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Fig. 1.
Observation of TGase crosslinking activity and distribution on unfixed tissue section
using substrate peptide
(A) Unfixed tissue section was incubated with each substrate peptide labeled with fluorescein
and then the sections were washed and observed under a fluorescence microscope. (B) The
fibrosis model in liver or kidney was produced by BDL (Day 7) and UUO (Day 3) surgery,
respectively. The in situ activities of TG1 and TG2 were visualized using FITC-labeled
substrate peptides (pepK5 and pepT26, respectively). Bar = 50 μ m.
Fig. 2.
Detection of possible substrates incorporated with each substrate peptide in liver
extracts
(A) Tissue extracts were incubated with each biotinylated substrate peptide, and resultant
proteins that incorporated the substrate peptide were subjected to detection using peroxidaseconjugated streptavidin on the membrane blotted after SDS-PAGE. (B) The liver extract at each
indicated day time point were incubated with either pepK5 or pepT26, and then the amount of
Lys-donor substrates incorporated with each biotinylated peptide on the blotting membrane was
detected using peroxidase-conjugated streptavidin. The sizes of the protein mass markers are
shown on the left.
Fig. 3.
Schematic diagram for purification and identification of possible lysine- or glutamine-
donor substrates
Tissue extracts were incubated with each biotinylated substrate peptide (A; pepK5 and pepT26)
and pentylamine (B; BPA), following the same procedure as outlined above for the in vitro
detection of activity (Fig. 2). Following the crosslinking reaction by the endogenous TGase in
the tissue extracts, these samples were applied to monoavidin resin for purification of
crosslinking products. The samples eluted by excess biotin were precipitated with TCA/acetone,
and then subjected to trypsin digestion for the identification of possible substrates using
MALDI-TOF/TOF mass spectrometer after fractionation using a reverse-phase Nano-HPLC in
a C18 column.
Fig. 4.
A comparative proteomic analysis of possible substrates incorporating pepK5, pepT26,
and BPA in liver and kidney fibrosis
11
From data in the previous our reports [17,18], the number and rate of identified proteins
incorporating pepK5, pepT26, and BPA in each sample from liver fibrosis (A) and kidney
fibrosis (B) were described as Venn diagram. The overlapped number and rate of identified
proteins were also indicated. (C) Comparison analyses of overlapped proteins between liver
(BDL) and kidney fibrosis (UUO) in pepK5, pepT26, and BPA-incorporated substrates were
also indicated as Venn diagram. These each overlapped proteins are listed in Tables 2-4.
12
Table 1. Members of the mammalian transglutaminase (TGase) family, tissue distribution,
biological functions, and amino acid sequence of our developed substrate peptide
TGase
Tissue distribution
Biological function
Sequence of substrate peptide
FXIII
Plasma, brain, bone
Blood clotting, bone growth
DQMMLPWPAVAL (pepF11)
TG1
Epithelia
Barrier function in epithelia
YEQHKLPSSWPF (pepK5)
TG2
Ubiquitous
Cell death, survival signal, cell
HQSYVDPWMLDH (pepT26)
adhesion, fibrosis, and so forth
TG3
Epidermis, hair follicle
Terminal differentiation of
PPPYSFYQSRWV (pepE51)
keratinocytes, hair follicles
TG4
Prostate gland
Semen coagulation (rodents)
Yet to be developed
TG5
Foreskin keratinocytes,
Epidermal differentiation
Yet to be developed
Formation of epidermis and hear
DDWDAMDEQIWF (pepY25)
female reproductive tissues,
skeletal muscle
TG6
Epidermis, testis, brain
follicle, neuronal development
TG7
Testis, kidney
Unknown
YSLQLPVWNDWA (pepZ3S)
13
Table 2.
The list of overlapped proteins between liver and kidney fibrosis in pepK5-
incorporated substrates.
TG1 (pepK5)
Liver sample from
Kidney sample from
each day after BDL (days)
each day after UUO (days)
Accession #
Name
Q8K0E8
Fibrinogen beta chain
+
Q8VCM7
Fibrinogen gamma chain
+
P01942
Hemoglobin subunit alpha
Q921I1
Serotransferrin
P68373
Q9CWF2
Table 3.
14
14
+
+
+
+
+
Tubulin alpha-1C chain
+
+
Tubulin beta-2B chain
+
+
The list of overlapped proteins between liver and kidney fibrosis in pepT26-
incorporated substrates.
TG2 (pepT26)
Liver sample from
Kidney sample from
each day after BDL (days)
each day after UUO (days)
Accession #
Name
14
Q6ZWV3
60S ribosomal protein L10
+
+
+
Q99LB2
Dehydrogenase/reductase
SDR family member 4
+
+
P26040
Ezrin
+
+
Q8R1M2
Histone H2A.J
Q9D2U9
Histone H2B type 3-A
+
+
Q921I1
Serotransferrin
+
+
P99024
Tubulin beta-5 chain
+
+
+
14
14
Table 4.
The list of overlapped proteins between liver and kidney fibrosis in BPA-
incorporated substrates.
TGase (BPA)
Liver sample from
Kidney sample from
each day after BDL (days)
each day after UUO (days)
Accession #
Name
P97351
40S ribosomal protein S3a
+
Q61147
Ceruloplasmin
P01029
Complement C4-B
P11276
Fibronectin
Q8BG05
+
+
+
+
+
+
Heterogeneous nuclear
ribonucleoprotein A3
+
+
Q9Z2X1
Heterogeneous nuclear
ribonucleoprotein F
+
+
Q6GSS7
Histone H2A type 2-A
+
+
Q8CGP1
Histone H2B type 1-K
+
+
P84244
Histone H3.3
+
+
P08071
Lactotransferrin
+
+
P26041
Moesin
+
+
P11247
Myeloperoxidase
+
+
Q8VDD5
Myosin-9
+
+
P62960
Nuclease-sensitive elementbinding protein 1
+
+
Q62446
Peptidyl-prolyl cis-trans
isomerase FKBP3
+
+
P35700
Peroxiredoxin-1
+
+
Q61656
Probable ATP-dependent RNA
helicase DDX5
+
+
P31725
Protein S100-A9
+
+
Q921I1
Serotransferrin
+
+
P99024
Tubulin beta-5 chain
+
+
15
14
14
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