Thymosin b4 and Tissue Transglutaminase. MolecularCharacterization of Cyclic Thymosin b4

Christine App • Jana Knop • Thomas Huff •

Heinrich Sticht • Ewald Hannappel

Published online: 23 August 2013

� Springer Science+Business Media New York 2013

Abstract Thymosin b4 is the prototype of b-thymosins

and is present in almost every mammalian cell. It is

regarded to be the main intracellular G-actin sequestering

peptide. Thymosin b4 serves as a specific glutaminyl sub-

strate for guinea pig transglutaminase. In the absence of an

appropriate additional aminyl donor an e-amino group of

thymosin b4 serves also as an aminyl substrate and an

intramolecular bond is formed concomitantly NH3 (17 Da)

is lost. The molecular mass of the product is 4,949.6 Da.

This is 16.3 Da less than the molecular mass of thymosin

b4 (4,965.9 Da). Digestion with endopeptidases and Edman

degradation of the fragments identified the exact position

of the ring forming isopeptide bond. In spite of 3 glutam-

inyl and 9 lysyl residues of thymosin b4 only one isopep-

tide bond between Lys16 and Gln36 was formed (cyclic

thymosin b4). These two amino acid residues are conserved

in all b-thymosins. Cyclic thymosin b4 still forms a com-

plex with G-actin albeit the stability of the complex is

about one fiftieth of the stability of the thymosin b4 9 G-

actin complex.

Keywords Cyclic thymosin b4 � Isopeptide bond �Thymosin b4 � Transglutaminase

Abbreviations

WH2 WASP-homology 2

EDC 1-ethyl-3-(3-

dimethylaminopropyl)carbodiimide

RP-HPLC Reversed phase-high pressure liquid

chromatographie

MALDI-TOF–MS Matrix-assisted laser ionization-time

of flight-mass spectrometry

1 Introduction

The family of the b-thymosins is highly conserved and

b-thymosins are present in many tissues of vertebrates [25].

Originally b-thymosins were defined by their isoelectric

points between 5 and 7 [28]. b-thymosins are a ubiquitous

family of more than 15 related molecules with highly con-

served amino acid sequences [22]. Thymosin b4 is the most

abundant member of the family. It can be detected in cells of

many vertebrates except of erythrocytes [10, 15, 16]. It was

shown that thymosin b4 is involved in many intra- and

extracellular processes. Extracellular thymosin b4 promotes

angiogenesis [9, 31, 43] and acts anti-apoptotic [49] and anti-

microbiotic [19]. Thymosin b4 stimulates directional migra-

tion of cells in vitro and in vivo [2, 30, 31, 43, 51]. Application

of thymosin b4 shortly after a myocardial infarction in animals

improves cardiac function and reduces the infarct size [2, 18,

42, 45]. Several studies on the injured eye or with cell lines

generated from eye tissue have shown that thymosin b4 pro-

motes wound healing and eases inflammatory response [46–

48, 50]. In 1991 it was determined that the G-actin seques-

tering protein Fx and thymosin b4 are identical [39]. Thy-

mosin b4 is the main intracellular G-actin sequestering peptide

[39, 40]. To prevent polymerization of G-actin to F-actin

thymosin b4 forms a 1:1 complex with G-actin [39]. Kd-values

for the ATP-G-actin: thymosin b4-complex have been deter-

mined by equilibrium centrifugation assays or ultrafiltration

between 0.5 and 2.5 lM [21, 25, 38, 41, 56].

C. App (&) � J. Knop � T. Huff � H. Sticht � E. Hannappel

Institute of Biochemistry, Emil-Fischer-Zentrum, Friedrich-

Alexander-University, Fahrstr.17, 91054 Erlangen, Germany

e-mail: thymosin@biochem.uni-erlangen.de

123

Protein J (2013) 32:484–492

DOI 10.1007/s10930-013-9507-0

Transglutaminases catalyze a calcium-dependent acyl

transfer reaction between the c-carboxamide group of a

peptide-bound glutamine residue and the primary amino

group of either a peptide bound lysine or polyamine [13].

The resulting bond is covalent, stable and resistant to

proteolysis. Transglutaminases have a broad specificity for

primary amine substrates, whereas the number of proteins

which serve as glutaminyl substrates is highly restricted. It

has been shown that thymosin b4 can serve as a specific

glutaminyl substrate of guinea pig transglutaminase in vitro

[20]. Factor XIIIa -a plasma transglutaminase- incorporates

thymosin b4 in fibrin and fibrinogen attached to a plastic

surface [24, 32], important factors in blood coagulation.

Transglutaminase can be used to attach cadaverine, for

instance Oregon green cadaverine [20] to thymosin b4. It

was shown that this derivatization does not influence the

biological activity of thymosin b4 [20].

In the last years the central actin-binding motif of thy-

mosin b4 was found to be repeated in other proteins such as

actobindin [4, 17, 55], ciboulot [3] and tetraThymosinb[54]. This binding motif was detected in a growing number

of proteins generally involved in actin-based motility act-

ing as a G-actin-binding element. Because of its occurrence

in WASP-proteins this motif was called the WH2 (WASP-

homology 2) domain [37]. Proteins with several WH2

domains are acting differently compared to thymosin b4

which has only one WH2 domain. TetraThymosinb for

instance is able to sequester multiple actin monomers

instead of the 1:1 complex formed by thymosin b4 [54].

Interestingly, the function of proteins with several WH2

domains like ciboulot [3] and tetraThymosinb [54] has

switched from a G-actin sequestering as of thymosin b4 to

F-actin promoting assembly similar to profilin.

In this work we analyzed if it is possible to generate

dimers or multimers of thymosin b4 by transglutaminase-

reaction to imitate a polypeptide with several WH2

domains. However, we found that thymosin b4 is rather

forming a cyclic form instead of polymers. This peptide is

still able to sequester G-actin with low affinity.

2 Experimental Procedures

2.1 Proteins and Reagents

Reagents were obtained from the following sources: thymosin

b4 was isolated from bovine spleen as described [25]; Li-

Chroprep RP-18 (40–63 lm), acetonitrile and trifluoroacetic

acid (TFA, Uvasol) from Merck (Darmstadt, Germany);

Guinea pig liver transglutaminase and 1-ethyl-3-(3-dimeth-

ylaminopropyl)carbodiimide (EDC) from Sigma; AsnC

endopeptidase was purchased by TaKaRa Biochemicals;

AspN endopeptidase from Roche; Fluorescamine from

Serva.

2.2 Thymosin b4 and Transglutaminase

500 lg thymosin b4 were dissolved in 20 ll bidistilled

water. 210 ll reaction buffer (0.2 M Tris; 10 mM CaCl2;

1 M EDTA; 1 mM DTT; pH 6) were added to this solution.

To start the reaction 0.1 U guinea pig liver transgluta-

minase was added. The transglutaminase was dried in

vacuo for storage. The final concentration of thymosin b4

in the reaction was 0.4 mM. Aliquots were taken to mon-

itor the time course of the reaction. The reaction was

stopped by the addition of 5 ll 10 % trifluoroacetic acid to

denaturate the enzyme.

2.3 RP-HPLC

The separation system used is based on a conventional

HPLC gradient system (D-6000; Merck/Hitatchi). The

solvents contained 0.1 % trifluoroacetic acid in water or in

40 % acetonitrile. The separation of the peptides or their

generated fragments was performed by a reverse-phase

column (Beckman ODS Ultrasphere column (5 lm,

4.6 mm 9 250 mm)). The detection of these peptides was

done by fluram post-column derivatization [23]. FluramTM

(fluorescamine) reacts at alkaline pH with primary amines

and forms highly fluorescent derivatives. It’s necessary for

this post column derivatization process to raise the pH

within the system. For that reason borate buffer has to be

added to the eluate after separation. This addition raises the

pH value for optimal reaction conditions of the peptide or

its fragments with the added flurescamine. The addition of

borate and flurescamine to the separated peptides or frag-

ments is controlled by a derivatization pump (655A-13;

Merck/Hitatchi). This pump has three channels to deliver

borate (0.3 M, pH 9.3; on two channels) and flurescamine

(0.02 % in acetone; on the remaining channel) to the probe.

After this addition the mix passes through an reaction coil

to give about 12 s reaction time. Afterwards the formed

fluorescent derivatives were detected by a fluorometer

(fluorescent detector L2485; Merck/Hitatchi). The fluo-

rometer signal was recorded on a computer using D-7000

HSM software (Merck). The flow rate was 0.75 ml/min.

The gradient was linear from 0 to 40 % acetonitrile in

30 min.

For preparative HPLC peptides were detected at 205 nm

(UV-detector L4000; Merck/Hitatchi). Detection via post-

column derivatization is not suitable because the reaction is

irreversible. Fractions were collected every minute using a

fraction collector. The gradient was linear from 0 to 100 %

acetonitrile in 120 min.

Thymosin b4 and Tissue Transglutaminase 485

123

2.4 MALDI-TOF

Prior to analysis by MALDI-TOF–MS the samples were

concentrated in vacuo. The MALDI-TOF MS was carried

out as described [20]. The instrument used was a BiflexTM

III MALDI-TOF mass spectrometer (Bruker Daltonics).

The technical equipment of the apparatus are a nitrogen

laser (k = 337 nm) and a reflectron. The analysis of laser-

desorbed positive ions takes place after acceleration by

19 kV in the reflection mode. A standard peptide mixture

was used for external calibration. On an average 30 indi-

vidual spectra formed a mass spectrum.

To obtain a final sample concentration of about 20 ng/ll

dried peptide samples were dissolved in suitable amounts

of 0.1 % trifluoroacetic acid containing 33 % acetonitrile.

1 ll of each sample was mixed with 2 ll of a saturated

solution of a-cyano-4-hydroxycinnamic acid (Sigma) in

0.1 % trifluoroacetic acid in 33 % acetonitrile. Then again

1 ll of this mixture was spotted onto a stainless steel

target.

2.5 Digestion with Various Enzymes

Thymosin b4 (50 lg) or cyclic thymosin b4 (42 lg) in

50 ll 0.2 M pyridine buffer was digested with 0.1 lg

AspN endoproteinase at room temperature. Monitoring of

the reaction was done by reversed phase HPLC analyses.

To stop the reaction 5 ll 10 % trifluoracetic acid were

added and products were separated by preparative HPLC.

50 lg thymosin b4 or 42 lg cyclic thymosin b4 were

incubated with 20 lU AsnC-endoproteinase in 100 ll

reaction buffer (50 mM sodium acetate, pH 5.0, 0.2 mM

DTT, 0.2 mM EDTA) for 16 h at room temperature. The

reaction was stopped by adding 5 ll 10 % TFA and

products were separated by preparative HPLC.

2.6 Edman Degradation

Lyophilised probes were diluted in 60 ll (40 % mercap-

toethanol and 1 % formic acid in water). 15 ll of each

probe from digest fractionations were applied to the

pulsed-liquid sequencer Procise model 494 (applied

biosystems).

2.7 Amino Acid Analysis

The method was caried out as described [14]. Peptides

(200–500 pmol) were hydrolyzed in 6 M HCl at 155 �C for

45–60 min. After hydrolysis the samples were lyophilized

and diluted in 10 ll 1 M sodium borate buffer, pH 9.3.

Afterwards the amino acids were determined by HPLC with

a LiChrospher RP-18e column. In this case a precolumn

derivatization with o-phthalaldehyde/3-mercaptopropionic

acid was applied according to Graser [12]. The precolumn

derivatization was started by addition of 5 ll of o-phthal-

dialdehyde/3-mercaptopropionic acid (10 mg o-phthalal-

dehyde in 1 ml methanol and 10 ll 3-mercaptopropionic

acid). After a derivatization time of 2.5 min, the reaction

was terminated by addition of 100 ll of solvent A. To

ensure the same sample amount in every analysis the

sample was injected via a 20 ll loop into the HPLC system.

The flow rate was 1.5 ml/min. The gradient was linear from

solvent A (12.5 mM Na2HPO4, 12.5 mM NaH2PO4, 2 %

tetrahydrofuran, 3 % acetonitrile) to 100 % solvent B

(30 % acetonitrile in 8.75 mM Na2HPO4, 8.75 mM

NaH2PO4) in 12 min.

2.8 Determination of the Kd-Value

Actin was prepared from bovine heart by the method of

Pardee and Spudich [36]. G-actin was stored in G-buffer at

0 �C. The concentration of actin was determined by amino

acid analysis after acid hydrolysis (6 M HCl, 155 �C, 1 h)

and precolumn derivatization with o-phthaldialdehyde/

3-mercaptopropionic acid [14]. Equilibrium centrifugation

assays and calculation of Kd-values were determined as

described [25]. Thymosin b4 or cyclic thymosin b4 were

diluted to a concentration of about 66 lM and supplemented

with a defined amount of the internal standard Phe-Ala.

175 ll of this solution were mixed with 75 ll of a dextran

solution (50 mg/ml) and either 500 ll G-buffer or G-actin-

buffer solution. The reference proteins lactalbumin, tryp-

sinogen, ovalbumine and bovine serum albumine were

dissolved at 10 mg / ml in 109 G-buffer without mercap-

toethanol. 20 ll of this solution were mixed with 160 ll

H2O and 20 ll Dextran solution for centrifugation. One-

third of the 12 centrifuge tubes were loaded with 150 ll of

the peptide/buffer solutions. The next 4 tubes were filled

with 150 ll of the peptide/actin solutions. The remaining 4

tubes were used for the molecular mass standard proteins

(150 ll solution each). All tubes were incubated for 15 min

at room temperature. For equilibrium centrifugation a

Beckman 70.1Ti rotor with home-made adaptors for 175 ll

polyallomer tubes (size 5 mm 9 20 mm, Beckman) in a

Beckman L70 ultracentrifuge was used. A run was per-

formed at 35000g and 4 �C. After 16 h the top 50 ll of the

tubes were removed and peptide, fragments or protein

content was determined. For determination the removed

50 ll were mixed with 5 ll 4 M perchloric acid and incu-

bated for 30 min at 4 �C: After centrifugation (20,000g for

5 min) the supernatant solution was carefully removed and

the precipitate was used for protein determination. The pH

of the supernatant solution was adjusted to 3–5 with 10 M

KOH. The precipitated KClO4 was removed after a 15 min

incubation on ice. An aliquot of this solution was analyzed

using RP-HPLC.

486 C. App et al.

123

2.9 Protein Determination

To determine the protein concentrations a modified 2,20-bicinchoninic acid assay was used [44]. The precipitated

protein was dissolved at a concentration in the range of

20-60 ng/ll in a dilution buffer (2 % Na2CO3�H2O,

0.16 % disodium tartrate, 0.4 % NaOH, 0.25 % NaHCO3

adjusted to pH 11.25 with NaOH). Samples with the

reference proteins were only diluted and not precipitated.

50 ll of the solubilized protein were added to 400 ll of

bicinchoninic acid reagent. The samples were incubated

for 30 min at 60 �C. Afterwards the probes were mea-

sured at 562 nm. All samples were done at least in

duplicate.

2.10 Cross-Linking with EDC

To perform cross-linking with EDC, a solution of 36 g/l in

H2O was prepared. 5 ll of this solution were added to

50 ll G-buffer (2 mM HEPES; 0.2 mM ATP; 0.2 mM

CaCl2; pH 8.0) containing 15 lM actin and 15 lM thy-

mosin b4 or cyclic thymosin b4. After 90 min incubation

another 5 ll of the EDC solution were added.

2.11 Falling Ball Viscosimetry

This method was used to determine the sequestering

activities of the peptides [6]. G-actin (1.4 lM) was prein-

cubated for 15 min with an equimolar amount of thymosin

b4 or cyclic thymosin b4 or a tenfold molar excess of cyclic

thymosin b4 in G-buffer at room temperature. To start the

polymerization MgCl2 (final concentration 4 mM) was

added to the solution. 50 ll of the solution were transferred

into a glass capillary (Ø 0.92 mm) and incubated for 4 h at

room temperature. As a measure for the viscosity served

the time a steel ball (Ø 0.794 mm) needed to cross 45 mm

of the filled capillary at an angle of 60�.

2.12 Modelling of Cyclic Thymosin b4

The conformation of the C-terminal half of thymosin b4

was retrieved from a complex crystal structure of G-actin

with a hybrid protein comprising gelsolin domain 1 and

residues 22–40 of thymosin b4 (PDB cide 1T44;[26]). After

truncation of the gelsolin moiety, the N-terminal residues

2–21 of thymosin b4 were added, and a helical conforma-

tion was modeled for residues 4–16 according to the NMR

spectroscopic data from Zabock et al. [58]. After genera-

tion of an isopeptide bond between the Lys16 and Gln36

sidechains, the structure was energy minimized showing

that this bond can be formed without any steric problems.

Modeling and energy minimization were performed using

Sybyl (Tripos Inc.).

3 Results

3.1 Generation of Cyclic Thymosin b4

It is known that thymosin b4 can serve as an aminyl sub-

strate [41] as well as a glutaminyl substrate [20] in reac-

tions catalyzed by transglutaminase. In the reaction

described here thymosin b4 lacks another substrate to react

with. 500 lg thymosin b4 were lyophilized and diluted in

reaction buffer. The reaction was started by the addition of

0.1 U transglutaminase and monitored by reverse phase

HPLC (Fig. 1). RP-HPLC data shows a decrease of thy-

mosin b4 and the simultaneous formation of a product at a

higher retention time. By adding 10 % trifluoric acid the

reaction was stopped. The formed product was isolated by

RP-HPLC using the preparative conditions described

above. The molecular mass of the isolated product was

determined by MALDI-TOF-MS (4,949.6 Da). It was

16.3 Da less than the molecular mass of thymosin b4

(4,965.9 Da) (Fig. 2). This corresponds to the loss of

ammonia and points to the formation of a cyclic form of

thymosin b4. No poly-thymosin b4 were detected in mass

spectrometry. Surprisingly, it was not possible to generate

a poly-thymosin b4 using tissue transglutaminase.

3.2 Molecular Characterization of Cyclic Thymosin b4

To elucidate if the newly formed product is a cyclic form

of thymosin b4 digestions of the isolated product with

various enzymes were performed. The endopeptidase AsnC

cleaves proteins C-terminal of asparagine. Thymosin b4

contains only one asparagine residue, thus thymosin b4

contains only one cleavage site, directly after Asn26. Two

fragments were generated by a cleavage of thymosin b4

with AsnC (3,109.7 and 1,871.7 Da; Fig. 3a). Whereas

Fig. 1 RP-HPLC analysis of the reaction of thymosin b4 catalyzed by

transglutaminase. 1 ll was taken from the reaction mix after

incubation for 0 and 3 h

Thymosin b4 and Tissue Transglutaminase 487

123

cleavage of the reaction product only generated one frag-

ment (4,964.6 Da) indicating that the two fragments were

still connected.

The other endopeptidase used was AspN. This enzyme

specifically cleaves N-terminally of aspartatic acid and

occasionally of glutamic acid. This digestion of thymosin

b4 is expected to generate three major fragments. The first

fragment runs from the N-terminus to Pro4. The second

fragment starts at Asp5 and ends at Phe12. These two

fragments are lost in the analysis because of their small

sizes. The third fragment starts at Asp13 and contains the

rest of the peptide. Within this fragment there is a Glu at

position 23 which can serve as an additional unusual

cleavage site. Cleavage of thymosin b4 with AspN pro-

duced one main product shown in Fig. 3 (3,531.4 Da). This

fragment runs from position 13 to 43. But in the case of the

cleavage of the reaction product two fragments with

identical amino acid composition were detected (data not

shown). The molecular mass of the two fragments (3,514.2

and 3,532.1 Da) generated from the reaction product dif-

fered by 18.1 Da indicating the cleavage of the peptide

bond between Gln23 and Glu24. After isolation of these

fragments of the reaction product Edman sequencing was

performed. It revealed undetectable amino acid residues at

the positions 16 and 36 (Fig. 3b). This indicates the for-

mation of an isopeptide bond between Lys16 and Gln36 of

thymosin b4. This confirms the observation that the intra-

molecular ring forming reaction is energetic more favor-

able than the intermolecular reaction leading to multimers

of thymosin b4.

It is known that thymosin b4 can serve as a substrate for

reactions catalyzed by factor XIIIa [24, 29], which plays an

important role in blood coagulation. We tried unsuccess-

fully to verify the existence of the cyclic peptide in vivo.

3.3 Interaction of Cyclic Thymosin b4 with G-Actin

After the identification of the isopeptide bond forming

residues it was tested if the cyclic peptide is still able to

sequester G-actin. The cyclic form of thymosin b4 was

Fig. 2 Mass spectrum of thymosin b4 and cyclic thymosin b4

determined by MALDI-TOF analysis. For the interaction experiments

the cyclic thymosin b4 was separated from thymosin b4 by RP-HPLC

and the purity of the eluate was verified by mass spectrometry

Fig. 3 a AsnC does specifically hydrolyze peptide bonds on the

carboxyl side of asparagine residues. Cleavage of thymosin b4 leads

to two fragments while in the cyclic form the two fragments are still

connected by an isopeptide bond. b AspN does perferentially

hydrolyze peptide bonds on the aminyl side of aspartate and

glutamate (rare). The amino acid sequence was determined by Edman

degradation and is depicted above the corresponding fragment.

Amino acid residues connected to another amino acid residue via an

isopeptide bond are missing in Edman sequencing and are shown as –

488 C. App et al.

123

isolated by RP-HPLC and the purity of the peptide was

determined by MALDI-TOF analysis before the following

experiments. First we studied the polymerization-inhibiting

capacity of the cyclic form of thymosin b4 (Table 1). For

polymerized actin the time of fall was 89.8 ± 8 s

(mean ± standard deviation, n = 5). The value decreased

to 10 ± 0.8 s (n = 6) in the presence of an equimolar

amount of thymosin b4. This equaled the value of the

buffer solution and corresponds to complete inhibition of

polymerization. For equal amounts of cyclic thymosin b4

and G-actin the values observed were 56.2 ± 5 s (n = 6).

The measurement at a two fold molar excess of cyclic

thymosin b4 over G-actin showed no significant difference

(56.6 ± 6.4 s, n = 3). Cyclic thymosin b4 can inhibit the

polymerization of actin but only at a tenfold molar excess

over G-actin (7.2 ± 0.8 s, n = 3).

For further analysis of this interaction we cross-linked

the samples with the zero-length cross-linker EDC. After

cross-linking we determined the cross-linking with a SDS-

PAGE-analysis using Pharmacia PhastGel Gradient 10–15.

The gel was stained Coomassie blue (Fig. 4). The staining

indicated that cyclic thymosin b4 can be cross-linked to

actin but with a very low efficiency. In western blot anal-

ysis we could show that the cyclic form of thymosin b4 is

still recognized by an antibody against thymosin b4 (data

not shown).

Determination of the Kd-value by equilibrium centri-

fugation showed a lower affinity to G-actin in the case of

the cyclic peptide (50.7 lM) in comparison to thymosin

b4 (0.7 lM). The obtained results are depicted in

Table 2.

4 Discussion

In this study we demonstrated that thymosin b4 when

incubated with transglutaminase forms an intramolecular

isopeptide bond. Using MALDI-TOF, amino acid analysis,

proteolytic fragmentation and Edman digestion we identi-

fied that Lys16 and Gln36 of thymosin b4 are involved in

the formation of the isopeptide bond. This cyclic variant of

thymosin b4 is still able to form a complex with G-actin.

This complex formation was shown in three independent

experiments. The cyclic form inhibits the salt-induced

polymerization of G-actin though it needed a tenfold molar

excess over G-actin to do so. The stability of the complex

of cyclic thymosin b4 formed with G-actin is only a fiftieth

of the stability of the complex formed with linear thymosin

b4. It was possible to cross-link cyclic thymosin b4 to

G-actin at a tenfold molar excess. The cross-linking reac-

tion with EDC has very stringent requirements [1] so it is a

reasonable method to proof the interaction between two

proteins.

It has been shown that extracellular thymosin b4 is

involved in angiogenesis [9, 31, 43], apoptosis [49], wound

healing [46–48, 50] and blood coagulation [16, 40, 59]. In

these processes, transglutaminases play important roles.

We demonstrated here that it is possible for transgluta-

minase to form a cyclic form of thymosin b4 in the absence

of other reaction partners. Unfortunately, we failed to

detect cyclic thymosin b4 in vivo. Therefore, we analyzed

saliva, wound fluid and a protein extract derived out of rat

liver using RP-HPLC and mass spectrometry experiments

with a high resolution. Thus the missing cyclic thymosin b4

in vivo might just be due to our experimental set up or to

other competing reactants for the transglutaminase reaction

in investigated cells. However, it is very challenging to

distinguish between thymosin b4 and its cyclic form using

Table 1 Falling-ball viscosimetry of G-actin with or without thymosin b4 or cyclic thymosin b4 (±SD, n = number of experiments)

G-buffer G-actin G-actin ?

thymosin b4

G-actin ? cyclic

thymosin b4

G-actin ? cyclic

thymosin b4

G-actin ? cyclic

thymosin b4

Molar ratio (G-actin: thymosins) 1:1 1:1 1:2 1:20

Time of fall [sec] 8 89.8 ± 8 n = 5 10 ± 0.8 n = 6 56.2 ± 5 n = 6 56.6 ± 6.4 n = 3 7.2 ± 0.8 n = 3

Fig. 4 Cross-linking of thymosin b4 and cyclic thymosin b4 to

G-actin by 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide. a puri-

fied G-actin without EDC, b equimolar concentrations of G-actin and

thymosin b4, c equimolar concentrations of G-actin and cyclic

thymosin b4, d tenfold molar excess of cyclic thymosin b4 over

G-actin. The protein mixture in the lanes b, c and d was cross-linked

by addition of EDC. Detection of the cross-link was performed by

coomassie staining

Table 2 Determination of the dissociation constant of the complex of

G-actin and thymosin b4 or cyclic thymosin b4 by equilibrium cen-

trifugation [25]

Complex of G-actin with Kd (lM)

Thymosin b4 0.7

Cyclic thymosin b4 50.7

Thymosin b4 and Tissue Transglutaminase 489

123

standard procedures because of the identical amino acid

sequence. Antibodies for instance recognize both variants

of the peptide. However, it has been shown recently that a

cyclic form of statherin, a 43 amino acids containing

peptide, can be found in saliva [5]. It was shown that about

1 % of this peptide can be found cyclized by transgluta-

minase 2. So maybe the total amount of cyclic thymosin b4

is just to low to be detected besides its native linear form.

Tissue transglutaminases are multifunctional enzymes

with various roles in cellular processes [53]. It has been

shown that transglutaminase 2 plays a key role in the

apoptotic death of cancer cells [57]. High activity of a

transglutaminase could possibly lead to a cyclation of

thymosin b4. This could be a mechanism to inhibit its

described anti-apoptotic effect [49]. Many neurodegener-

ative diseases in humans such as Alzheimer’s disease are

caused by the formation of protein aggregates. The avail-

able data show that transglutaminases are involved in

theses pathogenetic mechanisms [34]. It could be possible

to detect cyclic thymosin b4 as a side product of these

processes. The shown neuroprotective effect of thymosin

b4 [35] could be weakened by its cyclation.

In 1990 Safer et al. [40] proposed that the sequence17LKKTETQEK25 of thymosin b4 may be responsible for

actin sequestering, because of the high homology with the

known actin-binding sequence of actobindin [55]. In a

previous study we have demonstrated that truncation of up

to 13 N-terminal amino acid residues does not abolish

chemical cross-linking to G-actin. In contrast truncation of

the first 23 amino acid residues destroys EDC cross-linking

[25]. This supports the proposal by Safer and colleages.

Unexpectedly derivatization of Gln23 does not abolish

G-actin binding [20, 59]. This indicates that this amino acid

residue is not essential for actin sequestering. Indeed this

residue is not conserved in actobindin [55]. A model based

on the structural data of thymosin b4 [26, 58] showed that it

is sterically possible to form an isopeptide bond between

Lys16 and Gln36 of thymosin b4 (Fig. 5). In aqueous

solution thymosin b4 is rather unstructured [7]. NMR

studies have shown that thymosin b4 folds completely upon

binding to G-actin and displays a central extended region

flanked by an N- and C-terminal helix [8]. This versatility

of thymosin b4 to adopt different conformations might

allow promiscuous protein interactions and explain its

multiple functions [33]. The cyclic form of thymosin b4 has

lost this characteristic structural flexibility. By the forma-

tion of an intramolecular isopeptide bond the flexibility of

thymosin b4 is partially lost, in spite of this loss in flexi-

bility cylclic thymosin b4 is still able to interact with

G-actin. This loss might explain the destabilization of the

complex of cyclic thymosin b4 and G-actin. Thymosin b4

interacts with G-actin at three different points along the

subdomains 1–3 of actin [27] in an extended conformation

[41]. In cyclic thymosin b4 Lys16 is cross-linked to Gln36

(Fig. 5). This alters the conformation of the peptide and

results in a loss of flexibilty. For this reason it is not clear

how the cyclic form of thymosin b4 interacts with G-actin.

There are further investigations needed to have enough

data to propose a model of the interaction of G-actin and

the cyclized form of thymosin b4.

The glutaminyl substrate specificity has been studied by

the group of Gorman and Folk [11]. They have shown that

a Gln which is followed by an Lys at the position ?2 is

more likely to serve as glutaminyl substrate. Gln23 and

Gln36 of thymosin b4 are followed by such a Lys at

position ?2. Gln36 might be the only detected reacted

residue because of its spatial distance to Lys16. Trans-

glutaminases are not so selective in aminyl substrates [28,

59] but it might be that these two residues are in a

appropriate distance to bind in the reactive center of the

enzyme. The group of Touati et al. [52] has studied the

ability of a bacterial transglutaminase to form cyclic pep-

tides in vitro. It was shown with different artificial peptides

that the ring forming is depending on the amino acid

sequence and the length of the peptide. In all reactions the

cyclation was never complete (just 50 % of the peptide was

cyclized). This supports our observation that not all thy-

mosin b4 is cyclized in vitro when there are no other

reactants present. This poor efficency might also be a

reason for undectable cyclic thymosin b4 in vivo.

A reaction of thymosin b4 catalyzed by tissue trans-

glutaminase does not lead to an artifical WH2 domain

protein. Instead it leads to the formation of an

Fig. 5 Model of thymosin b4 and cyclic thymosin b4. Thymosin b4 in

green, cyclic thymosin b4 in purple; isopeptide bond forming residues

are labeled

490 C. App et al.

123

intramolecular bond involving Lys16 and Gln36. This

peptide is still able to sequester G-actin albeit the stability

of the complex is about one fiftieth of the stability of the

thymosin b4 9 G-actin complex.

Acknowledgments We would like to express our appreciation to

Doris Jaegers for excellent technical assistance and support. EH and

TH thank the Deutsche Forschungsgemeinschaft (DFG) for support.

Conflict of interest The authors declare no conflicts of interest.

References

1. App C, Knop J, Mannherz HG, Hannappel E (2012) Ann N Y

Acad Sci 1270:98–104. doi:10.1111/j.1749-6632.2012.06658.x

2. Bock-Marquette I, Saxena A, White MD, Dimaio JM, Srivastava

D (2004) Nature 432(7016):466–472. doi:10.1038/nature03000

3. Boquet I, Boujemaa R, Carlier MF, Preat T (2000) Cell

102(6):797–808

4. Bubb MR, Lewis MS, Korn ED (1991) J Biol Chem

266(6):3820–3826

5. Cabras T, Inzitari R, Fanali C, Scarano E, Patamia M, Sanna MT,

Pisano E, Giardina B, Castagnola M, Messana I (2006) J Sep Sci

29(17):2600–2608

6. Cooper JA, Pollard TD (1982). Methods Enzymol 85 Pt

B:182–210

7. Czisch M, Schleicher M, Horger S, Voelter W, Holak TA (1993)

Eur J Biochem 218(2):335–344

8. Domanski M, Hertzog M, Coutant J, Gutsche-Perelroizen I,

Bontems F, Carlier MF, Guittet E, van Heijenoort C (2004) J Biol

Chem 279(22):23637–23645. doi:10.1074/jbc.M311413200

9. Folk JE, Chung SI (1985) Methods Enzymol 113:358–375

10. Goodall GJ, Morgan JI, Horecker BL (1983) Arch Biochem

Biophys 221(2):598–601

11. Gorman JJ, Folk JE (1981) J Biol Chem 256(6):2712–2715

12. Graser TA, Godel HG, Albers S, Foldi P, Furst P (1985) Anal

Biochem 151(1):142–152

13. Greenberg CS, Birckbichler PJ, Rice RH (1991) FASEB J

5(15):3071–3077

14. Hannappel E, Kalbacher H, Voelter W (1988) Arch Biochem

Biophys 260(2):546–551

15. Hannappel E, Leibold W (1985) Arch Biochem Biophys

240(1):236–241

16. Hannappel E, van Kampen M (1987) J Chromatogr 397:279–285

17. Hertzog M, Yarmola EG, Didry D, Bubb MR, Carlier MF (2002) J

Biol Chem 277(17):14786–14792. doi:10.1074/jbc.M112064200

18. Hinkel R, El-Aouni C, Olson T, Horstkotte J, Mayer S, Muller S,

Willhauck M, Spitzweg C, Gildehaus FJ, Munzing W, Hannappel

E, Bock-Marquette I, DiMaio JM, Hatzopoulos AK, Boekstegers

P, Kupatt C (2008) Circulation 117(17):2232–2240. doi:10.1161/

CIRCULATIONAHA.107.758904

19. Huang LC, Jean D, Proske RJ, Reins RY, McDermott AM (2007)

Curr Eye Res 32(7–8):595–609. doi:10.1080/02713680701446653

20. Huff T, Ballweber E, Humeny A, Bonk T, Becker C, Muller CS,

Mannherz HG, Hannappel E (1999) FEBS Lett 464(1–2):14–20

21. Huff T, Hannappel E (1997) Anal Chim Acta 352:249–255

22. Huff T, Muller CS, Otto AM, Netzker R, Hannappel E (2001) Int

J Biochem Cell Biol 33(3):205–220

23. Huff T, Muller CSG, Hannappel E (1997) Anal Chim Acta

352:239–248

24. Huff T, Otto AM, Muller CS, Meier M, Hannappel E (2002)

FASEB J 16(7):691–696

25. Huff T, Zerzawy D, Hannappel E (1995) Eur J Biochem

230(2):650–657

26. Irobi E, Aguda AH, Larsson M, Guerin C, Yin HL, Burtnick LD,

Blanchoin L, Robinson RC (2004) EMBO J 23(18):3599–3608.

doi:10.1038/sj.emboj.7600372

27. Knop J, App C, Horn AHC, Iavarone F, Castagnola M, Hann-

appel E (2013) Biochemistry (Mosc). doi:10.1021/bi400664k

28. Low TL, Hu SK, Goldstein AL (1981) Proc Natl Acad Sci U S A

78(2):1162–1166

29. Makogonenko E, Goldstein AL, Bishop PD, Medved L (2004)

Biochemistry (Mosc) 43(33):10748–10756. doi:10.1021/

bi049253l

30. Malinda KM, Goldstein AL, Kleinman HK (1997) FASEB J

11(6):474–481

31. Malinda KM, Sidhu GS, Mani H, Banaudha K, Maheshwari RK,

Goldstein AL, Kleinman HK (1999) J Invest Dermatol

113(3):364–368. doi:10.1046/j.1523-1747.1999.00708.x

32. Mannherz HG, Dansranjavin T, Gremm D, Mazur A, Sokoll A,

Hannappel E, Jockusch BM (2008). Paper presented at the DGZ,

Marburg

33. Mannherz HG, Hannappel E (2009) Cell Motil Cytoskel. doi:10.

1002/cm.20371

34. Martin A, Giuliano A, Collaro D, De Vivo G, Sedia C, Serretiello

E, Gentile V (2013) Amino Acids 44(1):111–118. doi:10.1007/

s00726-011-1081-1

35. Morris DC, Chopp M, Zhang L, Lu M, Zhang ZG (2010) Neuro-

science 169(2):674–682. doi:10.1016/j.neuroscience.2010.05.017

36. Pardee JD, Spudich JA (1982) Methods Enzymol 85 Pt

B:164–181

37. Paunola E, Mattila PK, Lappalainen P (2002) FEBS Lett

513(1):92–97

38. Safer D, Chowrashi PK (1997) Cell Motil Cytoskel

38(2):163–171

39. Safer D, Elzinga M, Nachmias VT (1991) J Biol Chem

266(7):4029–4032

40. Safer D, Golla R, Nachmias VT (1990) Proc Natl Acad Sci U S A

87:2536–2540

41. Safer D, Sosnick TR, Elzinga M (1997) Biochemistry (Mosc)

36(19):5806–5816. doi:10.1021/bi970185v

42. Smart N, Bollini S, Dube KN, Vieira JM, Zhou B, Davidson S,

Yellon D, Riegler J, Price AN, Lythgoe MF, Pu WT, Riley PR

(2011) Nature 474(7353):640–644. doi:10.1038/nature10188

43. Smart N, Risebro CA, Melville AA, Moses K, Schwartz RJ,

Chien KR, Riley PR (2007) Nature 445(7124):177–182. doi:10.

1038/nature05383

44. Smith PK, Krohn RI, Hermanson GT, Mallia AK, Gartner FH,

Provenzano MD, Fujimoto EK, Goeke NM, Olson BJ, Klenk DC

(1985) Anal Biochem 150(1):76–85

45. Sopko N, Qin Y, Finan A, Dadabayev A, Chigurupati S, Qin J,

Penn MS, Gupta S (2011) PLoS ONE 6(5):e20184. doi:10.1371/

journal.pone.0020184

46. Sosne G, Albeiruti AR, Hollis B, Siddiqi A, Ellenberg D, Ku-

rpakus-Wheater M (2006). Exp Eye Res

47. Sosne G, Chan CC, Thai K, Kennedy M, Szliter EA, Hazlett LD,

Kleinman HK (2001) Exp Eye Res 72(5):605–608

48. Sosne G, Christopherson PL, Barrett RP, Fridman R (2005)

Invest Ophthalmol Vis Sci 46(7):2388–2395

49. Sosne G, Siddiqi A, Kurpakus-Wheater M (2004) Invest Oph-

thalmol Vis Sci 45(4):1095–1100

50. Sosne G, Szliter EA, Barrett R, Kernacki KA, Kleinman H,

Hazlett LD (2002) Exp Eye Res 74(2):293–299

51. Sosne G, Xu L, Prach L, Mrock LK, Kleinman HK, Letterio JJ,

Hazlett LD, Kurpakus-Wheater M (2004) Exp Cell Res

293(1):175–183

52. Touati J, Angelini A, Hinner MJ, Heinis C (2011) ChemBioChem

12(1):38–42. doi:10.1002/cbic.201000451

Thymosin b4 and Tissue Transglutaminase 491

123

53. Tucholski J, Johnson GV (2002) J Neurochem 81(4):780–791

54. Van Troys M, Ono K, Dewitte D, Jonckheere V, De Ruyck N,

Vandekerckhove J, Ono S, Ampe C (2004) Mol Biol Cell

15(10):4735–4748. doi:10.1091/mbc.E04-03-0225

55. Vancompernolle K, Vandekerckhove J, Bubb MR, Korn ED

(1991) J Biol Chem 266(23):15427–15431

56. Weber A, Nachmias VT, Pennise CR, Pring M, Safer D (1992)

Biochemistry (Mosc) 31(27):6179–6185

57. Yoo JO, Lim YC, Kim YM, Ha KS (2012) J Biol Chem

287(18):14377–14388. doi:10.1074/jbc.M111.326074

58. Zarbock J, Oschkinat H, Hannappel E, Kalbacher H, Voelter W,

Holak TA (1990) Biochemistry (Mosc) 29(34):7814–7821

59. Zoubek RE, Hannappel E (2007) Ann N Y Acad Sci

1112:442–450. doi:10.1196/annals.1415.031

492 C. App et al.

123