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

1

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

2

ABSTRACTTissue 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.

3

INTRODUCTIONTissue 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.

4

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 METHODSHuman 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

5

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-

6

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

RESULTSThe 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

7

transfected with the potent TG isoform as compared to the full length TGC and the PcDNA

controls, as shown in figure 4 .

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.

8

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

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

9

one day (24hours) and 98% after (2 days) 48 hours as compared to 31% and 52% in untreated

cells under the same time period.

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

10

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

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.

11

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.

12

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.

13