.gov/LocusLink/). Because of sample availability and considering the identity between humans and rodents, we decided to perform immunohistochemical analysis
on mouse and rat samples. As shown in Fig.
6, Txl-2 labeling is readily detected in
the cilia of the mouse lung airway epithelium. Preincubation of the
antibodies with human recombinant Txl-2 abolished the signal.
Similarly, Fig. 7 demonstrates the
presence of Txl-2 in rat testis seminiferous tubules, where the protein
is associated with the spermatid tail and manchette, a microtubular
structure assumed to participate in the elongation of the spermatid
head and storage of proteins for later delivery to the developing tail (32). Again, antibody preadsorbed with the human recombinant protein
gave no labeling, thus confirming the specificity of the antibodies and
their cross-reactivity in rodent samples. Surprisingly, a strong and
specific labeling in the testis blood vessels was also obtained.
Identical results were obtained with mouse testis sections (data not
shown).
The finding of Txl-2 protein in such specific cellular structures
prompted us to investigate in more detail its subcellular localization
because the possibility existed that Txl-2 is somehow associated with
MTs. Surprisingly, by immunogold electron microscopy, we found Txl-2 in
association with MTs both in lung and
testis: the Txl-2 signal was over MTs
that make up lung cilia as well as over the MT-containing spermatid
manchette and axoneme (Figs. 8 and 9). In
addition, in testis blood vessels Txl-2 colocalized with fibrillar
components of smooth muscle tissue (Fig.
10).
MT Binding Activity of Human Txl-2--
The immunolocalization
data strongly suggested that Txl-2 might be able to bind MTs. This
would constitute the first example of such a characteristic for
thioredoxins. To prove this possibility, we performed in
vitro MT binding assays using 35S-labeled in
vitro translated proteins. First, translated Txl-2 and MTs are fibrous cytoskeletal components of the eukaryotic cell
cytoplasm which serve to perform a wide variety of functions, such as
cell motility and division, organelle transport, and morphogenesis. In
addition, MTs are the main components of the complex and highly organized axonemal structures found in cilia and flagella (33). There
is evidence that some members of the NDP kinase family associate with
microtubular structures (34-38). In contrast, only four proteins containing a thioredoxin domain have been reported to be associated to
microtubular structures and in all cases in lower eukaryotes: LC14 and
LC15 proteins from Chlamydomonas flagellum (11) and the
dynein intermediate filaments IC1 from sea urchin sperm axoneme and IC3
of the ascidian, Ciona intestinalis (15, 39). The last two
proteins are composed of an N-terminal thioredoxin domain followed by
three NDP kinase domains. Similar domain structure has been found in
Sptrx-2 (14), a human protein exclusively expressed in the spermatozoon
fibrous sheath, a structure that surrounds the axoneme and outer dense
fibers of sperm tail principal piece.3 However, Sptrx-2 is
not likely to interact with sperm tail MTs despite the clear domain
homology with the IC proteins because they are kept apart from each
other by the outer dense fibers.
We report here a novel protein (Txl-2 and The electron microscopic study suggested that Txl-2 might represent the
first MT-binding thioredoxin family member, which would have a
considerable impact on its potential functions. This possibility was
approached by in vitro MT binding assays. These experiments,
using pure MTs, established definitively that At this stage we do not know whether Txl-2, At present we can only speculate on the mechanism of binding of
Txl-2/ As Txl-2 is also expressed in lung cilia the potential interacting
proteins or substrates are not likely to be testis-specific but rather
common to the microtubular infrastructure. The fact that Txl-2 contains
thioredoxin and NDP kinase domains, both thought to be catalytic
domains, in addition to its low abundance suggests that Txl-2 is more
likely to act as a signaling/enzymatic protein rather than to have a
structural role. The acquisition of a thioredoxin domain in flagellar
proteins occurs early in evolution and is most likely a consequence of
molecular or enzymatic requirements for a specific function in
flagellum movement (11). In this context, it has been reported recently
that the two thioredoxin domain-containing proteins from
Chlamydomonas flagella, DLC14 and DLC15 (11), might
regulate, by a redox mechanism, the ATPase activity of the flagella
outer dynein arm (48). In addition, Chlamydomonas flagella
p72 and sea urchin sperm axoneme IC1 proteins have been reported to
have NDP kinase activity and suggested to be suppliers of the GTP for
MT assembly (49). Interestingly, an exhaustive proteomic analysis of
human ciliary proteins has found nm23-H5, nm23-H7, and Txl-2 as the
only members of the NDP kinase family expressed in human axonemal
extracts, adding further support to our data here reported on axonemal
localization of Txl-2 (50). Based on this, a model could be proposed in
which Txl-2 is responsible for GTP generation in cilia and flagella axoneme in these organisms. Furthermore, the presence of an RCC1 signature in Txl-2 suggests that it could interact with Ran and supply
the GTP required for its enzymatic activity. Thus, it may be
anticipated that disruption of a Ran-RCC1-Txl-2 pathway might lead to
an alteration in the normal development of spermatozoa and other
phenotypes associated with defective cilia physiology. Txl-2 expressed
in bacteria is inactive in both the thioredoxin assay and the
autophosphorylation assay. A similar situation was found for the
closest Txl-2 homolog, Sptrx-2, as well as other thioredoxins and NDP
kinases (12, 14, 20). Taken together, the lack of enzymatic activity of
bacterially produced Txl-2, despite its interaction with thioredoxin
reductase, indicates that post-translational modifications that might
expose the active site upon conformational changes as well as
interaction with other proteins or additional cofactors (cell- or
tissue-specific) might be required for Txl-2 to function.
Alternatively, because Txl-2 binds to MTs, the weak interaction
detected in vitro with thioredoxin reductase may become
physiologically significant in vivo.
The prominent expression of Txl-2 in microtubular structures in lung
and testis may make it a candidate gene for diseases such as primary
ciliary dyskinesia (PCD) , an autosomal recessive disorder (OMIM
242650) characterized by a failure of proper ciliary and flagellar
movement whose clinical manifestations are chronic respiratory
infections, male infertility, and situs inversus (51, 52).
The motility of cilia and flagella is generated in the axoneme, which
has been estimated to be composed of more than 250 polypeptides (53).
The axoneme consists of a core of nine peripheral + two central MT
doublets connected by outer and inner dynein arms (composed of heavy,
intermediate, and light chains) plus other accessory proteins. Electron
microscopy studies of the sperm of patients affected by PCD reveal
multiple phenotypes with anomalies in both MT and dynein arm
organization, indicating that this is a genetically highly
heterogeneous disease (51). To determine whether any breakpoint in the
region where the human Txl-2 gene maps has been reported to be
associated with any of the PCD phenotypes, we screened the Mendelian
Cytogenetics Network Data base (www.wjc.ku.dk/data bases) and found one
translocation (46,XY, t (3, 8) (q23;p23)) with an associated trait of azoospermia/oligospermia. Interestingly, two more breakpoints in the
Txl-2 region are associated with hydrocephalus, a phenotype that is
assumed to be a consequence of malfunctioning of the cilia of ependymal
cells that facilitate circulation of the cerebral spinal fluid
(54).
In summary, we have identified and characterized a new protein composed
of thioredoxin and NDP kinase domains which binds directly to MTs. Its
axonemal localization in sperm flagella and lung cilia indicates that
it is a component of the axonemal machinery taking part in regulation
of ciliary and flagellar movement and that Txl-2 is a potential
susceptibility candidate gene involved in the PCD phenotype.
5Txl-2) has also been isolated. By
quantitative real time PCR we demonstrate that Txl-2 mRNA is
ubiquitously expressed, with testis and lung having the highest levels
of expression. Unexpectedly, light and electron microscopy analyses
show that the protein is associated with microtubular structures such
as lung airway epithelium cilia and the manchette and axoneme of spermatids. Using in vitro translated proteins, we
demonstrate that full-length Txl-2 weakly associates with microtubules.
In contrast,
5Txl-2 specifically binds with very high affinity brain microtubule preparations containing microtubule-binding proteins. Importantly,
5Txl-2 also binds to pure microtubules, proving that it
possesses intrinsic microtubule binding capability. Taken together,
5Txl-2 is the first thioredoxin reported to bind microtubules and
might therefore be a novel regulator of microtubule physiology.
INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-sheets externally surrounded by four
-helices (4). The
active site is located in a protrusion of the protein between the
2-strand and the
2-helix (4). The conserved active site sequence
and the three-dimensional structure of thioredoxin are the hallmarks of
the thioredoxin family.
-phosphates between nucleosides and deoxynucleoside di-
and triphosphates, playing a pivotal role in maintaining a balanced
pool of nucleotides (16, 17). In addition to the kinase function, nm23
proteins have been implicated in cell growth, cancer progression, and
development (17, 18). Similar to thioredoxins, humans have several NDP
kinases (termed nm23-H1 to H8, of which nm23-H8 is also known as
Sptrx-2). NDP kinases can also be classified into two groups based on
sequence alignment and phylogenetic analysis (19). Group I is composed of nm23-H1 to H4 which all share a similar genomic organization consisting of a hexameric three-dimensional structure and the classical
enzymatic activity of NDP kinases. Group II encompasses nm23-H5 to H8
genes, which are defined by a more divergent sequence, including a
sequence of the active site which is not strictly conserved.
Furthermore, nm23-H5, H7, and H8 have been shown to have a
tissue-specific distribution mainly in testis/spermatozoa (14, 20,
21).
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
5Txl-2 cDNA of human Txl-2 from the same
library. The amplification products were cloned in the pGEM-Teasy vector (Promega) and sequenced in both directions.
5Txl-2 in samples, plasmids containing either form were
quantified and serially diluted to prepare standard curves for analysis
at the same time as sample tissue cDNA. As a secondary control,
samples were also prepared containing various mixtures of Txl-2
full-length and
5Txl-2 plasmids. Each sample was amplified using the
Txl-2 primer and probe sets to determine the quantity of either total
or full-length Txl-2.
-D-galactopyranoside, and growth was
continued for another 3.5 h. Overexpressing cells were harvested
by centrifugation and frozen until use. The cell pellet was resuspended
in 40 ml of 20 mM Tris-HCl, 1 mM EDTA, and 150 mM NaCl plus protease inhibitor mixture at the
concentration recommended by manufacturer (Sigma). Lysozyme was added
to a final concentration of 0.5 mg/ml with stirring for 30 min on ice.
1% sarkosyl was added, cells were disrupted by a 10-min sonication, and the supernatant was cleared by centrifugation at 15,000 × g for 30 min and loaded onto a glutathione-Sepharose 4B
column (Amersham Biosciences). Binding to the matrix was allowed to
occur for 2 h at room temperature. Thrombin (5 units/mg of fusion
protein) was used to remove glutathione S-transferase by
incubation overnight at 4 °C. The resulting protein preparation was
then subjected to ion exchange chromatography using a HiTrap Q column
(Amersham Biosciences), and human Txl-2 was eluted using a gradient of
NaCl. For gel filtration chromatography, the Txl-2 preparation from ion
exchange chromatography was applied to a Superdex G-75 preparation grade column (Amersham Biosciences) under nondenaturing conditions, preequilibrated with the same buffer as the protein preparation. Protein concentration was determined from the absorbance at 280 nm
using a molar extinction coefficient of 24,310 M
1 cm
1, calculated with the
Protean Program included in the DNASTAR Software Package (DNASTAR Inc.,
Madison, WI). An identical protocol was used to purify the recombinant
5Trx-2 variant, and the protein concentration was determined using
the same molar extinction coefficient as that of the full-length form.
5Txl-2.
Immunodetection was performed with horseradish peroxidase-conjugated
donkey anti-rabbit IgG diluted 1/5,000 following the ECL protocol
(Amersham Biosciences).
20 °C. The tissue pieces were then
infiltrated and embedded in Lowicryl K4M at
35 °C. Lowicryl blocks
were polymerized by ultraviolet illumination for 24 h at
35 °C and an additional 48 h at
10 °C.
5Txl-2 to MT, both forms were
transcribed and translated in vitro in the presence of
[35S]cysteine using the TNT reticulocyte transcription
and translation system (Promega). Radiolabeled proteins were incubated
with polymerized brain MTs or with pure MTs at 30 °C for 15 min in
the presence of 2 mM AMP-PNP, 0.5 mM GTP, and
20 µM paclitaxel. MTs were pelleted at 30,000 rpm for 15 min at 22 °C. Supernatants were saved for SDS-PAGE analysis. Two
subsequent pelleting reactions were performed. Aliquots of both
supernatants and final pellets were boiled in SDS sample buffer and
analyzed by electrophoresis on 10% SDS-polyacrylamide gels. The gels
dried and exposed to KODAK BIOMAX film. Intensity of bands was
quantitated by image analysis. In the indicated experiments MTs were
preincubated with anti-Txl-2 antibodies or with human recombinant
Txl-2.
RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Nucleotide/protein sequence analysis
and protein domain organization of human Txl-2. A,
nucleotide and amino acid sequence of human Txl-2 gene and protein.
Numbers on the left indicate amino acids, and on
the right they indicate nucleotides. Thioredoxin and NDP
kinase active sites are boxed. The RCC1 signature is
double underlined, and the potential polyadenylation signal
in the 3'-UTR is underlined. Exon 5 (absent in the 5Txl-2
variant) within the ORF and the short interspersed repetitive element
repeat within the 3'-UTR are shadowed. B, domain
organization of Txl-2 and
5Txl-2 proteins. Numbers
indicate the amino acids flanking the thioredoxin, interface, and NDP
kinase domains.
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Fig. 2.
Homology alignment and phylogenetic analysis
of human Txl-2 with all the members of the NDP kinase family of
proteins. A, primary sequence comparison of the NDP kinase
domains of the human nm23/NDP kinase proteins. The numbering
at the top refers to the nm23-H1 sequence;
numbers on the left indicate the amino acid
residue for each respective full-length protein. Identical (empty
boxes) and conserved (gray boxes, according to Ref. 55)
residues within all sequences are indicated. The asterisk
denotes residues involved in catalysis and stability (19). Note the
amino acid insertion in Txl-2 where the RCC1 signature (MCSGPSHLLIL) is
located. B, phylogenetic tree of human NDP kinases. The
scale indicates the number of substitution events/100 bases.
The percentage bootstrap values (based on 1,000 replications) are given
on the nodes of the tree.
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Fig. 3.
Chromosomal localization and genomic
organization of human Txl-2 gene. The human Txl-2 gene is located
between the markers D3S1576 and D3S3586 at 144.46-145.33 cM from the
top of the linkage group of human chromosome 3 (based on deCODE high
resolution recombination map of human genome; see Ref. 56). By
comparing this location with other genes in the region we have mapped
Txl-2 gene to 3q22.3-q23 flanked by the genes DKFZP434A043
and MRAS. The human Txl-2 gene is organized in 11 exons, and
exon 5 (black) is absent in the 5Txl-2 variant.
5Txl-2 in the three tissues with highest mRNA content: testis, lung, and brain. As shown in Fig. 4B,
5Txl-2 is the predominant mRNA form in all
three cases. In testis and lung the ratio of
5Txl-2 to Txl-2 is
~60:40, whereas in brain this ratio is clearly shifted to a much
higher relative amount of the spliced variant, 90:10.
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Fig. 4.
Relative expression of Txl-2 mRNA in
human tissues. Real time PCR was used to quantify Txl-2 mRNA
using the standard curve method. Values were normalized to GAPDH.
A, bars represent the mean amount of total Txl-2
mRNA for each tissue as determined in triplicate samples.
B, real time PCR was used to determine the relative amounts
of full-length and alternatively spliced 5Txl-2 in human testis,
lung, and brain. All experiments were performed in triplicate, and
similar results were obtained in at least three separate experiments.
The value for testis was set as 100.
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Fig. 5.
Biochemical characterization of
recombinant human Txl-2. A, gel filtration chromatography of
human Txl-2. Elution profile of human Txl-2 applied to a Superdex 75 column in a volume of 5 ml, at a final concentration of 2 mg/ml, and a
flow rate of 5 ml/cm2/h. The inset shows the
SDS-PAGE on the fractions eluted from the Superdex 75 column
demonstrating the integrity of Txl-2. B, MALDI-TOF analysis
of human Txl-2. The spectrum shows the single and double charged ions
as well as the dimeric form of Txl-2. A decrease in the dimeric form is
obtained upon incubation with DTT. C, dominant negative
activity of human Txl-2. The dominant negative effect of full-length
Txl-2 on Trx-1 enzymatic activity was carried out in the presence of
NADPH and thioredoxin reductase. The assay conditions were identical to
those described previously (13) except that increasing amounts of Txl-2
protein were added to the mix prior to initiating the reaction.
Lysozyme was used as a control at the highest molar ratio. The reaction
was initiated by adding 5 µl of calf thymus thioredoxin reductase (50 A412 units) and stopped after 20 min by the
addition of 6 M guanidine HCl and 1 mM
5,5'-dithiobis(nitrobenzoic acid). The experiment was repeated three
times.
5Txl-2
variant (data not shown). We also determined whether Txl-2 behaves as
dominant negative, competing with Trx-1 for binding to thioredoxin
reductase. As shown in Fig. 5C, increasing amounts of
full-length Txl-2 weakly, but consistently, compete with human Trx-1 in
the thioredoxin reductase enzymatic assay, thus suggesting that the
absence of enzymatic activity of Txl-2 is not the result of a lack of
interaction with the reductase.
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Fig. 6.
Immunohistochemical localization of
Txl-2 protein in lung. Paraffin sections of mouse lung were
immunoperoxidase stained with affinity-purified anti-Txl-2 antibodies
diluted 1/20. A, large intrapulmonary airway; B,
small intrapulmonary airway. Both locations exhibit clear
immunostaining specifically localized to the cilia of ciliated
epithelial cells (arrows). No staining is observed in
nonciliated epithelial cells (arrowheads). C,
immunoreactivity is reduced substantially when Txl-2-blocking peptide
is included with the primary antibody. Bar, 10 µm.
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Fig. 7.
Immunohistochemical localization of Txl-2
protein in testis. Paraffin sections of rat seminiferous tubules
were immunoperoxidase stained with affinity-purified anti-Txl-2
antibodies diluted 1/100. A, survey section showing obvious
immunostaining of the arterioles (Ar). B, no
immunoreactivity is detectable when anti-Txl-2 is preincubated with
human recombinant Txl-2. C, at higher magnification, during
the spermatid elongation phase (steps 8-14), immunostaining was
apparent at the periphery of the spermatid nucleus and beyond
(arrows), suggesting labeling of the microtubular manchette.
D, at this magnification it is clear that the smooth muscle
(Sm) wall of the arteriole is immunoreactive. E,
fainter immunostaining of the sperm tails (arrows) in the
lumen is also apparent. Bar, 10 µm.
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Fig. 8.
Immunogold electron microscopy localization
of Txl-2 protein in lung. A, electron micrographs of
sections through mouse lung ciliated epithelium immunogold labeled with
anti-Txl-2. Labeling is found in close association with the MTs of the
axoneme. B, mouse cilia labeled with anti-Txl-2 preincubated
with human recombinant Txl-2. No immunogold labeling of the MTs
of the axoneme is evident. Bar, 0.2 µm.
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Fig. 9.
Immunogold electron microscopy
localization of Txl-2 protein in spermatids. Shown are electron
micrographs of sections through spermatid heads and tails immunogold
labeled with anti-Txl-2. A, elongating spermatid in step 10 showing labeling of the microtubular manchette (M).
Abbreviations: A, acrosome; Nr, nuclear ring of
the manchette; N, nucleus. B, spermatid in step
12 showing labeling of the manchette and of the axoneme (Ax)
C, spermatid tails in step 15 showing labeling of the
axoneme. R and L, ribs and longitudinal columns
of the fibrous sheath; An, anlagen of the fibrous sheath,
ODF, outer dense fibers. Bar, 0.2 µm.
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Fig. 10.
Immunogold electron microscopy localization
of Txl-2 protein in testicular arterioles. The testicular
arteriole wall shows immunoreactivity in smooth muscle (Sm)
cell. In contrast, the endothelium (En) does not appear to
be immunogold labeled. CT, connective tissue; M,
mitochondria; Lu, lumen. Bar, 0.2 µm.
5Txl-2
proteins were analyzed for binding to brain MTs containing myelin basic
proteins. As shown in Fig.
11A, upper panel,
full-length Txl-2 binds only weakly to MTs in this assay as
demonstrated by the presence of recombinant protein in both the MT
pellet (p; lane a, upper panel) as
well as in supernatant 2 (s2; lane c, upper
panel), a consequence of release of Txl-2 from MTs during the
course of the experiment. Preincubation of the in vitro
translated Txl-2 either with specific antibodies or unlabeled
recombinant protein did not result in a significant decrease of the
binding (lanes d and g, upper panel,
respectively). The appearance of two bands when the Txl-2 construct is
translated in vitro is probably a consequence of the use of
an internal methionine as translational start in the in
vitro system (Fig. 1A). In agreement with the immunoelectron microscopy data, which suggested that
5Txl-2 protein can colocalize with MTs,
5Txl-2 protein is clearly present in the
pellet fraction and not in the supernatant 2, demonstrating that
5Txl-2 is stably bound to MTs in this assay (Fig. 11A,
lower panel). Moreover, the binding is specific because
preincubation of
5Txl-2 with unlabeled recombinant protein or
anti-Txl-2 antibodies decreases binding to 49 and 28%, respectively,
of the levels achieved with the
5Txl-2 protein alone (lanes
d and g, lower panel, respectively). Because
brain MT preparations contain, in addition to MTs, MT-binding proteins
such as mitogen-activated proteins and kinesins, these experiments
demonstrate that Txl-2 associates with MTs but cannot distinguish
direct from indirect MT binding. To distinguish these two mechanisms we
compared MT binding for both Txl-2 and
5Txl-2 using pure MTs and
brain MTs. The results are shown in Fig. 11B. The data show
that
5Txl-2 binds with high affinity to pure MTs (lane i)
with very little material in the second supernatant (lane h). There is no significant difference in
5Txl-2 MT binding
using pure or brain MTs. This proves that
5Txl-2 MT binding is
direct and establishes
5Txl-2 as the first genuine MT-binding
thioredoxin. As with brain MTs, full-length Txl-2 associates
significantly more weakly with pure or brain MTs (lanes c
and f, respectively).
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Fig. 11.
Txl-2 and 5Txl-2
bind MTs in vitro with differential affinity.
A, 35S-labeled Txl-2 and
5Txl-2 were
synthesized by in vitro translation and incubated with rat
brain MTs. Binding reactions were pelleted through a cushion, washed,
and this procedure was repeated. Resulting supernatant 1 (s1), supernatant 2 (s2) and MT pellets
(p) were analyzed for Txl-2 and
5Txl-2 protein by
SDS-PAGE and autoradiography. Upper panel, Txl-2 binding
experiments; and lower panel,
5Txl-2 binding experiments.
Lanes a-c, proteins were incubated with brain MTs
(MT). Lanes d-f, Txl-2 and
5Txl-2 proteins
were analyzed for MT binding after preincubation of MTs with
recombinant Txl-2 protein. Lanes g-i, Txl-2 and
5Txl-2
proteins were analyzed for MT binding after preincubation of MTs with
anti-Txl-2 antibodies. Binding of Txl-2 and
5Txl-2 proteins relative
to untreated samples (arbitrarily set at 100%) is indicated as
percents. B, to distinguish direct from indirect MT
binding, 35S-labeled Txl-2 (lanes a-f) and
5Txl-2 (lanes g-l) were synthesized by in
vitro translation and incubated with purified rat brain MTs
(brain MT) or with pure MTs prepared by polymerization of
pure tubulin (pure MT). Pelleted MTs were washed, and
supernatants and pellets were analyzed as described for
A. s1, supernatant 1; s2,
supernatant 2; p, pellet. Note the strong binding of
5Txl-2 to pure MTs.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
5Txl-2, a splicing variant
lacking exon 5) consisting of an N-terminal thioredoxin domain followed
by one NDP kinase domain in close similarity to Sptrx-2 (14). Indeed, a
phylogenetic analysis of the separate domains places both proteins in
the same tree branch. However, a comparison of the expression pattern
of both proteins shows important differences. Sptrx-2 is a
testis-specific protein (14), whereas Txl-2 is more ubiquitous and has
lower overall expression levels, although it is more abundant in testis
and lung. Unexpectedly, an immunohistochemical analysis of
Txl-2/
5Txl-2 expression in these two tissues shows the protein to be
in close association with microtubular structures: cilia of the lung
airway epithelium and the manchette and axoneme of the spermatids.
Furthermore, Txl-2/
5Txl-2 is found within the fibrillar components
of the testis blood vessel smooth muscle.
5Txl-2 could bind
directly to MTs, without the aid of MT-associated proteins such as
kinesins or mitogen-activated proteins. In contrast the full-length
Txl-2 protein has a much reduced affinity for MT.
5Txl-2, or both
associate with MTs in lung and spermatids because the polyclonal antibodies raised against full-length Txl-2 recognize both forms. In
light of the quantitative RT PCR data and the in vitro MT
binding experiments, it appears most likely, however, that
5Txl-2
rather than Txl-2 is the major MT-binding form. The difference in
affinity may be explained in two ways. First, the MT interacting area
in
5Txl-2 may be disrupted by the presence of exon 5 in the Txl-2 form, or exon 5 sequences cause a change in Txl-2 conformation affecting MT binding. Further in vitro analysis of
5Txl-2
deletion mutants can address this point. Second, cell- or
tissue-specific factors might be required to modulate the MT binding
activity of
5Txl-2, which are not present in the in vitro
MT binding assay. Analysis of brain may help to resolve this
possibility because in that tissue
5Txl-2 is by far the major form.
Be that as it may, our results demonstrate for the first time that
genuine MT-binding members of the thioredoxin family exist.
5Txl-2 to MTs and its role in MT physiology. Cysteine residues in
tubulin are actively involved in regulating ligand interactions and MT
formation both in vivo and in vitro (40). Because
thioredoxins are considered to be general protein disulfide reductases,
it is conceivable that the cysteine residues at the active site of
5Txl-2 interact with tubulin sulfhydryl groups, therefore playing a
critical role as a regulator of MT stability and maintenance. Moreover,
because
5Txl-2 has four additional structural cysteines it is
plausible that disulfide bonding involving any of these residues could
play a role in the binding mechanism. Studies are in progress to
analyze the kinetics of
5Txl-2 binding to tubulin.
5Txl-2 has an RCC1 signature in its C-terminal NDP kinase
domain, a motif that is not present in any other member of the nm23 or
thioredoxin family. RCC1 is a chromatin-bound guanine nucleotide
exchange factor for the small nuclear GTPase Ran, a Ras-related protein
(41). RCC1 has been implicated in nuclear cytoplasmic transport,
mitotic spindle nucleation, and nuclear membrane formation (42).
Interestingly, both a somatic and a testis-specific form of Ran have
been described in mammals (43, 44). The testis-specific variant of Ran
is found in the spermatid manchette, a location where RCC1 has also
been found (42, 44). The spermatid manchette is a transient
microtubular structure that develops during spermiogenesis and caudally
surrounds the spermatid nucleus (45). The manchette has been proposed
recently to be a transient storage location for both signaling and
structural proteins involved in nucleocytoplasmatic trafficking or
eventually sorted to the centrosome and the developing spermatid tail
(32, 46). This sorting mechanism has been named intramanchette
transport, in close reference to the so-called intraflagellar transport
(46, 47). Both intramanchette and intraflagellar transport involve molecular motors (primarily kinesins and dyneins) mobilizing a multicomplex protein raft to which cargo proteins or precursors for the
assembly of the axoneme of a flagellum or a cilium are bound and
transported to the tip of the axoneme (46). Thus, the potential
association of Txl-2/
5Txl-2 with Ran by its RCC1 motif may be an
indirect mechanism of binding to MTs.
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ACKNOWLEDGEMENTS |
---|
We thank Prof. Niels Tommerup for invaluable help with the Mendelian Cytogenetic Network data base and Dr. Elias S. J. Arnér for critical reading of the manuscript and helpful suggestions.
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FOOTNOTES |
---|
* This work was supported by Swedish Medical Research Council Grants 03P-14096-01A, 03X-14041-01A, and 13X-10370, the Åke Wibergs Stiftelse, and the Karolinska Institutet to (A. M.-V.), by the Fundación Margit y Folke Pehrzon (to A. J.), and by grants from the Canadian Institutes of Health Research (to F. A. v. d. H. and R. O.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The on-line version of this article (available at
http://www.jbc.org) contains Table 1.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AF196568.
§§ To whom correspondence should be addressed: Center for Biotechnology, Dept. of Biosciences at NOVUM, Karolinska Institutet, Halsovagen 7, Huddinge S-14157, Sweden. Tel.: 46-8-608-3338; Fax: 46-8-774-5538; E-mail: anmi@biosci.ki.se.
Published, JBC Papers in Press, February 4, 2003, DOI 10.1074/jbc.M300369200
2 A. Karlsson, personal communication.
3 A. Miranda-Vizuete, K. Tsang, Y. Yu, A. Jiménez, M. Pelto-Huikko, P. Sutovsky, and R. Oko, unpublished results.
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ABBREVIATIONS |
---|
The abbreviations used are:
Trx, thioredoxin;
AMP-PNP, adenosine 5'-(,
-imino)triphosphate;
DTT, dithiothreitol;
GAPDH, glyceraldehyde-3-phosphate dehydrogenase;
GST, glutathione
S-transferase;
MALDI-TOF, matrix-assisted laser desorption
ionization time-of-flight;
MT, microtubule;
NDP kinase, nucleoside-diphosphate kinase;
ORF, open reading frame;
PCD, primary
ciliary dyskinesia;
RACE, rapid amplification of cDNA ends;
RCC1, regulator of chromosome condensation-1;
Sptrx, spermatid-specific
thioredoxin;
Txl-2, thioredoxin-like protein 2;
5Txl-2, splicing
variant lacking exon 5;
UTR, untranslated region.
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