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Presentation PPT The 55 kDa Tissue Transglutaminase Cross-Linking Active Isoform copy
1. The 55 kDa Tissue Transglutaminase
Cross-Linking Active Isoform TG
Induces Cell Death
Danstan Mogire
2. INTRODUCTION
• Tissue transglutaminase (T.G.C) is a protein enzyme with a variety of functions some of which
may include apoptosis (cell death). This experiments tries to prove 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.
• Tissue Transglutaminase is the most widely expressed member of the Transglutaminase family
and almost all cell types in the body express it to varying extents.
• Among its many supposed functions, it appears to play a role in wound healing, apoptosis,
and extracellular matrix development.
• The primary function of Tissue Transglutaminase is Transamidation
3. Transamidation
Deamination
Transamidation
Acyl Donors
Acyl
Acceptors
Reactions catalyzed by the transamidase activity of TGC
An isopeptide bond forms when the ε-
group of a peptide bound lysine is the
acyl-acceptor resulting crosslinking of
two proteins.
Water molecule as an acyl-acceptor to deaminate a
peptide-bound glutamine residue and convert it to a
glutamate residue.
A primary amine acts as acyl-acceptor, resulting to an isopeptide
bond which are resistant to disruptive physical or chemical
forces
The crosslink does not have to be an
isopeptide bond. A primary amine can act as
a crosslinker between two proteins.
Gundemir S, Colak G, Tucholski J, and Johnson G.V.W. (2012) Transglutaminase 2: A Molecular Swiss Army Knife Biochim Biophys Acta ; 1823(2): 406–419.
4. REGULATION
Activation of TGC has been shown
to be accompanied by large
conformational changes, switching
from a compact (inactive) to an
extended (active) conformation. As
shown in the figure on the left.
Compact/Inactive Extended /Active
The transamidating activity of TGC is regulated in cells by reversible conformational changes of
the protein. This which shifts TGC to the “open” (extended) conformation, thereby unmasking the
enzyme's active center, and inhibition by GTP, GDP, and ATP, which constrains it in the“closed”
(compact) conformation
It requires calcium as a cofactor for transamidation activity. Transcription is increased
by retinoic acid
5. Role In Disease
TGC is best known for its link with celiac disease. Anti-transglutaminase
antibodies (ATA) result in a form of gluten sensitivity.
TGC is also believed to be involved in several neurodegenerative disorders
including Alzheimer, Parkinson and Huntington diseases.
This conclusion was reached because TGC is the most prevalent neuronal
transglutaminase, and in the human brain increases in TGC expression have been
observed in a number of chronic or acute neuropathological conditions. Due to this up
regulation, it was hypothesized that TGC facilitates neuronal cell death in
neurodegenerative diseases.
TGC expression is elevated in multiple cancer cell types and is implicated in drug
resistance and metastasis due to its ability to promote mesenchymal transition and stem
cell like properties.
6. The Eight Isoforms of Transglutaminase
Name Gene Activity Chromosome
Factor XIII (fibrin-stabilizing
factor)
F13A1, F13B coagulation 6p25-p24
Keratinocyte
transglutaminase
TGM1 skin 14q11.2
Tissue transglutaminase TGC/TGM2 ubiquitous 20q11.2-q12
Epidermal
transglutaminase
TGM3 skin 20q12
Prostate transglutaminase TGM4 prostate 3p22-p21.33
TGM X TGM5 skin 15q15.2
TGM Y TGM6 unclear 20q11-15
TGM Z TGM7 testis, lung 15q15.2
7. Fig. 1 Genomic organization of the human TGC gene isolated from foreskin P1 genomic library shown in scale
(kb). Top: Genomic organization. Exons (13) are shown in solid boxes and labeled by Arabic numbers.
Introns(12) are represented by open boxes and indicated by Roman numerals.
Fraij BM and Gonzales R (1997) Organization and structure of the human tissue transglutaminase gene.
Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression, 1354: 65 - 71
Genomic Organization and Structure
8. • Difference between TG and TGC
Fraij BM (2014) The 55 kDa Tissue Transglutaminase Cross-Linking Active Isoform TG Induces Cell Death
WILEY PERIODICALS, INC. DOI 10.1002/mc.22134
9. EXPERIMENTAL METHOD
1. MDA cells were grown in 5% CO2 at 37 degree C, were grown in DMEM supplemented with 10%
FBS and Maitotoxine.
2. Grow the MDA cells in a flask using a media and applied Retinoic Acid through out keeping them in
incubator and changing the media within 2 to 3 days accordingly. Split the cells using Trypsin, centrifuge at
5 kps and collect them using a 5X Buffer in a tube..
10. EXPERIMENTAL METHODS: Column Chromatography
Gradient dependent column chromatography was performed using Ammonium
Bicarbonate or Tris as the mobile phase. Samples were collected manually.
• Protein separation
was obtained by
salt gradient.
Lower size
isoforms eluted
early at 0.1-0.2 M
NaCl and the Full
length TGC eluted
at 0.3-0.6 M NaCl.
• The NH4HCO3
showed a
significant
variation on the
salt gradient, and
was better than the
TRIS to isolate the
bands at ~55 KDa
11. EXPERIMENTAL METHODS: SDS/PAGE/ Western
Blot
7. After collecting the samples add 5X buffer, sonicate and run the Polyacrylamide gel
electrophoresis for 1 hr. and 15 min. Prepare the second step which is running the western blot.
After the bands are transferred on the membrane wash with TBS and add Primary antibody wash
for about 1 hr., wash with TBST (2X) and TBS (1X), and Secondary antibody, wash for about 2 hrs.
and develop using the color reagent to identify the bands.
12. EXPERIMENTAL METHODS
• We applied 50
ul of the sample
to identify the
bands.
• We identified
bands
approximately
around 55 KDa,
the red mark on
the figure above
shows the
position of the
isoforms of the
transglutaminas
e.
Protein expression was examined by Western
Blot
13. Results
The results on the left go on to 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
Apoptotic cells were quantitatively
identified with a hemocytometer
shown below
14. Conclusions and Discussion
• The results support our hypothesis, that the
cross- linking active TGC isoform is a potent
inducer of apoptosis.
• It helps to clarify the role of TGC isoforms in
transglutaminase activity.
• These results suggest that the 55 kDa TG
isoform may have significant roles in the
physiologic events of both healthy and diseased
tissues.
15. REFERENCES
• Gundemir S, Colak G, Tucholski J, and Johnson G.V.W. (2012) Transglutaminase 2: A Molecular Swiss Army Knife Biochim Biophys Acta ; 1823(2): 406–419.
Fraij BM and Gonzales R (1997) Organization and structure of the human tissue
transglutaminase gene. Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression,
1354: 65 - 71
Fraij BM (2014) The 55 kDa Tissue Transglutaminase Cross-Linking Active Isoform TG Induces Cell Death
WILEY PERIODICALS, INC. DOI 10.1002/mc.22134
