Báo cáo khoa học: Identification of preferred substrate sequences for transglutaminase 1 – development of a novel peptide that can efficiently detect cross-linking enzyme activity in the skin pot

To gain insight into the preferred substrate structure of TGase 1, we used a phage-displayed random peptide library to screen primary amino acid sequences that are preferentially selecte

transglutaminase 1 – development of a novel peptide

that can efficiently detect cross-linking enzyme

activity in the skin

Yoshiaki Sugimura1,*, Masayo Hosono1,*, Miyako Kitamura1, Tatsuya Tsuda2, Kiyofumi

Yamanishi2, Masatoshi Maki1and Kiyotaka Hitomi1

1 Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Japan

2 Department of Dermatology, Hyogo College of Medicine, Nishinomiya, Japan

Transglutaminase (TGase; EC 2.3.2.13) is a Ca2+

-dependent enzyme that catalyzes the formation of

isopeptide cross-links between the c-carboxyamide

group of glutamine residues and the e-amino group of

lysine residues [1,2] In addition, TGases catalyze the

incorporation of primary amines into glutamine

resi-dues (transamidation), or conversion of glutamine to

glutamic acid (deamidation) TGases comprise a family

of eight isozymes (Factor XIII and TGases 1–7) that

are distributed in a variety of tissues and have unique

substrate specificities Factor XIII and TGase 2 (tissue-type TGase), two of the major isozymes, are involved

in the stabilization of fibrin clots, and in apoptosis, extracellular matrix stabilization and wound healing, respectively [3–6] TGase 1 is expressed primarily in epithelial cells and plays an essential role in formation

of the skin epidermis [7–9]

In the outermost layer of the epidermis, the assembly

of a highly specialized structure, termed the cornified envelope (CE), is formed as a result of cross-linking of

Keywords

epidermis; keratinocyte; phage display; skin;

transglutaminase

Correspondence

K Hitomi, Department of Applied Molecular

Biosciences, Graduate School of

Bioagricultural Sciences, Nagoya University,

Chikusa, 464 8601 Nagoya, Japan

Fax: +81 52 789 5542

Tel: +81 52 789 5541

E-mail: hitomi@agr.nagoya-u.ac.jp

*These authors contributed equally to this

work

(Received 1 August 2008, revised

10 September 2008, accepted

18 September 2008)

doi:10.1111/j.1742-4658.2008.06692.x

Transglutaminase 1 (TGase 1) is an essential enzyme for cornified enve-lope formation in stratified squamous epithelia This enzyme catalyzes the cross-linking of glutamine and lysine residues in structural proteins

in differentiating keratinocytes To gain insight into the preferred substrate structure of TGase 1, we used a phage-displayed random peptide library to screen primary amino acid sequences that are preferentially selected by human TGase 1 The peptides selected as gluta-mine donor substrate exhibited a marked tendency in primary structure, conforming to the sequence: QxK⁄ RwxxxWP (where x and w represent non-conserved and hydrophobic amino acids, respectively) Using gluta-thione S-transferase (GST) fusion proteins of the selected peptides, we identified several sequences as preferred substrates and confirmed that they were isozyme-specific We generated GST-fused alanine mutants of the most reactive sequence (K5) to determine the residues that were crit-ical for reactivity Even in peptide form, K5 appeared to have high and specific reactivity as substrate In situ analysis of mouse skin sections using fluorescence-conjugated K5 peptide resulted in detection of TGase 1 activity with high sensitivity, but no signal was detected in a TGase 1-null mouse In conclusion, we were successful in generating a novel substrate peptide for sensitive detection of endogenous TGase 1 activity in the skin

Abbreviations

Bio-Cd, 5-(biotinamido)pentylamine; CE, cornified envelope; Dansyl-Cd, monodansylpentylamine; FITC, fluorescein isothiocyanate; GST, glutathione S-transferase; TGase, transglutaminase.

several structural proteins in keratinocytes [10–12] The

CE is located underneath the plasma membrane of

dif-ferentiated keratinocytes and is essential for barrier

function of the epidermis The CE comprises several

proteins, including involucrin, loricrin, trichohyalin and

small proline rich-proteins, that are preferred substrates

for TGases in the skin Of the eight TGase isozymes,

four (TGases 1, 2, 3 and 5) are expressed in

keratino-cytes [13–18], and are involved in cross-linking of the

structural proteins to form the CE at distinct

intracellu-lar sites [8,18] In the current model of TGase function,

cytoplasmic TGase 3 catalyzes the cross-linking of

several proteins, and these products are then stabilized

with a proteinaceous platform beneath the plasma

membrane, which is formed by membrane-anchored

TGase 1 [19] However, the precise mechanism by

which these enzymes coordinate the cross-linking of

various substrates is poorly understood Mice that lack

TGase 1 exhibit impaired skin barrier function due to

aberrant cornification [20] In humans, several types of

TGase 1 mutations have been identified in congenital

ichthyoses, such as non-bullous congenital

ichthyosi-form erythroderma and lamellar ichthyosis [21–25]

Thus, TGase 1 appears to be an essential and

non-redundant enzyme for CE formation in the

epi-dermis under functional regulation [26]

Generally, TGases display strict substrate specificity

towards peptide-bound glutamine residues and

rela-tively weak specificity with respect to lysine residues or

amine groups Hence, information about the primary

and⁄ or secondary structure of sequences surrounding

the reactive glutamine residue in substrates is

impor-tant for understanding substrate specificity in

TGase-mediated catalysis [27] Partial amino acid sequences

of substrates for TGase 2 and Factor XIII have been

identified [28] Although several protein substrates for

TGase 1 have been characterized, a consensus

sequence for the preferred substrate has not been

iden-tified [29–32]

Recently, we established a system for the

identifica-tion of preferred substrate sequences of TGases by

screening a phage-displayed random peptide library

[33,34] Using this system, we identified the primary

sequences of the various preferred, isozyme-specific

substrates of TGase 2 and Factor XIII These results

provide new insight into the substrate specificity of

TGases, and expand the range of application using the

enzyme reactions [35]

In the current study, we used this system to identify

preferred TGase 1-specific peptide sequences We

iden-tified several sequences that were preferentially selected

by human TGase 1 and exhibited a significant

tendency to serve as glutamine donor substrates One

peptide, K5, exhibited strong activity in both recombi-nant fusion protein and peptide forms Using this pep-tide, we were able to detect in situ TGase 1 activity in mouse skin sections with a high level of sensitivity and specificity

Results

Screening of candidate substrate sequences from

a random peptide library

In the presence of recombinant human TGase 1, phage clones were incubated with biotin-labeled primary amine (Bio-Cd) Phage clones that were covalently bound to Bio-Cd were selected by avidin affinity purifi-cation The selected clones were amplified and then subjected to four additional cycles of catalysis and panning as described previously [33] Sequence analysis

of the peptides encoded by the final clones revealed that 32 of the 56 clones contained glutamine residues (Fig 1) When the peptides were aligned based on the putative reactive glutamine residue, there were several significant similarities among the primary sequences of more than half of the clones: (1) tyrosine and gluta-mine were located at the N-terminus of the peptide, (2) there was a lysine or an arginine at position +2 (rela-tive to glutamine), (3) a hydrophobic amino acid was commonly observed at position +3, and (4) trypto-phan and proline were frequently located at positions +7 and +8, respectively

Among the clones, there was one frequently observed sequence (YEQHKLSSWPF) that was encoded by identical DNA sequences, but treated as

an individual clone

Evaluation of the selected sequences as recombinant peptide-fused GST proteins

To evaluate the ability of the selected peptide sequences

to serve as glutamine donor substrates, we measured the amount of monodansyl cadaverine (Dansyl-Cd) incorporated into recombinant peptide–GST(QN) fusion proteins (in which all the glutamine residues in glutathione S-transferase (GST) had been changed to asparagine) in the presence of TGase 1 The sequences marked with an asterisk in Fig 1 were selected for analysis The reaction products were separated by SDS–PAGE and then visualized by UV illumination (Fig 2)

GST(QN) fusion proteins of the K1, K2, K5, K13, K26, K51, K57 and K60 sequences exhibited signifi-cant levels of incorporation of Dansyl-Cd compared

to the other sequences examined All of the highly

reactive sequences had the similarities described above,

which suggested that our procedure was successful in

identifying the preferred sequences Of these highly

reactive sequences, we selected K5, K26, K51 and K60

for further analysis based on their levels of reactivity

Isozyme specificity of the peptide sequences

The four sequences with the highest reactivity were

further analyzed for isozyme specificity with respect to

TGases 1, 2 and 3, and Factor XIIIa (activated

Fac-tor XIII) GST(QN) fusion proteins of each peptide

were incubated with [3H]-putrescine in the presence of

each TGase Most of the peptide sequences exhibited

high reactivity with TGase 1, consistent with the

results of the Dansyl-Cd incorporation assay (Fig 3)

We did not observe cross-reactivity for any of the

peptides, with the exception of K60, which showed

sig-nificant incorporation in the presence of Factor XIIIa

K5 exhibited the lowest cross-reactivity to TGases 2

and 3, indicating the highest specificity Thus, we

selected K5 for further analysis

Substitution mutant analysis of the K5 peptide

sequence

To determine the role of each amino acid of K5 in

TGase 1-mediated catalysis, we generated alanine

substitution mutants for every residue of the 12-amino-acid peptide Wild-type and mutant peptides were produced as GST(QN) fusion proteins, and eval-uated for their reactivity in the presence of TGase 1

As shown in Fig 4, mutation of positions +2 (K), +3 (L), +5 (S), +6 (S), +7 (W), +8 (P) or +9 (F) to alanine decreased the level of incorporation of

Dansyl-Cd Substitutions at positions )2 (Y), )1 (E), +1 (H)

or +4 (P) had a smaller effect on the reactivity of the peptide

Assessment of the K5 sequence for reactivity and specificity in the peptide form

To assess the reactivity and specificity of K5 in the peptide form, biotinylated K5 (pepK5: YEQHK LPSSWPF), or K5 in which the reactive glutamine residue was replaced by asparagine (pepK5QN: YENHKLPSSWPF), were incubated with casein as a glutamine acceptor substrate in the presence of TGases

1, 2 or 3 For comparison, we also examined the reac-tivity of the TGase 2 preferred substrate peptide (pepT26: HQSYVDPWMLDH) [33] Incorporation of pepK5 into casein was observed in the presence of TGase 1 in a time-dependent manner, but pepT26 showed no apparent reactivity (Fig 5A) In contrast,

in the presence of TGase 2, pepT26 showed apparent incorporation into casein, whereas pepK5 reacted at

Fig 1 Alignment of candidate substrate peptide sequences Amino acid sequences of the peptides contained within the selected phage clones were aligned based on the putative reactive glutamine residues The glutamine residues, the hydrophobic amino acids at position +3 (relative to glutamine), and the most commonly observed amino acids are shaded Clones marked with an asterisk were evaluated as GST(QN) fusion proteins using the Dansyl-Cd incorporation assay.

much lower level (Fig 5B) However, at a higher

con-centration of the peptide (above 0.1 mm), pepK5

showed cross-reactivity to TGase 2 (data not shown)

pepK5QN was not incorporated into casein, also

act-ing as glutamine-donor substrate, in the presence of

either TGase 1 or TGase 2 This result indicates that

the lysine residue in the sequence of K5 does not

participate in the enzymatic reaction

To determine whether K5 cross-reacts with TGase 3,

we compared the incorporation rates of pepK5 in the

presence of TGases 1 or 3, as a preferred peptide

sequence for TGase 3 has not yet been established

(Fig 5C) In the presence of TGase 3, the

incorpora-tion of pepK5 was similar to control reacincorpora-tions that did

not contain any enzyme, whereas there was a signifi-cant increase in incorporation in the presence of TGase 1 pepK5QN also failed to react with casein in the presence of TGase 3 (data not shown) These results indicate that the K5 peptide sequence acts as a glutamine donor substrate in an isozyme-specific manner, even in the peptide form

Detection of in situ TGase 1 activity in skin

As pepK5 appeared to be a highly preferred and spe-cific substrate for TGase 1, we examined whether this peptide could be used to detect endogenous TGase 1 activity in the skin epidermis (Fig 6A) In the presence

of CaCl2 in the reaction mixture, we detected specific incorporation of fluorescein isothiocyanate (FITC)-labeled K5 peptide (FITC-pepK5) (1 lm) into sub-strate proteins in the epidermis, mainly at the cell periphery of the upper spinous and granular layers This staining pattern of reactivity corresponded well with the localization of TGase 1 by immunostaining with monoclonal anti-mouse IgG No signals were detected when we used FITC-pepK5QN mutant pep-tide, or in the presence of EDTA, which indicated that the cross-linking reaction was catalyzed specifically by TGase 1 To further evaluate the specificity of the assay for TGase 1, skin sections from a TGase 1 knockout mouse were also examined No signals were detected in TGase 1-null sections in the presence or absence of Ca2+, which indicates that FITC-pepK5 specifically detects endogenous TGase 1 activity

We also compared the reactivity of FITC-pepK5 and FITC-cadaverine, which has been used previously for detection of in situ TGase activity, over a range of concentrations (0.1–100 lm) As shown in Fig 6B, endogenous TGase 1 activity was detected at 0.1–1 lm FITC-pepK5, but no apparent signal was observed with FITC-cadaverine at the same concentrations In the presence of higher concentrations of cadaverine, the staining pattern became somewhat ambiguous in mouse skin sections, possibly because of cross-reac-tivity with other TGases These results suggest that FITC-pepK5 detects endogenous TGase 1 activity with greater sensitivity than FITC-cadaverine

Discussion

In terminally differentiating stratified epithelial cells, isopeptide cross-linking of keratinocyte proteins into organized protein structures lead to the formation of the CE Although the cooperative action of several TGases in the keratinocytes plays a role in this pro-cess, the precise role of each isozyme in cross-linking

K1

K2

K5

K11

K13

K26

K29

K42

K60

K51

K56

K57

K59

K70

Ctrl

DMC

CBB 0 0.5 1 2 5 10 15 20 (min)

Fig 2 Evaluation of the reactivity of selected peptides as peptide

GST fusion proteins Incorporation of Dansyl-Cd into peptide–

GST(QN) fusion proteins of the selected amino acid sequences in

the presence of TGase 1 The numbers prefixed with K (K1, K2, K5,

etc.) for each peptide sequence correspond to the ID numbers of

the clones (Fig 1) At the indicated times, the reaction products

were separated by 12.5% SDS–PAGE and then illuminated by UV

light Unreacted (0 min) peptide–GST(QN) fusion proteins were

stained with Coomassie brilliant blue and are shown on the left

of the fluorograph Ctrl, GST(QN) fusion protein of a peptide that

does not contain a glutamine residue (K3, SPLAYVAPWSNL).

Dimethylcasein (DMC) was examined in parallel as a positive control.

of the target substrates remains elusive TGase 1 has

unique substrate specificity, which results in a distinct

pattern of in vitro cross-linked substrate proteins

com-pared to other TGases [32,36,37] A better

understand-ing of the preferred substrate structure for TGase 1 is

essential to evaluate substrate reactivity, and also to

develop isozyme-specific regulatory molecules

In the current study, preferred substrate sequences

for TGase 1 selected through screening of a

phage-dis-played peptide library exhibited different tendencies

compared to those for Factor XIIIa and TGase 2

[33,38] The preferred substrate sequence for TGase 1 conformed to the consensus motif QxR⁄ KwxxxWP, whereas those for TGase 2 and Factor XIIIa conform

to the motifs QxPwDP and QxxwxWP, respectively A hydrophobic amino acid at +3 relative to the gluta-mine residue is present in the substrate motifs of all three TGases Based on previous studies, the preferred sequence for TGase 2 contains the sequence QxP, which has been shown to be an in vivo transamidation motif [39] In the case of TGase 1, most of the selected substrates contained QxK or QxR This motif is fre-quently observed in involucrin, trichohyalin and small proline-rich proteins, and has been implicated as a putative cross-linking site in these proteins [32,36,37] Sequences homologous to the motif QxR⁄ KwxxxWP are present in the C-terminal regions of involucrin (QQKQEVQWP) and loricrin (QQKQAPTWP), which are the main components of the CE In the developing TGase substrate database (TRANSDAB; http:// genomics.dote.hu/wiki), several sequences containing the reactive glutamine residue have been listed for TGase 1 as well as other isozymes When these sequences were compared with our proposed consensus sequence, half of the sequences were found to contain QxK or QxxK, which is partially consistent with the obtained motif

Interestingly, the TGase 1 substrate motif was simi-lar to that of Factor XIIIa, QxxwxWP Among TGase family members, the primary structures of Factor XIII and TGase 1 display the most homology, which sug-gests that the catalytic domains of these two enzymes may recognize similar substrate structures

Most of the highly reactive peptide sequences identi-fied in this study (K1, K2, K5, K26, K57 and K60) contained the TGase 1 substrate consensus sequence The peptide sequences K5, K26 and K51 exhibited less

WT –2A –1A QN +1A +2A +3A +4A +5A +6A +7A +8A +9A

0

0.2

0.4

0.6

0.8

1

1.2

1.4

Fig 4 Assessment of the contribution of each amino acid residue

of K5 to substrate recognition Alanine substitution mutants of K5

were produced as GST(QN) fusion proteins, and then incubated

with Dansyl-Cd for 10 min in the presence of TGase 1 The reaction

products were separated by SDS–PAGE, followed by UV irradiation.

The fluorescence intensity was analyzed by SCION imaging

soft-ware for quantification The relative values shown are normalized to

the intensity for the reaction of wild-type Data represent the

means of duplicate samples Numbers ( )2A to +9A) indicate the

position of substitution mutations WT, peptide in which there were

no amino acid substitutions; QN, peptide in which the glutamine

residue was changed to asparagine The mutations that resulted in

an apparent decrease in the reactivity are shaded in darker gray.

3 H]-putrescine incorporated (

4 dpm)

0 1 2 3 4 5 6 7 8 9 10

Fig 3 Cross-reactivity of the preferred

sequences with TGase 2, TGase 3 and

Factor XIIIa GST(QN) fusion proteins of

peptides K5, K26, K51 and K60, and the

control sequence (WGHTIYHLHPTI [33])

(Ctrl), were incubated for 10 min with

1.0 m M [ 3 H]-putrescine in the presence of

TGase 1, TGase 2, TGase 3 or Factor XIIIa.

Dimethylcasein (DMC) was used in parallel

to adjust the enzymatic activities Data are

the means ± SD of triplicate assays.

cross-reactivity with other TGases, and K60 exhibited

some reactivity towards Factor XIIIa These results

indicate that the consensus motif identified in this

study defines a unique structure of preferred substrates

of TGase 1 This information should be useful for pre-dicting substrate reactivity and designing regulatory molecules that can interact with the active site of the enzyme

K5 (YEQHKLPSSWPF) showed the highest level of reactivity and specificity as a substrate for TGase 1 Of note, the K5 sequence was the most frequently selected clone during screening, which suggests that (a) this sequence is a highly preferred substrate as compared

to other sequences, and⁄ or (b) the phage clone that contained this sequence was efficiently amplified in bacteria

Mutational analysis of each amino acid residue in the K5 sequence demonstrated that the consensus TGase 1 motif, as well as additional C-terminal amino acids, is important in substrate activity This was expected because the other selected peptides had conserved amino acid residues among analyzed sequences It would be interesting to explore whether shorter peptides can function as substrates with similar levels of reactivity

The reactivity and specificity of K5 for TGase 1 was maintained even in peptide form Initially, we expected that the lysine residue of K5 would react with the glu-tamine residue of pepK5 itself or with casein during the catalytic reaction However, pepK5QN, in which the glutamine was mutated but lysine was not, failed

to react with casein (Fig 2), which indicates that there

is no reactivity of K5 as a glutamine acceptor Although there is far less selectivity of TGase for glu-tamine acceptor substrates in general, this result indi-cates that the lysine residue in the pepK5 is not strongly reactive Thus, pepK5 represents a potentially valuable tool for determining the specific partners that are favored substrates during TGase 1 catalysis When we examined the ability of pepK5 to detect endogenous TGase 1 activity in mouse skin sections (Fig 6), Ca2+-dependent incorporation of FITC-pepK5 into glutamine acceptor substrates in keratino-cytes was seen, and this activity was undetectable in sections from TGase 1-null mice As observed in immunostaining analysis, TGase 1 protein localized to the peripheral regions of the keratinocytes in the gran-ular and upper spinous layers, consistent with previous reports [40,41], and the K5-reactive proteins most likely co-localize with TGase 1 at these sites These results indicate that the K5 peptide acts as a sensitive and precise probe to detect in situ activity of endoge-nous TGase 1 In the current study, K5 did not exhibit cross-reactivity to TGase 3, skin-specific TGase In the

in vitro assay with TGase 2, K5 reacted to a small extent at higher enzyme activity or higher peptide

0

0.2

0.4

0.6

0.8

1

1.2

0

0.2

0.4

0.6

0.8

1

1.2

K5 K5QN T26

K5 K5QN T26

Reaction time (min)

Reaction time (min)

TGase 1

A

B

C

TGase 2

0

0.2

0.4

0.6

0.8

1

1.2

-TGase

TGase 1 TGase 3

Reaction time (min) Fig 5 Analysis of the reactivity of the K5 peptide with casein in

the presence of TGases The time-dependent incorporation of

bioti-nylated peptides (pepK5, pepK5QN and pepT26) into casein was

examined in the presence of TGase 1 (A) and TGase 2 (B) The

amount of reaction product was determined by microtiter assay,

and is represented as absorbance at 492 nm The square, circle

and triangle represent pepK5, pepK5QN and pepT26, respectively.

(C) Time-dependent incorporation of pepK5 measured in the

pres-ence of TGase 1 (closed circle) or TGase 3 (open circle) Reaction

mixtures that did not contain enzyme were analyzed in parallel

(tri-angle) as a negative control Data represent the means ± SD of

triplicate samples.

concentration However, in the skin sections, there was

no significant TGase 2 activity because FITC-labeled

T26 showed no signal (data not shown)

To date, detection of cross-linked TGase products

using tissue sections has utilized an FITC-labeled

primary amine (FITC-cadaverine) or FITC-labeled

substrate peptides [42,43] The pattern of TGase

activ-ity that we observed was consistent with that seen

in the skin using FITC-cadaverine [42], although

cadaverine is not an isozyme-specific probe, and thus

detects total TGase activity in keratinocytes However,

in our study, the sensitivity of K5 was approximately

100–1000 times higher than that of FITC-cadaverine

(Fig 6B) Thus, the method that we have established

for the detection of TGase 1 exhibits superior

specific-ity and reactivspecific-ity

TGase 1 expression is not limited to the skin

epider-mis It is also expressed in the kidney, hair follicle and

liver [40,42] However, the precise roles of TGase 1 in

these tissues are unknown In addition, in the epithelial

cells, activated TGase 1 is concentrated at adherent

junctions, but the substrates and biological significance

of the enzyme are not fully resolved [40] A specific

and sensitive detection probe, such as FITC-labeled

pepK5, would be useful for investigating the role of TGase 1-mediated cross-linking reactions in keratino-cytes and in other cells and tissues

In ongoing experiments, we are attempting to char-acterize the reactivity and to identify the preferred substrate sequences of other skin TGases, as our devel-oped screening system is applicable to other TGases [33,44] Recombinant TGase 5 is poorly expressed as a soluble active form [17,45], thus we could not examine its reactivity in this study However, it is unlikely that the selected peptides cross-react with TGase 5, as there was no obvious reactivity of FITC-pepK5 in skin sec-tions from a TGase 1 knockout mouse Furthermore, the cross-linking products containing pepK5 localized primarily at the periphery of the cell, which is distinct from the location of TGase 5 [17,18]

In conclusion, we have identified several preferred substrate sequences for TGase 1 Our findings provide valuable insight into substrate specificity in the cross-linking processes of the epidermis, and possibly other epithelial tissues as well Specific and sensitive detec-tion of TGase 1 activity using FITC-labeled K5 peptide could be used for clinical diagnosis of con-genital ichthyoses Additional studies, particularly

A

B

Fig 6 Detection of in situ TGase 1 activity

in mouse skin sections (A) FITC-labeled

peptides (1 l M ), pepK5 (upper) or pepK5QN

(lower), were incubated with mouse skin

sections in the presence of CaCl 2 or EDTA.

TGase 1 activity in skin sections from a

TGase 1 knockout mouse was also

exam-ined (right) Sections analyzed by

immuno-staining using monoclonal anti-mouse

TGase 1 IgG (TGase1 mAb) and by

hema-toxylin and eosin staining are also shown

(left columns) (B) Detection of TGase 1

activity in wild-type mouse skin sections

using increasing concentrations

(0.1–100 l M ) of FITC-labeled pepK5 or

FITC-cadaverine Scale bar = 50 lm.

co-crystallization of TGase 1 with the K5 peptide, are

required to refine the TGase 1 reaction mechanism,

and to develop specific regulatory molecules

Experimental procedures

Transglutaminases

Human recombinant TGases 1 and 3 were obtained by

expression and purification from baculovirus-infected insect

cells, as described previously [46,47] For activation of

TGase 3, the zymogen was proteolyzed by treatment with

dispase Guinea pig liver TGase 2 was kindly provided by

K Ikura (Graduate School of Science and Technology,

Kyoto Institute of Technology, Japan) [48] Human Factor

XIII (Fibrogammin P; ZLB Behring, Marburg, Germany)

was activated to Factor XIIIa by treatment with bovine

thrombin (Sigma, St Louis, MO, USA)

Screening of a phage-displayed peptide library

Screening was carried out as previously described, using an

M13 Ph.D.-12 phage-display system (New England Biolabs

Inc., Ipswich, MA, USA) [33] Briefly, approximately

1.5· 1011

(1st round panning) or 1· 1012)13 (2nd to 5th

round panning) phage clones were incubated at 37C with

TGase 1 (10 ngÆlL)1) in 10 mm Tris⁄ HCl pH 8.0, 150 mm

NaCl⁄ Tris buffer containing 1 mm dithiothreitol, 5 mm

CaCl2and 5 mm Bio-Cd [EZ-link

5-(biotinamido)pentyl-amine] (Pierce Biotechnology, Rockford, IL, USA) After

termination of the catalytic reaction by addition of EDTA,

the phage particles were precipitated in the presence of

poly-ethyleneglycol and NaCl with salmon sperm DNA as a

carrier Next, the phage clones that covalently incorporated

Bio-Cd were selected by affinity chromatography using

mono-avidin gel (SoftLink soft release avidin resin;

Pro-mega Corp., Madison, WI, USA) After washing for 3 min

with NaCl⁄ Tris containing 0.1% Tween-20 and 2 mm

EDTA and then with NaCl⁄ Tris twice, the bound phage

particles were eluted by NaCl⁄ Tris containing 5 mm biotin

The entire eluate was used to infect Escherichia coli ER2738

host bacteria to amplify the phage The phage particles were

concentrated by precipitation with polyethyleneglycol–NaCl

and then used for subsequent rounds of amplification After

panning five times in all, the DNA sequences of the peptides

in the selected phage clones were determined

Construction of the expression vector for GST

fusion proteins

The vector plasmid pET24d-GST(QN) was used to express

modified GST in which all the glutamine residues had been

substituted by asparagine residues, fused to a peptide at the

N-terminus [33] The DNA of each phage was isolated, and

the sequences of the 12-mer peptides were amplified by PCR Amplified PCR products were digested and inserted into pET24d-GST(QN) For generation of peptide mutants

in which each amino acid was substituted by alanine or asparagine, PCR-based mutagenesis was carried out Either E coli BL21(DE3)LysS or BL21(DE3)LysE were transformed with expression plasmids, and expression in bacteria was induced by the addition of isopropyl b-d-thio-galactoside Recombinant proteins were purified using TALON metal affinity resin according to the manufac-turer’s instructions (BD Biosciences, San Jose, CA, USA) The concentration of the purified protein was determined

by the Bradford method (Bio-Rad Laboratories Inc., Hercules, CA, USA)

Evaluation of preferred sequences using recombinant proteins

The reactivities of recombinant GST(QN) fusion proteins were evaluated by incorporation of Dansyl-Cd (Sigma), a fluorescence-labeled pentylamine Recombinant protein (200 ngÆlL)1) and 0.5 mm Dansyl-Cd were incubated in NaCl⁄ Tris buffer containing 5 mm CaCl2and 1 mm dithio-threitol in the presence of TGase 1 (0.75 ngÆlL)1) Dim-ethylcasein (200 ngÆlL)1) was used as a positive control The reaction mixture was incubated at 37C and then sep-arated by 12.5% SDS–PAGE A fluorograph of the gel was obtained by UV irradiation (254 nm) to visualize the amount of incorporated Dansyl-Cd To quantify the results, the fluorescence intensity of each product was analyzed using scion imaging software (Scion Corp., Frederick, MD, USA)

For incorporation of radiolabeled putrescine, [3 H]-putres-cine (1.0 mm) was incubated in NaCl⁄ Tris buffer containing

5 mm CaCl2and 1 mm dithiothreitol with the recombinant proteins (1 lgÆlL)1) in the presence of TGase 1 (37.5 ngÆlL)1), TGase 2 (2.5 ngÆlL)1), TGase 3 (8 ngÆlL)1)

or Factor XIIIa (12.5 ngÆlL)1), so that similar amounts of incorporation into dimethylcasein (1 lgÆlL)1) were achieved The reaction mixture was precipitated by addition

of the 10% trichloroacetic acid and then subjected to cen-trifugation at 10 000 g for 15 min at 4C The precipitate was dissolved and radioactivity was determined using a scintillation counter

Evaluation of synthetic peptides as a substrate

The 12-amino-acid peptide pepK5 (YEQHKLPSSWPF) was synthesized and biotinylated at the N-terminus A mutant peptide in which glutamine was substituted by

(YENHKLPSSWPF) and biotinylated Biotinylated pepT26 (HQSYVDPWMLDH) was used as the TGase 2 preferred substrate peptide for comparison The peptides were

dissolved in dimethylsulfoxide (Me2SO) To evaluate the

activity and specificity of the peptides, a microtiter plate

assay was performed as described previously, with some

modifications [33] Briefly, 200 ngÆlL)1bovine casein

(Nac-alai Tesque Inc., Kyoto, Japan) was incubated with each

peptide (0.01 mm peptide) in the presence of TGase 1

(1.9 ngÆlL)1) or TGase 2 (2.0 ngÆlL)1) in NaCl⁄ Tris buffer

containing 5 mm CaCl2 and 1 mm dithiothreitol at 37C

When comparing the reactivities of TGase 1 and TGase 3,

the molar concentration of recombinant enzyme in each

reaction mixture was the same (20 nm: TGase 1,

1.8 ngÆlL)1; TGase 3, 1.5 ngÆlL)1)

At the indicated times, 50 lL of the reaction mixture was

transferred to a 96-well microtiter plate (Maxisorp; Nunc,

New York, NY, USA) The microtiter plate was incubated

for 1 h at 37C to allow casein in the reaction mixture to

attach to the plate After blocking with skim milk and

washing with 0.1 m Tris buffer (pH 8.0), streptavidin–

peroxidase (Rockland Immunochemicals Inc., Gilbertsville,

PA, USA) was added, and the plates were incubated for

1 h at 37C The plates were washed in 0.1 m Tris buffer

with and without 0.01% Triton-X 100, and conjugation of

streptavidin–peroxidase to the biotinylated peptide was

detected by the addition of H2O2 and o-phenylenediamine

An equal volume of 2.5 n H2SO4 was added, and the

absorbance at 492 nm was measured

Detection of in situ TGase 1 activity in mouse

skin sections

Fluorescence-labeled peptides (pepK5 and

FITC-pepK5QN) were synthesized, and skin sections were

pre-pared from wild-type and TGase 1-null mice using standard

methods [20] The frozen sections were dissected into 3-lm

slices and frozen until use

Sections were dried and then blocked with 1% BSA in

NaCl⁄ Piat room temperature The sections were incubated

for 90 min with a solution containing 100 mm Tris⁄ HCl

pH 8.0, 5 mm CaCl2 or 1 mm EDTA, and 1 mm

dithio-threitol, in the presence of labeled peptide or

FITC-cadaverine (Sigma) After washing with NaCl⁄ Pithree times

for 5 min, anti-fading solution was added to the sections,

which were then covered with a cover glass

For hematoxylin and eosin staining, tissue sections were

fixed and stained using standard methods For

immuno-staining, skin tissue sections were analyzed using a

monoclo-nal anti-mouse TGase 1 IgG [40] The skin sections were

fixed in a solution containing methanol and acetone (1 : 1)

and then blocked in NaCl⁄ Pi containing 0.1% BSA The

sections were incubated with the antibody at 37C for

90 min, and the immunoreactive proteins were detected

using biotinylated secondary antibody (anti-rat IgG) and

FITC-avidin (Zymed, San Francisco, CA, USA)

Fluores-cence was analyzed by confocal laser-scanning microscopy

(Carl Zeiss, Oberkochen, Germany)

Acknowledgements

We are grateful to Dr K Ikura (Graduate School of Science and Technology, Kyoto Institute of Technol-ogy, Japan) for providing guinea pig liver TGase 2,

Dr T Yoshimura (Graduate School of Bioagricultural Sciences, Nagoya University, Japan) for technical advice on analysis of the tissue sections, and Dr

H Shibata of our laboratory for his valuable sugges-tions This work was supported by Grant-in-Aid for Scientific Research (C) number 19580103 to K.H., a grant from the Cosmetology Foundation to K.H., and

a Grant-in-Aid for Young Scientists Research number

186701 to Y.S Y.S is a research fellow at the Japan Society for the Promotion of Science

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