From the Department of Psychiatry and Behavioral Neurobiology, University of Alabama at Birmingham, Birmingham, Alabama 35294-0017
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ABSTRACT |
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Tissue transglutaminase is a calcium-dependent transamidating enzyme that has been postulated to play a role in the pathology of expanded CAG repeat disorders with polyglutamine expansions expressed within the affected proteins. Because intranuclear inclusions have recently been shown to be a common feature of many of these codon reiteration diseases, the nuclear localization and activity of tissue transglutaminase was examined. Subcellular fractionation of human neuroblastoma SH-SY5Y cells demonstrated that 93% of tissue transglutaminase is localized to the cytosol. Of the 7% found in the nucleus, 6% copurified with the chromatin-associated proteins, and the remaining 1% was in the nuclear matrix fraction. In situ transglutaminase activity was measured in the cytosolic and nuclear compartments of control cells, as well as cells treated with the calcium-mobilizing agent maitotoxin to increase endogenous tissue transglutaminase activity. These studies revealed that tissue transglutaminase was activated in the nucleus, a finding that was further supported by cytochemical analysis. Immunofluorescence studies revealed that nuclear proteins modified by transglutaminase exhibited a discrete punctate, as well as a diffuse staining pattern. Furthermore, different proteins were modified by transglutaminase in the nucleus compared with the cytosol. The results of these experiments clearly demonstrate localization of tissue transglutaminase in the nucleus that can be activated. These findings may have important implications in the formation of the insoluble nuclear inclusions, which are characteristic of codon reiteration diseases such as Huntington's disease and the spinocerebellar ataxias.
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INTRODUCTION |
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Numerous adult onset neurodegenerative diseases are caused by unstable, expanded CAG trinucleotide repeats within the coding region of an affected gene, which result in the synthesis of disease-specific proteins with expanded polyglutamine domains. Although there are numerous hypotheses concerning the contribution of these proteins with expanded glutamine repeats to the pathogenesis of the diseases, the mechanisms remain unknown (1-4). Recent studies have demonstrated that intranuclear inclusions are a common feature of most of these codon reiteration diseases and that the disease-related protein forms the inclusions (5, 6). However, the underlying mechanisms causing the formation of these inclusions are not clear.
Previously it had been hypothesized that tissue transglutaminase may be
involved in the pathological process of the CAG repeat diseases (7).
Tissue transglutaminase is both a signal transducing GTP-binding
protein (8) and a transamidating enzyme (9). As a member of the
transglutaminase family, tissue transglutaminase catalyzes a
calcium-dependent acyl transfer reaction between the -carboxamide group of a peptide bound glutamine residue and either an
-amino group of peptide bound lysine yielding a isopeptide bond
or the primary amino group of a polyamine resulting in a (
-glutamyl)-polyamine bond (9). Tissue transglutaminase is found
within neurons (10, 11) and has been implicated in a variety of
processes including apoptosis (12) and axonal growth and regeneration
(13, 14). Because the polypeptide bound glutamine is the primary
determining factor for a transglutaminase substrate, Green (7)
hypothesized that there may be a threshold effect and that the addition
of glutamine residues beyond a certain number may allow the mutant
protein to be modified by transglutaminase and result in the formation
of cross-linked products. Investigators have since demonstrated that
peptides containing glutamine repeats are substrates for tissue
transglutaminase (15) and that transglutaminase cross-links expanded
polyglutamine domains with glyceraldehyde-3-phosphate dehydrogenase
resulting in inactivation of the enzyme (16).
In an earlier investigation of GTP-binding proteins in rabbit liver nuclei, tissue transglutaminase was tentatively identified as a nuclear GTP-binding protein (17). However, in this previous study the in situ activity of the transglutaminase was not examined. Considering these and other findings, the purpose of this study was to determine the specific nuclear localization of tissue transglutaminase and the in situ activation of nuclear tissue transglutaminase.
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EXPERIMENTAL PROCEDURES |
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Cell Culture-- Human neuroblastoma SH-SY5Y cells were grown as described previously (18). To induce expression of tissue transglutaminase, cells were grown in the low serum medium containing 20 µM retinoic acid (18). All experiments were carried out 6-9 days after the addition of retinoic acid.
Subcellular Fractionation of SH-SY5Y Cells-- Cells were fractionated into cytosolic and nuclear fractions, and the nuclei were further fractionated into Triton X-100-soluble, nuclear chromatin and nuclear matrix compartments as described previously (19). Protein concentrations were determined using the BCA assay (Pierce).
Immunoblotting--
To evaluate the expression level of tissue
transglutaminase in cell fractions, samples were electrophoresed on 8%
SDS-polyacrylamide gels and transferred to nitrocellulose. The blots
were probed with monoclonal antibody 4C1, which recognizes tissue
transglutaminase (20); monoclonal antibody 5H1 (21), which recognizes
-tubulin; or monoclonal antibody MAB052 (Chemicon), which recognizes
histone proteins. After incubation with primary antibody, blots were
washed and probed with the appropriate horseradish
peroxidase-conjugated secondary antibody, washed again, and then
developed with the enhanced chemiluminescence (ECL) system (Amersham
Pharmacia Biotech).
In Situ Transglutaminase Activity Assay-- SH-SY5Y cells were labeled with 2 mM 5-(biotinamido)pentylamine (Pierce), a biotinylated polyamine, for 60 min prior to treatment with 1 nM maitotoxin for 15 min. The cells were subsequently harvested and fractionated into nuclear and cytosolic components (19), and the protein concentrations were determined. Transglutaminase activity was quantitated by measuring the presence of incorporated 5-(biotinamido)pentylamine into proteins by a microplate assay as described by Zhang et al. (18). To visualize the proteins into which the 5-(biotinamido)pentylamine had been incorporated, samples (10 µg of protein) were electrophoresed on 8% SDS-polyacrylamide gels, transferred to nitrocellulose, and probed with horseradish peroxidase-conjugated streptavidin (Pierce). The blots were developed as described above.
Immunocytochemistry-- SH-SY5Y cells were replated onto poly-D-lysine-coated coverslips. Cells were preincubated for 60 min with 2 mM 5-(biotinamido)pentylamine and subsequently treated with 0.5 nM maitotoxin for 20 min. Tissue transglutaminase was localized with the anti-tissue transglutaminase monoclonal antibody CUB 7402 (NeoMarkers) and Texas Red-conjugated goat anti-mouse IgG (Jackson ImmunoLabs), and transglutaminase activity was detected by FITC-streptavidin1 staining (18).
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RESULTS |
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Tissue Transglutaminase Co-purifies with the Chromatin and
Associated Proteins--
To determine the subcellular localization of
tissue transglutaminase, cells were separated into cytosolic and
nuclear fractions, and then the nuclear fraction was separated further
into the Triton X-100 fraction containing lipids and soluble proteins,
the chromatin and associated protein fraction, and the nuclear matrix
fraction. Equal amounts of protein (10 µg) from each fraction were
electrophoresed and immunoblotted with a monoclonal antibody to tissue
transglutaminase (Fig. 1). To confirm
that the isolated nuclei were free from cytoplasmic contamination, the
fractions were immunoblotted for -tubulin, a predominant cytosolic
protein. These results revealed that
-tubulin was found almost
exclusively in the cytosolic fraction (Fig. 1). The fractions were also
immunoblotted for histones to verify the extraction and isolation of
the nuclear fractions. As expected, histone immunoreactivity was
present primarily in the chromatin fraction. The presence of histone
immunoreactivity in the other fractions was virtually undetectable
(Fig. 1). Tissue transglutaminase immunoreactivity was found in the
cytosolic, chromatin, and nuclear matrix fractions (Fig. 1). Tissue
transglutaminase immunoreactivity in each fraction was determined
quantitatively and adjusted for the amount of protein in each fraction.
Results from two independent experiments revealed that 93% of total
tissue transglutaminase was found in the cytosol, 6% was extracted in
the chromatin fraction, and 1% co-purified with the nuclear
matrix.
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Nuclear Tissue Transglutaminase Is Active in Situ--
To
determine if tissue transglutaminase in the nucleus could be activated,
an in situ transglutaminase assay was used (18). The
biotinylated polyamine 5-(biotinamido)pentylamine was used as a probe
for endogenous tissue transglutaminase activity. Transglutaminases react with glutamine residues in substrate proteins, and then the
enzyme-substrate intermediate reacts with an appropriate nearby primary
amine. This can be the primary amino group of a polyamine resulting in
the covalent incorporation of the polyamine into the protein by a
(-glutamyl)polyamine bond (9). Therefore control and treated cells
that had been preincubated with 5-(biotinamido)pentylamine were
separated into cytosolic and nuclear fractions, and incorporation of
this polyamine derivative into proteins was used as a measure of
in situ transglutaminase activity (22). Fig.
2A shows that the basal
in situ activity of transglutaminase in the nucleus was less
than the activity in the cytosol; however, the difference was not
significant (p = 0.056, n = 5 separate
determinations). To activate tissue transglutaminase, cells were
incubated with 1 nM maitotoxin, which activates both
voltage-sensitive and ligand gated calcium channels (23). In SH-SY5Y
cells, 1 nM maitotoxin increases intracellular calcium
concentrations approximately 10-fold to 700 nM but does not
result in loss of viability during the time course of the experiment
(18). In addition, it has been well documented that increasing the
cytoplasmic calcium concentration results in an increase in the nuclear
calcium concentration (24, 25). Treatment with 1 nM
maitotoxin resulted in a significant increase in the in situ
transglutaminase activity in both the cytosolic and nuclear fractions
(Fig. 2A). To examine the transglutaminase-induced modifications of nuclear and cytosolic proteins, fractions were blotted
with horseradish peroxidase-conjugated streptavidin to identify
proteins modified with the biotinylated polyamine. A representative
blot of this data is shown in Fig. 2B. These findings show
that there are different proteins modified by transglutaminase in the
nucleus compared with the cytosol.
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Cytochemical Analysis of Nuclear Transglutaminase Activity-- To further assess the nuclear activity of tissue transglutaminase, a cytochemical approach was used. SH-SY5Y cells were incubated with 5-(biotinamido)pentylamine and then treated with 0.5 nM maitotoxin for 20 min. Control and maitotoxin-treated cells were fixed and immunostained with a monoclonal antibody to tissue transglutaminase and also were probed with FITC-conjugated streptavidin to localize polyamine-modified proteins as a measure of endogenous transglutaminase activity. Transglutaminase was present in control cells (Fig. 3, A and C), but transglutaminase activity was very low (Fig. 3, B and C). However, treatment with maitotoxin resulted in a significant increase in transglutaminase activity (as determined by the presence of proteins that had been polyaminated by transglutaminase and visualized with streptavidin-FITC) that was readily evident in the nucleus (Fig. 3, E and F). In addition, an increased presence of transglutaminase in the nucleus was often observed in the maitotoxin-treated cells (Fig. 3D), indicating that transglutaminase may translocate to the nucleus in response to elevated intracellular calcium levels. Tissue transglutaminase and proteins that had been modified by transglutaminase showed nuclear co-localization (Fig. 3F). Furthermore, proteins that had been modified by transglutaminase exhibited a discrete punctate as well as a diffuse staining pattern in the nucleus (Fig. 3E).
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DISCUSSION |
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One pathogenic process that has been proposed to contribute to the neurodegeneration in Huntington's disease, as well as other CAG trinucleotide repeat diseases, is the homodimerization or heterodimerization of the mutant polyglutamine-containing proteins with subsequent stabilization by transglutaminase resulting in the formation of poorly soluble protein aggregates (7, 15, 16). This hypothesis has attained additional support with the recent discovery of intranuclear inclusions, which are common to many codon reiteration diseases and are composed of aggregates of the disease-specific protein (5, 6). A previous study presented preliminary evidence that tissue transglutaminase was present in rabbit liver nuclei (17). However, this study focused on the GTP binding properties of tissue transglutaminase and did not examine the in situ transamidating activity of the enzyme. Considering the potential pathophysiological importance of nuclear tissue transglutaminase, it was of importance to verify the localization of tissue transglutaminase in the nucleus and to determine whether it had transamidating activity in situ. In this study, we clearly demonstrate that tissue transglutaminase localizes to specific nuclear fractions and can be activated in situ. Interestingly, when cells were treated with maitotoxin to increase the intracellular calcium concentration, a distinct punctate as well as diffuse pattern of transglutaminase-modified proteins were readily evident in the nucleus (Fig. 3E). The identity of the nuclear proteins that have been polyaminated by transglutaminase in the present study are unknown. However, it is intriguing that cotransfection of ataxin-1 containing 30 glutamines and leucine-rich acidic nuclear protein into COS-7 cells resulted in a nuclear distribution similar to that observed for nuclear transglutaminase substrates in this study (26).
The physiological function of nuclear tissue transglutaminase is not
known. It has been suggested previously that its G protein activity may
be important in the activation of nuclear phospholipase C (17).
Interestingly, phospholipase C- is found in the nucleus (25, 27),
and this is the isoform of phospholipase C, which is likely activated
by tissue transglutaminase in its capacity as a signal transducing G
protein (28). It has also been demonstrated recently that core histones
are excellent substrates of tissue transglutaminase, and the
modification of histones by either cross-linking or incorporation of
polyamines has been proposed to play both physiological and
pathological roles in nuclear function (29). In relation to codon
reiteration diseases, it was shown that expanded polyglutamine domains
tightly bind to glyceraldehyde-3-phosphate dehydrogenase; however, the
enzyme is only inactivated when it is cross-linked to the polyglutamine
domain by transglutaminase (16). These findings are extremely
intriguing given the recent findings of Sawa and co-workers (30). They
demonstrated that during the cell death process in several cell lines,
glyceraldehyde-3-phosphate dehydrogenase is translocated to the nucleus
and becomes tightly bound, resisting extraction by DNase or salt
treatment. Sawa et al. hypothesized that the inability of
glyceraldehyde-3-phosphate dehydrogenase to be extracted from the
nucleus could be due to covalent cross-linkage to another protein (30).
Because transglutaminase localizes to the nucleus and is often
up-regulated during apoptosis (31), it may involved in the nuclear
modification of glyceraldehyde-3-phosphate dehydrogenase during the
cell death process. In addition tissue transglutaminase is also in a
position to contribute to the formation of the intranuclear inclusions
in the codon reiteration diseases. Further research is required to
elucidate the putative roles(s) of nuclear transglutaminase in the cell
death process, as well as in the pathology of codon reiteration
diseases.
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ACKNOWLEDGEMENTS |
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We thank Jared Ordway, Dr. Richard Jope, and Dr. Peter Detloff for comments on the manuscript.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant AG12396.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.
To whom correspondence should be addressed: 1720 7th
Ave., South, SC1061, Dept. of Psychiatry and Behavioral Neurobiology,
University of Alabama at Birmingham, Birmingham, AL 35294-0017. Tel.:
205-934-2465; Fax: 205-934-3709; E-mail: gvwj{at}uab.edu.
1 The abbreviation used is: FITC, fluorescein isothiocyanate.
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REFERENCES |
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