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The Effects of the 55 kDa Tissue Transglutaminase Cross-
Linking Active Isoform TG on Inducing Apoptosis
Senior Paper in Partial Fulfillment of Requirements for Graduation in Biology
Department of Biology, Chemistry and Environmental Health Sciences
Benedict College, Columbia SC
Fall 2014
By; Danstan .A. Mogire
Mentor: Dr. Bassam Fraij
Instructor: Dr. Rush Oliver

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TABLE OF CONTENTS
I. Title Page 1
II. Abstract 3
III. Introduction 4
IV. Materials and Methods 5
V. Results 7
VI. Discussion 10
VII. References 12

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ABSTRACT
Tissue transglutaminase ( T.G 2 or T.G.C) is a protein enzyme which is widely
distributed and is considered the most common of the tissue transglutaminase family of enzymes,
and it is credited with a variety of functions such as adhesion, migration, growth, survival,
apoptosis, differentiation, and extracellular matrix organization. In turn, the impact of TG2 on
these processes implicates this protein in various physiological responses and pathological states,
contributing to wound healing, inflammation, autoimmunity, neurodegeneration, vascular
remodeling, tumor growth and metastasis, and tissue fibrosis.( Nurminskaya MV and Belkin,
AM ,Cellular Functions of Tissue Transglutaminase Int Rev Cell Mol Biol. 2012 ; 294: 1–97.
doi:10.1016/B978-0-12-394305-7.00001-X).One other controversial function of T.G 2 include
apoptosis (cell death) whereby the cross-linking function of transglutaminase enzymes has been
shown to play a role in cell death(Fraij, Bassam M. "The 55 kDa tissue transglutaminase
cross‐linking active isoform TG induces cell death." Molecular carcinogenesis (2014).) The cells
used in this experiment were human cancer cells (MCF7 and T47D) and since this cells have
relatively low levels of TG2 inside them; they were transfected with the cross-linking 55KDa
active TG isoform or its precursor the 80kDa full length TGC. The increased frequency of
apoptosis correlated with the increase in transglutaminase expression and highest rate of
apoptosis were found in cells transfected with the TG isoform as compared to full length TGC
(Fraij. MB, The 55 kDa Tissue Transglutaminase Cross-Linking Active Isoform TG Induces Cell
Death). .This experiments tries to prove and clarify that tissue transglutaminase plays a
significant role in cell apoptosis by playing some role in the formation of the apoptotic bodies
since some traces of T.G.C could be detected after apoptosis.

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INTRODUCTION
Tissue transglutaminase is an 80 kDa protein polypeptide enzyme that is found to be
expressed ubiquitously across almost all the cells of the body at varying extents. The
multifunctional role of TGC both playing a role in various cell activities especially in the
pathology of a number of diseases. Its role in transamidation or deamination as shown in figure
1.1, GTpase/G-protein, ATPase, protein disulphide isomerase, and kinase activities have been
reported to either correlate with the induction of cell death or conversely the promotion of cell
survival. These two opposite functions can be explained by the presence of numerous tissue
transglutaminase or its isoforms in either case. Transamidations are calcium-dependent reactions;
Ca2+ concentration regulates transamidation activity, catalyzed by transglutaminase enzymes,
and contributes in cell death and it has been assumed that cross-linking function of
transglutaminase also plays a significant role in apoptosis
Figure 1.

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TG2 acting as transglutaminase catalyzes several types of posttranslational modifications of
proteins. (1) Protein cross-linking. TG2-mediated transamidation reactions proceed via
formation of an Nε (γ-glutamyl) lysine isopeptide bond between the acceptor Glycine residues
of the protein 1 and deprotonated Lys donor residue of the protein 2. TG2 displays specificities
toward both their Glycine and Lysine substrates. (2) Protein aminylation. TG2-mediated
Transamination reactions occur via incorporation of an amine (H2NR) into the Glycine residue
of the acceptor protein. Diamines and polyamines may act as a tether in a bis-glutaminyl
adduct between two protein molecules. (3) Deamination of proteins. TG2-mediated
hydrolysis reactions in the absence of amine co-substrates convert the Glycine residues of the
reactive protein into the Glutamate residues. Electron movements are shown by curved arrows.
The de novo formed covalent bonds are shown by curved lines.
This paper proposes that TG is the isoform of TGC that is responsible for triggering
apoptosis due to strong transamidation activity and reputing the previous assumption of TGC
being the apoptotic trigger.
MATERIALS AND METHODS
Human breast cancer cell lines (MCF7 and T47D) obtained from American Type Culture
Collection were grown in Dulbecco’s Modified Eagle’s Medium(DMEM ) a cell culture medium
containing amino acids, salts, glucose and vitamins which had been supplemented by fetal
bovine serum (10 % FBS) and contained 100 µg/ml streptomycin and 100 units/ml penicillin.
All-trans-retinoic-acid(ATRA )/tretinoin was then added 24 hours after the first plating from
a10Mm stock solution prepared in 100% ethanol to a final concentration of 1 μM. The cell

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culture was then covered by aluminum foil to protect the UV sensitive retinoic acid against light.
The control cells were treated with maitotoxin prepared in dimethyl sulfoxide (DMSO) to create
a final media concentration of 0.03% DMSO. The cells were grown in a six-well plate and grown
until they were 90-95% confluent. MCF7 and T47D cells which express low amounts of
transglutaminase, were transfected with eukaryotic expression vectors PcDNA, containing
inserts of the cross-linking 55 kDa active isoform TG or its precursor the 80 kDa full-length
TGC.The cells were the incubated overnight at 37 0C in a 5% CO2 incubator after which the
media was again changed to DMEM with 10% FBS or to DMEM without serum.
The control cells were treated with 0.03% DMSO while the transfected cells were then
changed into a serum free media induced with 1 μM calcium ionophore A23187 prepared in
DMSO to produce a final media concentration of 0.03% DMSO.the cells were then lysed in cell
lysis buffer (40mM Tris (pH 7.5) 150mM NaCl, 1 mM EDTA, 1Mm PMSF, and 1 µg/mL of
aprotinin and leupeptin). The MCF7 and T47D cells were then cultured in DMEM with 10%
FBS and incubated with 1 μM retinoic acid prepared in dimethyl sulfoxide (DMSO) or ethanol
for 72 hours for retinoic acid induction. The cells were then harvested and stained with
propidium iodide (PI) and Annexin V. RA was prepared in dimethyl sulfoxide (DMSO) or
ethanol.
Overexpression effects of transglutaminase isoforms were then investigated by
examining the MCF7 and T47D cells transfected with TG, TGC, and pcDNA3.1 plasmids. The
TG and TGC protein expression was examined by Western blot analysis shown in Figure 3. Cell
lysates (15 µg of protein) were subjected to a 5%(w/v)stacking gel and a 10%(w/v) separating
gel. The separated proteins were then electro blotted into immobilon-P membrane and blots were
probed with rabbit polyclonal antibodies to TG (1:6,000) followed by incubation with GAM-

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HRP (1:3,000 Bio-Rad Q6) Reactivity was visualized by the HRP color development . Trypan
blue was used to evaluate cell growth and viability used in conjunction with flow cytometry for
quantification of apoptosis. The Annexin V-FITC, PI Apoptosis Detection Kit 1 was used to
quantitatively identify apoptotic cells. (Fraij, Bassam M. "The 55 kDa tissue transglutaminase
cross‐linking active isoform TG induces cell death." Molecular carcinogenesis (2014)).
Figure 3
RESULTS
The results show evidence of a higher rate of cell death with the increase in
transglutaminase expression both in retinoic acid treated cells and in transient transfected cells.
We demonstrate for the first time that the highest rates of apoptosis were found in cells
transfected with the potent TG isoform as compared to the full length TGC and the PcDNA
controls, as shown in figure 4 .

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Figure 4;TG induced MCF7 and T47D cancer cells had a higher rate of apoptosis in transfected
human breast cancer cells as compared to the full length TGC and the PcDNA controls.
The Calcium ionophore (A231827), which is a transamidation trigger was found to be a cell
death promoter, whereas, cystamine, an active TG inhibitor, blocked the apoptosis effect of over-
expression of active TG. These results indicate that TG-dependent irreversible cross-linking of

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intracellular proteins, a function given previously to TGC, represents an important biochemical
event in the induction of the structural changes featured cells dying by apoptosis. See Figure 5
Figure 5
The results went on to assert that it is the TG isoform that triggers the process of apoptosis and
invalidates the previous misconception that TGC was the apoptosis ERT inducing factor. The TG
induced cells showed four times as much crosslinking activity compared to cells transfected with
TGC. TG cells transfected with the Calcium Ionophore A23187 led to a 70% cell death in just
one day (24hours) and 98% after (2 days) 48 hours as compared to 31% and 52% in untreated
cells under the same time period.

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DISCUSSION
T.G.C shares the same overall four-domain tertiary structure and several conserved
secondary structure elements with other mammalian TGs (Grenard et al., 2001; Liu et al., 2002;
Lorand and Graham, 2003; Nemeset al., 2005). Unlike closely related TG1, TG3, and Factor
XIIIA (FXIIIA) TGs, TGC does not require proteolysis for activation. In humans, it is encoded
by a single TGC gene located on chromosome 20q11–12.
Several distinctive features, including its ubiquitous and regulated expression, its
localization in multiple cellular compartments, and its multiple enzymatic and nonenzymatic
activities, underscore its enormous complexity and set this fascinating protein apart from other
TGs. Moreover, the intricate compartment-dependent regulation of its transamidating activity
and the non-covalent interactions unique for this TG profoundly impact multiple cell functions
and, therefore, are likely to contribute to a number of pathological states. Several lines of future
research will likely be central for the elucidation of pathophysiological functions of this protein.
Tissue transglutaminase is particularly notable for being the auto antigen in celiac
disease, a lifelong illness in which the consumption of dietary gluten causes a pathological
immune response resulting in the inflammation of the small intestine. If you have celiac disease,
you have an allergy to gluten, the protein found in wheat, rye, and barley. Celiac disease is an
autoimmune disorder, which means that your body attacks itself. This process damages the lining
of the small bowel, reducing its ability to absorb nutrients, The sensitivity to gluten can also
cause pain in the abdomen, anemia, fatigue, muscle and joint pain, gas, diarrhea, vomiting,
weight loss, and malnutrition.
T.G.C is also believed to be involved in several neurodegenerative disorders
including Alzheimer, Parkinson and Huntington diseases. Such neurological diseases are
characterized in part by the abnormal aggregation of proteins due to the increased activity of

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protein crosslinking in the affected brain. Additionally, specific proteins associated with these
disorders have been found to be in vivo and in vitro substrates of TGC. Although TGC is up
regulated in the areas of the brain affected by Huntington's disease, a recent study on mice
showed that increasing the levels of TGC does not affect the onset or progression of the disease
In conclusion, the hypothesis that was eventually proved by the results was that , it was
the active isoform TG that trigger apoptosis through the transamidation activity and that the
protein –polymer formation was as a result of the protein cross linking activity of TG and finally
that this protein-polymer formation was connected with increased rates of apoptosis.

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REFERENCES
1. Nurminskaya MV and Belkin, AM ,Cellular Functions of Tissue
Transglutaminase Int Rev Cell Mol Biol. 2012 ; 294: 1–97. doi:10.1016/B978-0-
12-394305-7.00001
2. Fraij, Bassam M. "The 55 kDa tissue transglutaminase cross‐linking active
isoform TG induces cell death." Molecular carcinogenesis (2014
3. Chen J, Kanopleva M, Multani A, Pathak S, Mehta K. (2004).Drug resistant
breast cancer MCF-7 cells are paradoxically sensitive to apoptosis. J Cell Physio
200: 223–234.
4. Chen JS, Mehta K. (1999). Tissue transglutaminase: an enzyme with a split
personality. Int J Biochem Cell Biol 31:817–836.
5. Mehta K, Fok J, Miller FR, Koul D, Sahin AA. (2004).Prognostic significance of
tissue transglutaminase expression in drug-resistant and metastatic breast cancer.
Clin CancerRes 10: 8068–8076.