(Received for publication, October 3, 1996, and in revised form, February 4, 1997)
From the Department of Cell and Molecular Biology and the Feinberg Cardiovascular Research Institute, Northwestern University Medical School, Chicago, Illinois 60611
N-(
-glutamyl)lysine
cross-links, connecting various peptide chain segments, are frequently
the major products in transglutaminase-catalyzed reactions. We have now
investigated the effectiveness of these enzymes for hydrolyzing the
:
linkage. Branched compounds were synthesized, in which the
backbone on the
-side of the cross-bridge was labeled with a
fluorophor (5-(dimethylamino)-1-naphthalenesulfonyl or 2-aminobenzoyl)
attached through an
-aminocaproyl linker in the N-terminal position,
and the other branch of the bridge was constructed with Lys methylamide
or diaminopentane blocked by 2,4-dinitrophenyl at the
N
position. Hydrolysis of the
cross-link could be followed in these internally quenched substrates by
an increase in fluorescence. In addition to the thrombin and
Ca2+-activated human coagulation Factor XIIIa,
cytosolic transglutaminases from human red cells and from guinea pig
liver were tested. All three enzymes were found to display good
isopeptidase activities, with Km values of
10
4 to 10
5 M.
Inhibitors of transamidation were effective in blocking the hydrolysis
by the enzymes, indicating that expression of isopeptidase activity did not require unusual protein conformations. We
suggest that transglutaminases may play a dynamic role in biology not only by promoting the formation but also the breaking of
N-(
-glutamyl)lysine
isopeptides.
Apart from obvious differences in substrate specificities for the
-carbonyl groups of endo-Lys and Arg residues by papain (EC
3.4.22.2) and for the
-carbonyl groups of certain endo-Gln residues
by transglutaminases (EC 2.3.2.13), considerable kinetic and
mechanistic similarities exist between these two families of enzymes.
Both operate by acylation-deacylation pathways, with a Cys thiol in the
catalytic center assisted by a His residue (1-6). However, because of
the exceptional specificities of transglutaminases for amines mimicking
the
-amino groups of Lys side chains in proteins (7-9), this group
of enzymes shows a unique ability for generating protein-to-protein
N
-(
-glutamyl)lysine cross-links, a
post-translational reaction of major biological significance.
Transglutaminases are known to participate in various clotting
phenomena (7, 10-16), in the assembly of extracellular matrices (17)
and of intracellular polymeric structures in cells under
Ca2+ stress (18-22), and in apoptosis (23).
While a great deal of attention has been paid to the amine transferase
activities of transglutaminases (3, 4, 24), i.e. to the
production of N-(
-glutamyl)lysine
bridges and the incorporation of small molecular weight amines into
proteins, the isopeptide breaking potential of the enzymes has not yet
been explored. Since lack of availability of appropriate substrates may
have been a main reason, we embarked on synthesizing
-branched
peptides with built-in features, which would facilitate the application
of fluorescence methodologies for kinetic studies. Two cytosolic
transglutaminases of different properties, isolated from human red
blood cells (HTg)1 and from guinea pig
liver (GTg) respectively, and a recombinant form of the human
coagulation Factor XIIIa (rA2*) were employed as enzymes.
Organic Synthesis
Reagents, solvents, and blocked amino acid derivatives were purchased from Aldrich, Sigma, and Bachem Bioscience Inc. TLC (thin-layer chromatography) was performed on Whatman K6F-Silica gel glass plates (0.25 mm) using the following solvent systems (v/v): (A) chloroform/methanol/glacial acetic acid (10:3:1); (B) chloroform/methanol/2-propanol (10:4:4); (C) n-butanol/glacial acetic acid/water (15:6:5); (D) 1-propanol/water (7:3); (E) propanol/water/concentrated ammonium hydroxide/ethanol (7:4:2:3). Plates were viewed under UV light (at 254 nm and 366 nm for detection of UV absorbing and fluorescent moieties, respectively) or were developed by ninhydrin (0.25% in 1-butanol for N-deblocked peptides) or by hypochlorite (10%) followed by starch/KI spray for N-blocked peptides (26). Melting points were determined with a Büchi apparatus and are uncorrected. After acid hydrolysis, amino acid analyses were kindly carried out by Dr. Thomas J. Lukas of the Department of Molecular Pharmacology and Biochemistry, Northwestern University Medical School, Chicago, IL. Elemental analyses were performed by G. D. Searle Laboratories, Skokie, IL.
Peptide Coupling
To a stirred and cooled (0 °C) 0.5-0.8 M solution of the pertinent Boc amino acid in dry DMF were added equimolar amounts of 1-hydroxybenzotriazole and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride. The mixture was stirred at 0 °C for 40 min and then added to a solution of the trifluoracetate salt of the peptide benzyl ester (obtained by acidolytic deblocking of the Boc-peptide benzyl ester described below) in dry DMF, which was pre-neutralized with N-methylmorpholine. The reaction mixture was stirred at 0 °C for 1 h and then at room temperature for 18-36 h. The mixture was evaporated under reduced pressure to remove DMF, and the residue was stirred with 3% sodium bicarbonate for 15 min. The precipitated product was filtered off, or separated by centrifugation, washed with 5% NaHCO3, water, cold 0.5 N HCl, water and dried in vacuo in a P2O5 desiccator for ~18 h. When necessary, the product was reprecipitated from DMF-water. Alternatively, the product was extracted with ethyl acetate, and the organic extract was washed as above, dried over anhydrous Na2SO4, and the solvent evaporated to give the product.
Deblocking of the Boc Group
To 1.0 mmol of the Boc-peptide benzyl ester was added 2 ml of 50% trifluoroacetic acid in anhydrous dichloromethane. The solution was allowed to stand at room temperature for 1 h, and excess trifluoroacetic acid was removed by adding fresh dichloromethane to the mixture followed by evaporation under reduced pressure, then by addition of anhydrous ether to the residue. The precipitated trifluoroacetic acid salt of the peptide benzyl ester was filtered off, washed with anhydrous ether, and dried under vacuum in a desiccator for 2 h before proceeding with the coupling reaction.
Deblocking of the Benzyl Ester Group
The blocked peptide benzyl ester was dissolved in a mixture of DMF/ethanol/water in the approximate volume ratio of 10:20:2 at 50 °C and hydrogenated in presence of 10% Pd/C catalyst for 3.5 h at 50 °C with stirring. After cooling to room temperature, water and 1 N NH4OH to pH 9 were added and the mixture filtered to remove the catalyst through a Celite filter pad. The filtrate was evaporated under reduced pressure, the residue was taken up in a minimum volume of absolute ethanol, and about 10 volumes of anhydrous ether added and cooled to 0 °C. The precipitated product was filtered off, washed with anhydrous ether, and dried in vacuo.
Compounds Used as First Substrates
Dns-Eaca-Gln-Gln-Ile-Val (I)This was synthesized as described in Ref. 27.
Dns-Eaca-Glu-[This was synthesized by a multistep procedure as
follows. First, Boc-Glu--benzyl ester was coupled to
mono-Z-cadaverine (24), essentially according to the general procedure
above and the product isolated from an ethyl acetate extract of the
reaction mixture to give an 80% yield of
Boc-Glu[
-(Cad-Z)]-
-benzyl ester, melting point (m.p.)
68-71 °C, RF 0.91 (B), 0.92 (C), 0.87 (D), and
0.89 (E). Catalytic hydrogenation of this intermediate followed by
reaction with N-(benzyloxycarbonyloxy)succinimide in
presence of NaHCO3 according to the general procedures gave
Boc-Glu-[
-(Cad-Z)] in 75% yield, RF 0.34 (B),
0.80 (C), 0.66 (D), and 0.72 (E). This was coupled by the above general
procedure with the trifluoracetate salt of Gln-Ile-Val-O-Bzl
(28) to give an 83% yield of
Boc-Glu[
-(Cad-Z)]-Gln-Ile-Val-O-Bzl, m.p.
229-231 °C, RF 0.91 (A), 0.89 (C), 0.82 (D), and
0.89 (E). This material was deblocked to remove the Boc group and
coupled with equimolar amount of Dns-Eaca according to the general
procedures to give the fluorescent peptide intermediate,
Dns-Eaca-Glu[
-(Cad-Z)]-Gln-Ile-Val-O-Bzl, yield 81%,
m.p. (dec.) 238-240 °C, RF 0.94 (A), 0.88 (C),
0.86 (D), and 0.91 (E). This was catalytically hydrogenated to give
Dns-Eaca-Glu-(
-Cad)-Gln-Ile-Val, RF 0.26 (A), 0.31 (C), 0.55 (D), and 0.63 (E). This was then reacted in 50% aqueous
ethanol with a 40% molar excess of 2,4-dinitrofluorobenzene and
NaHCO3. The reaction mixture was stirred at 0 °C for
0.5 h and at room temperature for 14 h while protected from
light. About 15 drops of concentrated Na2CO3
solution were added to the mixture to raise the pH to 9-9.5 (for
decomposing excess 2,4-dinitrofluorobenzene), and ethanol was
evaporated under reduced pressure. After adding 5 ml of water, the
solution was centrifuged to remove a small amount of precipitate. The
clear supernatant was separated, cooled (0 °C), and acidified to pH
2.5 with cold 2 N HCl with stirring. The yellow precipitate
was collected by centrifugation, washed with cold water (3 × 3 ml), and finally washed with ether (5 × 5 ml). The precipitate
was triturated with ethanol-ether (1:10) to remove the contaminating
2,4-dinitrophenol and dried in vacuo to give a 40% yield of
the titled quenched fluorescent peptide, Dns-Eaca-Glu[
-(Cad-Dnp)]-Gln-Ile-Val, as a yellow solid, m.p. (dec.) 223-225 °C, RF 0.77 (A), 0.74 (C), 0.69 (D), and 0.83 (E). 1H NMR
(Me2SO-d6) was in agreement with the
structure. Amino acid analysis gave Glu 2.17 (2), Ile 0.92 (1), and Val
0.91 (1).
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This was synthesized essentially as the
-Cad-Dnp analog peptide. First, Z-Lys(
-Boc) was coupled with
methylamine hydrochloride in presence of N-methylmorpholine
by the general method to give Z-Lys(
-Boc)-NHCH3, yield
92%, m.p. 125-127 °C, RF 0.94 (B), 0.87 (C),
0.77 (D), and 0.85 (E). Removal of Boc group followed by coupling with
Boc-Glu-
-O-Bzl gave a 94% yield of
Boc-Glu-[
-
-(
-Z-Lys-NHCH3)]-
-O-Bzl, m.p. 162-164 °C, RF 0.92 (B), 0.86 (C), 0.82 (D), and 0.87 (E). Further reactions similar to those described for
peptide II gave
Dns-Eaca-Glu-[
-
(
-Z-Lys-NHCH3)]-Gln-Ile-Val-O-Bzl in about 60% yield, m.p. (dec.) 234-236 °C, RF
0.9 (B), 0.76 (C), 0.79 (D), and 0.89 (E). This was catalytically
hydrogenated and reacted with 2,4-dinitrofluorobenzene by a similar
procedure as above to give the titled quenched fluorescent peptide
(III), yield 21%, yellow solid, m.p. (dec.) 216-218 °C,
RF 0.15 (B), 0.73 (C), 0.68 (D), and 0.84 (E). Amino
acid analysis gave Glu 2.0 (2), Val 0.79 (1), and Ile 0.81 (1).
Starting with 2-aminobenzoic acid (anthranilic
acid), Boc-Abz was prepared according to the literature procedure (29),
m.p. 152-154 °C, m.p. (literature) 149-150 °C,
RF 0.79 (B), 0.93 (C), 0.76 (D), and 0.77 (E).
Coupling of this with -aminocaproic acid by the general procedure
gave a 74% yield of Boc-Abz-Eaca, RF 0.79 (B), 0.79 (C), 0.74 (D), and 0.66 (E). Further reactions similar to those carried
out for the synthesis of II above gave a 32% yield of
Boc-Abz-Eaca-Glu-[
-(Cad-Dnp)]-Gln-Ile-Val, yellow solid, m.p.
(dec.) 225-227 °C, RF 0.72 (C), 0.68 (D), and
0.8 (E). This was finally deblocked to remove the Boc group according
to the general procedure above to give a 66% yield of the
trifluoroacetate salt of the titled compound, IV, as a
yellow solid m.p. (dec.) 230-232 °C, RF 0.64 (C), 0.56 (D), and 0.74 (E). Amino acid analysis gave Glu 2.37 (2), Ile
0.86 (1), and Val 0.76 (1).
Coupling of dansylcadaverine (Cad-Dns) with
Boc-Glu--O-Bzl followed by hydrogenation according to the
published procedure (30) gave Boc-Glu[
-(Cad-Dns)], which was
subsequently coupled by the general procedure with the trifluoroacetate
salt of Gln-Ile-Val-O-Bzl (28) to give a 76% yield of
Boc-Glu[
-(Cad-Dns)]-Gln-Ile-Val-O-Bzl, m.p.
210-212 °C, RF 0.94 (A), 0.94 (B), 0.91 (C),
0.86 (D), and 0.91 (E). Catalytic hydrogenation of this material gave the titled fluorescent peptide, V, yield 84%, m.p. (dec.) 212-214 °C, RF 0.8 (A), 0.3 (B), 0.84 (C), 0.74 (D), and 0.88 (E). Amino acid analysis gave Glu 2.1 (2), Ile 0.88 (1),
and Val 0.98 (1).
The above peptide intermediate was deblocked to
remove Boc group and coupled with pGlu-Ala according to the general
procedures to give
pGlu-Ala-Glu[-(Cad-Dns)]-Gln-Ile-Val-O-Bzl, yield 81%, m.p. (dec.) 276-278 °C, RF 0.1 (B), 0.64 (C),
0.68 (D), and 0.79 (E). Finally, hydrogenation, followed by
recrystallization, gave a 20% yield of the titled peptide,
VI, m.p. (dec.) 259-261 °C, RF 0.18 (A), 0.1 (B), 0.62 (C), 0.65 (D), and 0.73 (E). Amino acid analysis
gave Glu 3.23 (3), Ala 1.1 (1), Ile 0.83 (1), and Val 0.83 (1).
First, Dns-Lys-NHCH3
(XIII) described below was coupled with
Boc-Glu-O-Bzl and the product hydrogenated to give an 80%
yield of Boc-Glu-[-
-(
-Dns-Lys-NHCH3)], as an
amorphous solid, RF 0.29 (B), 0.73 (c), 0.66 (D),
and 0.78 (E). This was coupled by the general procedure with the
trifluoroacetate salt of Gln-Ile-Val-O-Bzl (see Ref. 28) to
give a 60% yield of
Boc-Glu-[
-
-(
-Dns-Lys-NHCH3)]-Gln-Ile-Val-O-Bzl,
m.p. (dec.) 206-208 °C, RF 0.93 (B), 0.81 (C),
0.82 (D), and 0.89 (E). Hydrogenation of this material gave the titled
fluorescent peptide, VII, yield 89%, m.p. (dec.)
210-212 °C, RF 0.18 (B), 0.74 (C), 0.68 (D), and
0.79 (E). Amino acid analysis gave Glu 2.24 (2), Ile 0.87 (1), and Val
0.88 (1).
First, Boc-Gly-Pro was coupled to
Leu-O-Bzl by the general procedure to give a 80% yield of
Boc-Gly-Pro-Leu-O-Bzl, m.p. 118-120 °C, RF 0.93 (B), 0.9 (C), 0.82 (D), and 0.88 (E). This
peptide was deblocked to remove Boc group and coupled to
Boc-Gln-Ile-Val (31) to give a 54% yield of
Boc-Gln-Ile-Val-Gly-Pro-Leu-O-Bzl, m.p. 226-228 °C,
RF 0.93 (B), 0.8 (C), 0.77 (D), and 0.87 (E). This
was subsequently deblocked to remove Boc group and coupled to
Boc-Glu-[-
-(
-Dns-Lys-NHCH3)] as described under compound VII above to give a 71% yield of
Boc-Glu-[
-
-(
-Dns-Lys-NHCH3)]-Gln-Ile-Val-Gly-Pro-Leu-O-Bzl, m.p. (dec.) 209-212 °C, RF 0.9 (B), 0.79 (C),
0.77 (D), and 0.91 (E). Hydrogenation of this material gave the titled
fluorescent peptide, VIII, yield 90%, m.p. (dec.)
216-218 °C, RF 0.23 (B), 0.66 (C), 0.68 (D), and
0.84 (E). Amino acid analysis gave Glu 2.25 (2), Gly 0.94 (1), Leu 1.01 (1), Pro 1.09 (1), Ile 0.79 (1), and Val 0.76 (1).
This was synthesized by a procedure similar to
that for peptide II above. First, Dns-Eaca was coupled with
alanine benzyl ester and the product hydrogenated to give a 95% yield of Dns-Eaca-Ala, m.p. 82-85 °C, RF 0.36 (B),
0.78 (C), 0.65 (D), and 0.74 (E). This was then coupled to
Glu[-(Cad-Z)]-Gln-Ile-Val-O-Bzl to give a 80% yield of
Dns-Eaca-Ala-Glu-[
-(Cad-Z)]-Gln-Ile-Val-O-Bzl, m.p.
(dec.) 258-261 °C, RF 0.89 (B), 0.83 (C), 0.79 (D), and 0.88 (E). Amino acid analysis gave Ala 1.18 (1), Glu 2.3 (2),
Ile 1.0 (1), and Val 0.86 (1). This was hydrogenated and reacted with
2,4-dinitrofluorobenzene by a procedure similar to that above to give
the titled quenched fluorescent peptide, IX, yield 11%,
yellow solid, m.p. (dec.) 235-237 °C, RF 0.3 (B), 0.68 (C), 0.62 (D), and 0.86 (E). Amino acid analysis gave Ala
1.03 (1), Glu 2.39 (2), Ile 1 (1), and Val 0.9 (1).
Boc-Ala-Gln-Gln-Ile-Val-O-Bzl (28) was deblocked with trifluoroacetic acid to remove the Boc group and coupled to Boc-Pro to give Boc-Pro-Ala-Gln-Gln-Ile-Val-O-Bzl, yield 91%, m.p. (dec.) 270-272 °C, RF 0.78 (A), 0.77 (C), 0.73 (D), and 0.8 (E). This was deblocked with trifluoroacetic acid and coupled to Dns-Eaca to give Dns-Eaca-Pro-Ala-Gln-Gln-Ile-Val-O-Bzl, yield 88%, m.p. (dec.) 270-272 °C, RF 0.81 (A), 0.55 (B), 0.63 (C), 0.57 (D), and 0.82 (E). Catalytic hydrogenation of this material gave the titled fluorescent peptide, X, yield 77%, m.p. (dec.) 248-250 °C, RF 0.44 (A), 0.08 (B), 0.55 (C), 0.55 (D), and 0.72 (E). Amino acid analysis gave Glu 2.03 (2), Pro 1.29 (1), Ala 1.28 (1), Ile 0.82 (1), and Val 0.92 (1).
Amine Compounds Used as Second Substrates for Studying the Enzyme-catalyzed Formation of Isopeptides
Dnp-Cadaverine (XI)This was prepared according to the published procedure (24).
The
Z-Lys(-Boc)-NHCH3 intermediate was catalytically
hydrogenated and reacted with 2,4-dinitrofluorobenzene to give
Dnp-Lys(
-Boc)-NHCH3, yield 79%, a yellow solid, m.p.
149-151 °C, RF 0.96 (B), 0.88 (C), 0.80 (D), and
0.86 (E). This was deblocked with trifluoroacetic acid according to the
general procedure to give an 80% yield of the initially hygroscopic
trifluoroacetate salt, which was converted to the hydrochloride
derivative by adding 1N HCl in 2-propanol followed by anhydrous ether
to give the HCl salt of Dnp-Lys-NHCH3, yellow solid, m.p.
135-137 °C, RF 0.4 (B), 0.49 (C), 0.57 (D), and
0.59 (E).
This was prepared similarly to the Dnp analog, with dansyl chloride replacing the reagent in the preparation to give the HCl salt of Dns-Lys-NHCH3, yield 76%, m.p. 194-196 °C, RF 0.08 (B), 0.38 (C), and 0.58 (E).
Dbc-Cadaverine (XIV)First,
4-(4-dimethylaminophenylazo)benzoic acid sodium salt was coupled to
Boc-cadaverine hydrochloride (32) in presence of two equivalents of
N-methylmorpholine by a slightly modified peptide coupling
method. A 2-fold molar excess of 1-hydroxybenzotriazole was used in the
coupling, and the reaction was carried out at room temperature for
24 h and the product isolated from an ethyl acetate extract of the
reaction mixture to give dabcylcadaverine-Boc, a bright red solid,
yield 71%, m.p. 159-161 °C, RF 0.97 (A), 0.94 (C), 0.87 (D), and 0.93 (E). 1H NMR
(Me2SO-d6) was in agreement with the
structure. This was then deblocked with 50% trifluoroacetic acid in
anhydrous dichloromethane as described above to give a 77% yield of
dabcylcadaverine trifluoroacetate as a red solid, m.p. 177-179 °C,
RF 0.33 (A), 0.55 (C), 0.21 (D), and 0.65 (E). The
absorption spectrum showed maxima at 470 nm ( = 23, 610; pH 7.5) and
502 (
= 45, 860; pH 1.0).
Reference Compound: Boc-Glu-Gln-Ile-Val-Gly-Pro-Leu (XV)
The intermediate in the synthesis of VIII above,
Boc-Gln-Ile-Val-Gly-Pro-Leu-O-Bzl was deblocked by removing
the the Boc group and was then coupled to Boc-Glu--O-Bzl
to give a 76% yield of
Boc-Glu-(
-O-Bzl)-Gln-Ile-Val-Gly-Pro-Leu-O-Bzl,
m.p. 237-239 °C, RF 0.94 (B), 0.87 (C), 0.80 (D), and 0.86 (E). This was hydrogenated according to the general
procedure to give a 76% overall yield of
Boc-Glu-Gln-Ile-Val-Gly-Pro-Leu (XV), m.p. (dec.)
249-252 °C , RF 0.06 (B), 0.65 (C), 0.64 (D),
and 0.66 (E). Amino acid analysis gave Glu 2.26 (2), Gly 1.04 (1), Pro
1.14 (1), Val 0.8 (1), Ile 0.83 (1), and Leu 1.24 (1).
Enzymatic Studies
Guinea pig liver transglutaminase (GTg; Refs. 33 and 34) and
human red blood cell transglutaminase (HTg; Refs. 35 and 36) were
purified as described previously and stored at 80 °C. Protein
concentrations for GTg were measured by absorbance at 280 nm
(
1 cm1% = 15.8). Protein
concentrations for HTg were determined with the BCA protein assay
(Pierce), utilizing bicinchoninic acid with bovine serum albumin for
standard. Calculations were based on molecular weights of 76,600 for
GTg (37) and 80,000 for HTg (35).
Transglutaminase activity was measured in a CytoFluor model 2300 (Millipore, Bedford, MA), upgraded to a model 2350, by monitoring the rate of increase in fluorescence during the transglutaminase-catalyzed incorporation of dansylcadaverine (38) into N,N-dimethylcasein (excitation filter = 360 ± 40 nm; emission filter = 490 ± 40; sensitivity 6). Incubations were carried out at 37 °C in a Millipore 96-well low fluorescence CytoPlate in 125-µl reaction mixtures which comprised 50 mM Tris-HCl, pH 7.5, 0.1 mM dansylcadaverine, 2 mg/ml N,N-dimethylcasein, 1 mM dithiothreitol, 1 mM CaCl2, and 0-0.124 µM GTg or HTg.
Recombinant human factor XIII A subunit (rA2; Ref. 39), a
gift from Dr. Paul D. Bishop (Zymogenetics, Seattle, WA), was converted
to rA2 by incubating 4-8 µM rA2
with 32-64 NIH units/ml human
-thrombin (a gift from Dr. J. W. Fenton III, New York State Department of Health, Albany, NY) in 50 mM N-methylmorpholine, pH 7.5, for 30 min at
room temperature. Thrombin activity was then quenched by the addition
of a 4-fold excess of hirudin (128-256 units/ml; Sigma). Protein
concentration for rA2 was determined using absorbance at
280 nm (
1 cm1% = 14.9).
Thin Layer and High Performance Liquid Chromatography (TLC and HPLC)
Mixtures of 100 µl comprising 50 mM
N-methylmorpholine-HCl, pH 7.5, ionic strength 0.1 (adjusted
with NaCl), 100 µM
Boc-Glu[-
-(
-Dns-Lys-NHCH3)]-Gln-Ile-Val-Gly-Pro-Leu (VIII) or
-Dns-Lys-NHCH3 (XIII),
0.65 µM GTg, 1 mM DTT, and either 1 mM CaCl2 or 1 mM EDTA were
incubated at 37 °C for 120 min, when 2 µl of 100 mM
EDTA were added to stop the reaction; 25-µl samples were spotted
(2 × 10 mm) on a Polygram TLC plate (0.1-mm Polyamide-6, 20 × 20 cm; Macherey & Nagel, Alltech Associates, Deerfield, IL), and
separation was accomplished in an ascending mode in aqueous 1%
pyridine, pH 5.4, for 60 min (40). The dried plate was photographed
under UV light (366 nm).
HPLC separations were also performed on the same mixtures.
Approximately 50 µl of sample was mixed with 60 µl of 0.6 M perchloric acid and centrifuged (2 min, 15,600 × g), and 100 µl was injected onto an Ultrasphere
C8 column (Beckman, Fullerton, CA) using gradients formed
with H2O (containing 0.1% trifluoroacetic acid) and MeCN (containing 0.1% trifluoroacetic acid): from injection to 20 min, linear increase of MeCN to 20%; 20 to 22 min, isocratic 20% MeCN; 22 to 32 min, linear increase of MeCN to 30%; at 32 min, MeCN was
eliminated (0.1 min) and the column was then re-equilibrated with 0.1%
trifluoroacetic acid in H2O for 15 min. Peaks were detected by absorbance at 220 nm and by fluorescence (exc = 338 nm;
em = 500 nm), recorded on a Hewlett-Packard 3390A
integrator and collected with a Foxy 200 fraction collector (ISCO,
Lincoln, NE) set for detection of slope. Collected fractions were
concentrated on a Savant (Farmingdale, NY) Speed-Vac concentrator.
Monitoring Transglutaminase-catalyzed Isopeptide Formation or the Breaking of Isopeptide Bonds by Changes in Fluorescence
Conditions for individual experiments are specified in the
figure legends. Fluorescence measurements in the CytoFluor instrument were carried out with an excitation filter = 360 ± 40 nm and
an emission filter = 590 ± 35 nm at sensitivities of 7 or 8. Incubations were set up in a CytoPlate at 37 °C in 150-µl reaction
mixtures, which, in addition to the specified substrates and
inhibitors, comprised 50 mM buffer (either Tris-HCl,
N-methylmorpholine-HCl or sodium acetate:acetic acid), 1 mM DTT, 1 mM CaCl2, and one of the
transglutaminases or rA2. Ionic strength was adjusted with
NaCl as specified. For monitoring isopeptide formation, fluorescence readings were expressed as a percentage of the initial fluorescence measured in the absence of enzyme. Fluorescence readings for the breaking of the isopeptide bonds were corrected for the initial background fluorescence in the absence of enzyme.
Inhibition of the rA2*-catalyzed hydrolysis of
Abz-Eaca-Glu-[-(Cad-Dnp]-Gln-Ile-Val (IV) by
1,3,4,5-tetramethyl-2[(2-oxopropyl)thio]imidazolium chloride
(L-682,777 (41); prepared in Me2SO and stored at
20 °C; kindly provided by Dr. Andrew M. Stern of Merck Research
Laboratories, West Point, PA) was measured on a SLM (Urbana, IL) model
8000C spectrofluorometer (excitation wavelength = 320 nm, emission
wavelength = 410 nm, high voltage = 400 V, gain = 100) at
37 °C. GTP and GTP
S (Sigma) were used as equimolar mixtures with
MgCl2.
Three representative preparations from the family of
transglutaminases, exhibiting different kinetic and physical
properties, were employed in this study. The cytosolic
transglutaminases were purified from human red cells and from guinea
pig liver, whereas the activated form of human fibrin stabilizing
factor (Factor XIIIa = A2*) was generated by
treatment with thrombin and Ca2+ from the recombinant
placental rA2 zymogen. The findings presented in Fig.
1 serve as evidence that transglutaminases can be
effective in catalyzing the cleavage of the :
isopeptide bond. In
panel A (lane 2), thin layer chromatographic
separation was used to demonstrate the production of fluorescent
-Dns-Lys-NHCH3, marked as P1, during
the liver transglutaminase-catalyzed hydrolysis of the branched peptide
substrate:
Boc-Glu-[
-
(
-Dns-Lys-NHCH3)]-Gln-Ile-Val-Gly-Pro-Leu (VIII). No release of P1, identified by the
mobility (RF ~ 0.6) of the reference compound
XIII in lane 3, occurred with the enzyme when
Ca2+ was replaced by EDTA, as in lane 1.
Analysis by HPLC, presented in panel B, confirmed the
formation of
-Dns-Lys-NHCH3 as the
fluorescent product P1 (graph 2)
eluting at the position (~14.5 min) of the reference compound
XIII (graph 3), while the amount of the starting
fluorescent substrate S (i.e. compound
VIII, eluting at ~36.5 min) diminished by about 60%
(compare graphs 1 and 2). Monitoring by
absorbance at 220 nm revealed the production of a non-fluorescent peak,
representing the second product of hydrolysis in the
Ca2+-containing enzymatic mixture, with an elution time
(~32.5 min) corresponding to that of the
Boc-Glu-Gln-Ile-Val-Gly-Pro-Leu reference (XV; data not
shown). These findings established the catalytic potential of
transglutaminases for hydrolyzing the isopeptide bond. However, it
became obvious that a more penetrating evaluation of branched
substrates would require a different analytical approach, and, with
this in mind, fluorescence quenching procedures were explored first for
the enzyme-catalyzed conventional reaction of a Gln residue-containing
acceptor with a primary amine as the donor substrate.
Isopeptide Formation Monitored by the Quenching of Fluorescence
Acceptor substrates were synthesized with a
fluorescent N-terminal blocking group (Dns), whereas the donor
substrates (cadaverine or Lys-NHCH3) contained some
quenching moiety (Dnp or Dbc) in the N-terminal position. Affinities of
the new substrates for the enzymes were sufficiently favorable so that
they could be employed at low enough concentrations for minimizing
bimolecular quenching in the starting mixtures. However, as the
coupling reaction for forming the :
product progressed with the
addition of enzymes, a significant drop in fluorescence ensued. This
was attributed to the intramolecular quenching effect exerted by the
Dnp or Dbc group on the Dns fluorophore concomitant with forming the
isopeptide linkage. This approach, tested for a variety of substrate
pairs, proved to be highly sensitive for measuring the rate of
isopeptide formation. Catalysis by the human red cell and the guinea
pig liver enzyme was explored either with Dnp-cadaverine
(XI), Dbc-cadaverine (XIV), or
-Dnp-Lys-NHCH3 (XII) as donor, in conjunction
with Dns-Eaca-Gln-Gln-Ile-Val (I) as the acceptor substrate.
Figs. 2 and 3 illustrate the findings for
the tissue type of transglutaminases. Fig. 2 pertains to the reaction
of the Dns-labeled acceptor (I) with
Dnp-Lys-NHCH3 (XII) as promoted by guinea pig
liver transglutaminase, whereas Fig. 3 presents the data for the human
red cell enzyme-catalyzed reaction between I and
Dbc-cadaverine (XIV). Dns-Eaca-Pro-Ala-Gln-Gln-Ile-Val (X) could be used as first substrate either with
Dbc-cadaverine (XIV) or with Dnp-cadaverine (XI)
as second substrate for following the coupling reactions catalyzed by
rA2* (data not shown). These results guided our synthetic
work for designing
-branched peptides for examining the isopeptide
breaking potentials of transglutaminases.
Transglutaminase-catalyzed Breaking of the Isopeptide Bond
We
synthesized a number of intramolecularly quenched compounds containing
an isopeptide linkage (described under "Materials and Methods") and
followed the actions of the enzymes by the increase in fluorescence
resulting from the release of the quenching moiety (Dnp) attached to
the leaving amine group. On the side of the backbone the substrates
carried either a Dns or an Abz fluorophor in an N-terminal
position.
Figs. 4 and 5 show the progression curves
for the hydrolysis of the
Dns-Eaca-Glu-[-
(
-Dnp-Lys-NHCH3)]-Gln-Ile-Val
(III) (10
4 M) substrate with the
guinea pig liver and of compound II with the human red blood cell
enzyme, respectively. Approximately twice as much GTg (0.43 µM) was used for demonstrating the hydrolytic cleavage of
compound III (0.1 mM) in Fig. 4 than the
concentration of the enzyme (0.21 µM) for generating the
highest rate of cross-bridge formation in the coupling reaction between
compounds (I; 0.24 mM) and XII (0.5 mM), depicted by solid triangles (
) in Fig. 2. Because
of inherent ambiguities in comparing rate constants for the reaction of
a single substrate (as in the experiment in Fig. 4) with those for a
two-substrate reaction (as in Fig. 2), we did not attempt to draw
kinetic comparisons between these different enzymatic processes.
Some of the -branched peptides also satisfied the more restrictive
specificity requirement of human Factor XIIIa. Fig.
6 presents our data by for the hydrolysis of
Dns-Eaca-Ala-Glu[
-(Cad-Dnp)]-Gln-Ile-Val (IX) by
rA2* and Fig. 7 for the reaction of the same
enzyme with Abz-Eaca-Glu[
-(Cad-Dnp)]-Gln-Ile-Val (IV).
Because of the limitations of the CytoFluor plate reader, fluorescence of the Abz-blocked compound (
exc = 320 nm and
em = 410 nm), could only be monitored in the
spectrophotofluorimeter. Fig. 7 shows that the hydrolytic activity of
rA2* on the
-branched peptide could be abolished by an
active site-directed inhibitor of Factor XIIIa:
1,3,4,5-tetramethyl-2-[(2-oxopropyl)thio]imidazolium chloride (41).
At the same concentration (10
4 M), this
compound also inhibited totally the hydrolysis of
Dns-Eaca-Glu[
-(Cad-Dnp)]-Gln-Ile-Val (II) by the human
red cell transglutaminase (5.5 × 10
7 M)
measured in the CytoFluor under similar conditions (data not shown).
Hydrolysis of Dns-Eaca-Glu[-(Cad-Dnp)]-Gln-Ile-Val (II)
by the human red cell transglutaminase proceeded fastest in the pH
6.5-7.5 range (ionic strength ~ 0.1), with reaction rates falling
off on either side of this pH range (Fig. 8). For the guinea pig liver enzyme, the apparent optimum for the hydrolysis of the
same substrate was in the pH 5.5-7 range (data not shown).
Tissue transglutaminases are negatively regulated by GTP for
incorporating amines into protein substrates (42-44). We tested the
effects of equimolar GTP·Mg2+ complexes on the
isopeptidase activities of the enzymes. As illustrated in Fig.
9, GTP was found to exert a very strong inhibitory
effect on the hydrolysis of Dns-Eaca-Glu[-(Cad-Dnp)]-Gln-Ile-Val
(II) by the human red cell enzyme (I50 ~ 5-10 × 10
6 M GTP). However, even the
~90% inhibition caused by 2 × 10
5 M
GTP·Mg2+ at low Ca2+ (10
3
M), could be overcome substantially at higher
concentrations of Ca2+ (Fig. 10). This
finding supports the concept (43) that the binding of GTP causes a
reduction in the Ca2+ sensitivity of transglutaminase. The
inhibitory effect of GTP
S was indistinguishable from that of GTP
(data not shown).
Transglutaminases are known to catalyze the hydrolysis of protein or
peptide-bound Gln to Glu residues (45), and also to hydrolyze
nitrophenyl and thiocholine esters (3, 4, 24). The hydrolytic nature of
transglutaminases is brought even more to the forefront by the examples
provided in this paper for the breaking of isopeptide linkages, with
high affinities for the -branched substrates (e.g.
Km ~ 10
5 M for substrate
II by the human red cell enzyme; Fig. 5). Inhibition by the
active-site directed blocking agent:
1,3,4,5-tetramethyl-2-[(2-oxopropyl)thio]-imidazolium chloride
(Fig. 7) and also by GTP or GTP
S (Figs. 9 and 10), which modulate
the Ca2+ sensitivities of cytosolic transglutaminases, show
that the same functional domains are involved in the expression of
isopeptidase activities as in the well studied transamidating reactions
promoted by these enzymes.
Claims have been made (46, 47) and refuted (48) for having isolated an
isopeptidase, called destabilase, from leech saliva with specificity
for hydrolyzing the
N-(
-glutamyl)lysine bonds between
the
-chains of solubilized fibrin. The primary structure of
destabilase, derived from the cDNA clone, does not share
significant homology with transglutaminase (49). Based on the findings
described in the present paper, it may be suggested that if a select
group of enzymes exists with properties that would uniquely define them
as isopeptidases, they would probably also display transamidating
activities, the characteristic attributes of transglutaminase. One of
the enzymes, Factor XIIIa, which was shown in our
experiments to exhibit isopeptidase activity (Figs. 6 and 7), has been
reported to actually hydrolyze the cross-link formed between
2-plasmin inhibitor and fibrinogen, and to a lesser extent also the cross-link between the inhibitor and fibrin (50, 51).
Altogether, our findings with the cytosolic enzymes suggest that
transglutaminases may play a more dynamic role in cell biology than
hitherto envisaged, not only by catalyzing the formation but also the
breaking of N
-(
-glutamyl)lysine
bonds.