- Email: [email protected]
[25] Horseshoe crab transglutaminase
378
BLOOD COAGULATION FACTORS AND INHIBITORS
[25]
HEXXH
HR2a HR2b HT -2 Ht-d H2
HR1B FIG. 3. Gross structure of snake venom metalloendopeptidases. HR2a, HR2b, HT-2, Ht-d and H2-proteinase are the venom low-molecular-mass hemorrhagic and nonhemorrhagic metalloendopeptidases. HR1B is composed of three domains: the metalloproteinase domain, the disintegrin-like domain, and an unknown cysteine-rich region. The putative zinc ligands and active site are shown (HEXXH). Sugar chains (O) linked to Asn-residues are indicated.
activities, such as fibrinogenase, 8 serpin inactivator, 48'49 and several procoagulants,9'10 are members of this newly identified venom metalloendopeptidase family. 48 T. Kurecki, M. Laskowski, Sr., and L. F. Kress, J. Biol. Chem. 253, 8340 (1978). 49 L. F. Kress, T. Kurecki, S. K. Chan, and M. Laskowski, J. Biol. Chem. 254, 5317 (1979).
[25] H o r s e s h o e C r a b T r a n s g l u t a m i n a s e
By FUMINORI
TOKUNAGA a n d SADAAKI IWANAGA
Introduction The hemocytes circulating in horseshoe crab (Limulus) hemolymph contain a coagulation system that participates both in hemostasis and in defense against invading microorganisms) This coagulation system is highly sensitive to gram-negative bacterial endotoxins, which are lipopolysaccharides (LPS). 2-4 In 1973, Lorand and Campbell-Wilkes discovered
1 j. Levin and F. B. Bang, Bull. Johns Hopkins Hosp. 115, 265 (1964). 2 S. Iwanaga, T. Morita, T. Miyata, T. Nakamura, and J. Aketagawa, J. Protein Chem. 5, 255 (1986). 3 T. Muta, R. Hashimoto, T. Miyata, H. Nishimura, Y. Toh, and S. Iwanaga, J. Biol. Chem. 265, 22426 (1990). 4 T. Muta, T. Miyata, Y. Misumi, F. Tokunaga, T. Nakamura, Y. Toh, Y. Ikehara, and S. Iwanaga, J. Biol. Chem. 266, 6554 (1991).
METHODS IN ENZYMOLOGY,VOL.223
Copyright© 1993by AcademicPress, Inc. All rightsof reproductionin any formreserved.
[9.5]
HORSESHOE CRAB TRANSGLUTAMINASE
379
the existence o f Ca2+-dependent transglutaminase (TGase) in L i m u l u s p o l y p h e m u s a m e b o c y t e s and showed the TGase-catalyzed incorporation of [14C]putrescine into/3-1actoglobulin and a-casein. 5 Later, Chung et al. 6 confirmed this fact and suggested that TGase participates in the crosslinking o f coagulin gel. H o w e v e r , no further studies have been made on the purification and characterization o f L i m u l u s TGase, because o f an extremely unstable e n z y m e in the lysate. In 1989, Roth et al. 7 reported that T G a s e (factor XIII-like) activity in either native or gelled L i m u l u s lysate was negative, and that stable gels formed after coagulation of limulus lysate by bacterial endotoxin were not covalently cross-linked. T G a s e [EC 2.3.2.13] constitutes a family of Ca2+-dependent e n z y m e s that catalyze an acyl transfer reaction in which the 7-carboxamide group of the peptide-bound Gln residue acts as acyl donor and the primary amino group of either the peptide-bound L y s residue or the free amine can serve as acyl acceptor. 8 In mammals, TGase is widely distributed in blood plasma, platelets, placenta, keratinocyte, and tissues such as liver, prostate gland, and epidermis. 8 TGase is classified into three g r o u p s - - p l a s m a T G a s e (factor XIII),9 type I membrane-bound TGase (keratinocyte type), ~0 and type II cytosolic TGase (liver type). H Moreover, one of the major e r y t h r o c y t e membrane proteins, band 4.2, is related to the T G a s e family, although it does not show any T G a s e activity. 12 Recently, we have succeeded in isolating TGase from the h e m o c y t e lysate of the Japanese horseshoe crab ( T a c h y p l e u s tridentatus), providing the first demonstration o f invertebrate TGase.13 This chapter describes the purification, properties, and c D N A sequence of L i m u l u s TGase.
5 Campbell-Wilkes, Ph.D. Dissertation (Examiner, L. Lorand), Northwestern University, Univ. Microfilms, 73-30, 763, Ann Arbor, MI (1973). 6 S. I. Chung, R. C. Seid, and T.-Y. Liu, Thromb. Haemostasis 38, 182 (1977). 7 R. I. Roth, J. C.-R. Chen, and J. Levin, Thromb. Res. 55, 25 (1989). 8 j. E. Folk and J. S. Finlayson, Adv. Protein Chem. 31, 1 (1977). 9 A. Ichinose, B. A. McMullen, K. Fujikawa, and E. W. Davie, Biochemistry 25, 4633 (1986). 10M. A. Phillips, B. E. Stewart, Q. Qin, R. Chakravarty, E. E. Floyd, A. M. Jetten, and R. H. Rice, Proc. Natl. Acad. Sci. U.S.A. 87, 9333 (1990). I1 K. Ikura, T. Nasu, H. Yokota, Y. Tsuchiya, R. Sasaki, and H. Chiba, Biochemistry 27, 2898 (1988). lz L. A. Sung, S. Chien, L.-S. Chang, K. Lambert, S. A. Bliss, E. E. Bouhassira, R. L. Nagel, R. S. Schwartz, and A. Rybicki, Proc. Natl. Acad. Sci. U.S.A. 87, 955 (1990). 13F. Tokunaga, M. Yamada, T. Muta, M. Hiranaga-Kawabata, S. Iwanaga, A. Ichinose, and E. W. Davie, Thromb. Haemostasis 65, 936 (1991).
380
BLOOD COAGULATION FACTORS AND INHIBITORS
[25]
Assay Method
Principle TGase activity was determined by the fluorescence of monodansylcadaverine (DCA) incorporated into N,N-dimethylcasein as described in the method of Lorand and Gotoh. 14
Reagents and Procedure The reaction mixture, containing 20/zl of 50 mM Tris-HC1 buffer, pH 7.5, 20 ~1 of 100 mM CaCI2, 20/zl of 100 mM dithiothreitol (DTT), 100 /xl of 2 mM DCA (Sigma Chemical Co., St. Louis, MO), 200/zl of 0.4% (w/v) dimethylcasein (Sigma), and the sample, in a total of 400 ttl, is incubated at 37° for 30 min. After incubation, 400/zl of 10% trichloroacetic acid is added to terminate the reaction and the mixture is centrifuged at 15,000 rpm for 15 min. The precipitate is washed three times with 1 ml of ethanol/ethyl ether (1 : I, v/v) and dried by Speed-Vac concentrator (Savant Instruments, NY). The dried precipitate is dissolved in 1 ml of 50 mM Tris-HC1, pH 8.0, containing 8 M urea and 0.5% SDS. Fluorescent intensity is then measured with excitation at 355 nm and emission at 525 nm. DCA dissolved in the same solution to give a concentration of 10 /xmol/liter is used as a standard. One unit of the enzyme activity, defined as amine incorporation unit per minute (AIU), is calculated as described. 14 Purification Procedure Japanese horseshoe crab (T. tridentatus) hemocytes are collected as described.15 Briefly, the fresh hemocyte pellet is homogenized three times with 300 ml of 50 mM Tris-acetate buffer, pH 7.5, containing 1 mM EDTA (TAE) using a Physcotron homogenizer (Nihon-Seimitsu Kogyo, Ltd., Tokyo) in a sterilized centrifugation tube; it is then centrifuged at 8000 rpm for 30 min. The lysate (890 ml) thus obtained from 32.4 g of hemocytes (wet weight) is dialyzed at 4 ° for 12 hr against 20 liters of 50 mM TAE buffer. This procedure is very important to stabilize the Limulus TGase. The dialyzed lysate is centrifuged at 8000 rpm for I hr and the supernatant is then applied to a CM-Sepharose CL-6B column (3.5 x 22 cm) equilibrated with 50 mM TAE buffer. The column is washed with 700 ml of the equilibrated buffer and proteins are eluted with TAE buffer containing 2 14 L. Lorand and T. Gotoh, this series, Vol. 19, p. 770. 15 T. Nakamura, T. Morita, and S. Iwanaga, J. Biochem. (Tokyo) 97, 1561 0985).
[25]
HORSESHOE CRAB TRANSGLUTAMINASE
381
M NaCI. The breakthrough fractions containing TGase activity are pooled and dialyzed overnight against 50 mM TAE buffer. The dialyzate is brought to 50% saturation of ammonium sulfate and the resulting precipitate is collected by centrifugation at 8000 rpm for 30 min. The pellet thus obtained is dissolved in 460 ml of 50 mM TAE buffer and dialyzed overnight against same buffer. The dialyzed solution is applied to a DEAE-cellulose (DE52) column (2.0 x 14.5 cm) equilibrated with 50 mM TAE buffer. The column is washed with 300 ml of the equilibration buffer and the elution is carried out with a linear salt gradient with 500 ml each of 50 mM TAE buffer and the same buffer containing 0.3 M NaCl. As shown in Fig. l, TGase is eluted in the fractions with a conductivity of 8 mmho. These fractions (indicated by a bar) are collected, dialyzed overnight against 50 mM TAE buffer, and concentrated by Diaflo ultrafiltation (Amicon, Danvers, MA). The concentrated sample is applied to a Sephacryl S-300 column (3.0 x 94 cm) equilibrated with 50 mM TAE buffer and eluted 3O A
V
gradient
2.0"
E
.\ ..-.
o oo
4) 0 (¢l
/\
1.o
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25
,
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"15
5
<
""
o
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0.0
.
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.
.
.
.
i
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.
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.
.
i
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50 Fraction
.
number
(8.0ml/tube)
FIG. 1. Chromatography of Limulus TGase fraction on a DEAE-cellulose column (2.0 x 14.5 cm). The breakthrough fractions obtained from a CM-Sepharose CL-6B column were pooled and fractionated by 50% saturated (NH4)2SO4, and the resulting precipitate was dissolved in 50 mM TAE buffer and dialyzed overnight against the same buffer. The dialyzed solution was then applied to a DEAE-cellulose column equilibrated with 50 mM TAE buffer. The elution was performed with a linear salt gradient from 0 to 0.3 M NaCl in the equilibration buffer. Fractions of 8 ml were collected at 4°, and the fractions indicated by a bar were pooled. The absorbance of the eluate at 280 nm (O) and the TGase activity ( e ) are shown.
382
[25]
BLOOD COAGULATION FACTORS AND INHIBITORS
30
T 0.3 v
E t-
°'t
v
"20
E
O GO ¢M
0.2 . m
° m
0 C ¢g 0
10 0.1
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m
.Q
O
~t
I--
0.0 5~,_,_,_,_.~z~aam. . . .
0
:,,(,:,:,;~
50 Fraction
"---
number
100 (3.0 ml/tube)
150
FIG. 2. Gel filtration of T G a s e fraction on a Sephacryl S-300 c o l u m n (3.0 × 94 cm). T h e pooled fraction obtained from Fig. 1 was concentrated a n d applied to a Sephacryl S-300 column. T h e elution w a s carried out with 50 m M T A E buffer and fractions o f 3.4 ml were collected at 4 °, a n d the fractions indicated by a bar were pooled. The a b s o r b a n c e o f eluate at 280 n m (O) a n d the T G a s e activity ( 0 ) are shown.
with the same buffer (Fig. 2). The pooled TGase-containing fraction (indicated by a bar) is applied to a DEAE-Cosmogel (7.5 x 50 mm) column (Nakalai Tesque Co.) linked to high-performance liquid chromatography. The column is equilibrated with 50 mM Tris-acetate, pH 7.5, and the elution is performed with a linear gradient from 0 to 0.4 M NaCI in the same buffers for 120 min at a flow rate of 1.0 ml/min, as shown in Fig. 3. The pooled TGase-containing fraction is dialyzed against 50 mM Tris-acetate, pH 7.5, and the dialyzed fraction is finally applied to a zincchelating Sepharose 6B (1.6 × 25 cm) column equilibrated with 50 mM Tris-acetate, pH 7.5. The proteins are eluted with a linear gradient of histidine (0-30 mM) in 50 mM Tris-acetate, pH 7.5. In each of the procedures, the protein concentrations are determined by a dye-binding method using bovine serum albumin as a standard (Bio-Rad, Richmond, CA). Table I shows the summary of the purification of TGase from the hemocyte lysate. The yield of purified TGase is about 1.6 mg from 32.4 g of hemocytes (wet weight, collected from 10 horseshoe crabs) and 156-fold purification is achieved. The purified TGase shows a single band
[9-5]
HORSESHOE CRAB TRANSGLUTAMINASE
383
I I
1.25
A
0.4
i
1.0
r-
E
z
o 0.75
..f" Is. 0.a
A
i A
al
0.2 6 z
~ o.5 m o pJ
~ 0.25
~
0.1
so~sssi~s'ssJ~
o B 150
5 1oo
t~ w t~ o I-
50
0
m
30 60 Retention tlme (mln)
t
m 9O
FIG. 3. Chromatography of TGase fraction on a DEAE-Cosmogel column (7.5 x 50 mm). The pooled fraction obtained from Fig. 2 was dialyzed against 50 mM TAE buffer and the dialyzed solution was applied to a DEAE-Cosmogel column connected to highperformance liquid chromatography. The elution was performed with a linear salt gradient to 0.4 M NaC1 in 50 mM Tris-acetate, pH 7.5, at a flow rate of 1.0 ml/min. The fractions indicated by a bar were collected manually• (A) Absorbance of eluate at 280 nm; (B) shaded bars indicate the TGase activity. o f 86 k D a u n d e r n o n r e d u c e d S D S - P A G E (Fig. 4), a n d it g a v e h i g h e r m o l e c u l a r m a s s in t h e p r e s e n c e o f 2 - m e r c a p t o e t h a n o l .
Properties T h e e x t i n c t i o n c o e f f i c i e n t o f Limulus T G a s e (1% s o l u t i o n in 50 m M T A E b u f f e r ) is e s t i m a t e d to b e 21.7 at 280 nm. T h e s a m p l e s h o u l d b e s t o r e d at 4 °, n o t in a f r o z e n s t a t e , b e c a u s e f r e e z e - t h a w i n g p r o c e d u r e s u n s t a b i l i z e this e n z y m e . Limulus T G a s e s h o w s C a 2 ÷ - d e p e n d e n t a c t i v i t y like m a m m a l i a n t i s s u e T G a s e a n d 10 m M C a 2÷ is r e q u i r e d f o r m a x i m u m
384
BLOOD COAGULATION FACTORS AND INHIBITORS
[25]
TABLE I Limulus TGAsE
P U R I F I C A T I O N OF
Volume (ml)
Step Hemocyte lysate CM-Sepharose CL-6B Ammonium sulfate precipitation DEAE-cellulose Sephacryl S-300 DEAE-Cosmogel Zinc-chelating Sepharose 6B a
890 1000 225 175 27.0 12.0 13.0
Total protein (rag) 1335 462 191 17.1 9.2 2.7 1.6
Total activity (AIU) ~
Specific activity (AIU/mg)
8055 8425 2690
6.0 18.3 14.0
4541 3109 2305 1528
266 340 851 938
Purification (fold)
Yield (%)
1.0 3.0 2.3
100 105 33.3
44.3 56.6 142 156
56.4 38.6 28.6 19.0
Amine incorporation u n i t )
1
2
94 kDa -.~ 67 kDa
43 kDa
30 kDa - ~
20.1 kDa - I ~ Fro. 4. S D S - P A G E of purified Limulus TGase. The purified Limulus TGase was electrophoresed in the absence (lane 1) and presence (lane 2) of 2-mercaptoethanol. S D S - P A G E was carried out using a 6-15% gradient gel. After electrophoresis, the gel was stained with Coomassie brilliant blue.
[25]
HORSESHOE CRAB TRANSGLUTAMINASE
385
TABLE II EFFECTOF VARIOUSINHIBITORSON ACTIVITY OF Limulus TGASE Inhibitor
Remaining activity (%)
None 100 mM hydroxylamine 1 mM ICH2COOH 1 mM N-ethylmaleimide 1 mM CuCl 2 5 mM EDTA 5 mM diisopropyl fluorophosphate
100 0 6 2 0 0 100
activity. 5 Interestingly, however, dithiothreitol, which enhances tissue TGase activity, shows little effect on Limulus TGase activity. Table II shows the inhibitor spectrum of Limulus TGase. At concentrations of 1 to 5 mM, CuCl2, EDTA, and SH-blocking reagents such as iodoacetic acid and N-ethylmaleimide strongly inhibit the Limulus TGase activity. Furthermore, hydroxylamine shows inhibitory effect, but diisopropyl fluorophosphate (DFP) has no effect. The Limulus TGase activity is also inhibited by high concentrations (>0.5 M) of NaC1. A micromolar amount of GTP is known to inhibit guinea pig liver TGase] 6 but nucleoside 5'-triphosphates such as ATP, CTP, GTP, and UTP have no effect on the Limulus TGase activity up to final concentrations of I mM in the presence of 5 mM CaC12. cDNA and Amino Acid Sequence We have succeeded in cloning the cDNA encoding Limulus TGase and have determined the entire amino acid sequence using recombinant DNA technique. As shown in Fig. 5, Limulus TGase consists of 764 amino acid residues with a calculated molecular mass of 87,1 I0 Da, which is in good agreement with the value estimated on SDS-PAGE. Based on a sequence similarity with other mammalian tissue TGases, Cys-343 is deduced to be a catalytic site. Although there are five potential carbohydrate attachment sites (Asn-X-Ser/Thr) at positions of 12, 196, 206, 578, and 611, no hexosamines are detected by amino acid analysis, indicating that Limulus TGase is a simple protein.
t6 K. E. Achyuthan and C. S. Greenberg, J. Biol. Chem. 262, 1901 (1987).
386 i~ I
I
BLOOD COAGULATION FACTORS AND INHIBITORS O O
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[25]
HORSESHOE CRAB TRANSGLUTAMINASE
387 0
!:
io
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388
BLOOD COAGULATION FACTORS AND INHIBITORS
[25]
The entire sequence of Limulus TGase shows a significant similarity to mammalian TGase family sequences as follows: guinea pig liver TGase (32.7%), ll human factor XIIIa subunit (36.9%), 9 human keratinocyte TGase (39.9%), l° and human erythrocyte band 4.2 (23.7%). 12 However, Limulus TGase has a unique NH2-terminal cationic 60-residue extension with no homology to the mammalian TGases. The function of this extension is not known.
Fie. 5. The composite nucleotide sequence (upper) and the deduced amino acid sequence (lower) of Limulus TGase. A polyadenylation signal (AATAAA) is double underlined. The amino acid residues that have been confirmed by sequencing of purified peptides are single underlined. The amino acid residue suspected to be an active Cys residue is circled.
BLOOD COAGULATION FACTORS AND INHIBITORS
[25]
HEXXH
HR2a HR2b HT -2 Ht-d H2
HR1B FIG. 3. Gross structure of snake venom metalloendopeptidases. HR2a, HR2b, HT-2, Ht-d and H2-proteinase are the venom low-molecular-mass hemorrhagic and nonhemorrhagic metalloendopeptidases. HR1B is composed of three domains: the metalloproteinase domain, the disintegrin-like domain, and an unknown cysteine-rich region. The putative zinc ligands and active site are shown (HEXXH). Sugar chains (O) linked to Asn-residues are indicated.
activities, such as fibrinogenase, 8 serpin inactivator, 48'49 and several procoagulants,9'10 are members of this newly identified venom metalloendopeptidase family. 48 T. Kurecki, M. Laskowski, Sr., and L. F. Kress, J. Biol. Chem. 253, 8340 (1978). 49 L. F. Kress, T. Kurecki, S. K. Chan, and M. Laskowski, J. Biol. Chem. 254, 5317 (1979).
[25] H o r s e s h o e C r a b T r a n s g l u t a m i n a s e
By FUMINORI
TOKUNAGA a n d SADAAKI IWANAGA
Introduction The hemocytes circulating in horseshoe crab (Limulus) hemolymph contain a coagulation system that participates both in hemostasis and in defense against invading microorganisms) This coagulation system is highly sensitive to gram-negative bacterial endotoxins, which are lipopolysaccharides (LPS). 2-4 In 1973, Lorand and Campbell-Wilkes discovered
1 j. Levin and F. B. Bang, Bull. Johns Hopkins Hosp. 115, 265 (1964). 2 S. Iwanaga, T. Morita, T. Miyata, T. Nakamura, and J. Aketagawa, J. Protein Chem. 5, 255 (1986). 3 T. Muta, R. Hashimoto, T. Miyata, H. Nishimura, Y. Toh, and S. Iwanaga, J. Biol. Chem. 265, 22426 (1990). 4 T. Muta, T. Miyata, Y. Misumi, F. Tokunaga, T. Nakamura, Y. Toh, Y. Ikehara, and S. Iwanaga, J. Biol. Chem. 266, 6554 (1991).
METHODS IN ENZYMOLOGY,VOL.223
Copyright© 1993by AcademicPress, Inc. All rightsof reproductionin any formreserved.
[9.5]
HORSESHOE CRAB TRANSGLUTAMINASE
379
the existence o f Ca2+-dependent transglutaminase (TGase) in L i m u l u s p o l y p h e m u s a m e b o c y t e s and showed the TGase-catalyzed incorporation of [14C]putrescine into/3-1actoglobulin and a-casein. 5 Later, Chung et al. 6 confirmed this fact and suggested that TGase participates in the crosslinking o f coagulin gel. H o w e v e r , no further studies have been made on the purification and characterization o f L i m u l u s TGase, because o f an extremely unstable e n z y m e in the lysate. In 1989, Roth et al. 7 reported that T G a s e (factor XIII-like) activity in either native or gelled L i m u l u s lysate was negative, and that stable gels formed after coagulation of limulus lysate by bacterial endotoxin were not covalently cross-linked. T G a s e [EC 2.3.2.13] constitutes a family of Ca2+-dependent e n z y m e s that catalyze an acyl transfer reaction in which the 7-carboxamide group of the peptide-bound Gln residue acts as acyl donor and the primary amino group of either the peptide-bound L y s residue or the free amine can serve as acyl acceptor. 8 In mammals, TGase is widely distributed in blood plasma, platelets, placenta, keratinocyte, and tissues such as liver, prostate gland, and epidermis. 8 TGase is classified into three g r o u p s - - p l a s m a T G a s e (factor XIII),9 type I membrane-bound TGase (keratinocyte type), ~0 and type II cytosolic TGase (liver type). H Moreover, one of the major e r y t h r o c y t e membrane proteins, band 4.2, is related to the T G a s e family, although it does not show any T G a s e activity. 12 Recently, we have succeeded in isolating TGase from the h e m o c y t e lysate of the Japanese horseshoe crab ( T a c h y p l e u s tridentatus), providing the first demonstration o f invertebrate TGase.13 This chapter describes the purification, properties, and c D N A sequence of L i m u l u s TGase.
5 Campbell-Wilkes, Ph.D. Dissertation (Examiner, L. Lorand), Northwestern University, Univ. Microfilms, 73-30, 763, Ann Arbor, MI (1973). 6 S. I. Chung, R. C. Seid, and T.-Y. Liu, Thromb. Haemostasis 38, 182 (1977). 7 R. I. Roth, J. C.-R. Chen, and J. Levin, Thromb. Res. 55, 25 (1989). 8 j. E. Folk and J. S. Finlayson, Adv. Protein Chem. 31, 1 (1977). 9 A. Ichinose, B. A. McMullen, K. Fujikawa, and E. W. Davie, Biochemistry 25, 4633 (1986). 10M. A. Phillips, B. E. Stewart, Q. Qin, R. Chakravarty, E. E. Floyd, A. M. Jetten, and R. H. Rice, Proc. Natl. Acad. Sci. U.S.A. 87, 9333 (1990). I1 K. Ikura, T. Nasu, H. Yokota, Y. Tsuchiya, R. Sasaki, and H. Chiba, Biochemistry 27, 2898 (1988). lz L. A. Sung, S. Chien, L.-S. Chang, K. Lambert, S. A. Bliss, E. E. Bouhassira, R. L. Nagel, R. S. Schwartz, and A. Rybicki, Proc. Natl. Acad. Sci. U.S.A. 87, 955 (1990). 13F. Tokunaga, M. Yamada, T. Muta, M. Hiranaga-Kawabata, S. Iwanaga, A. Ichinose, and E. W. Davie, Thromb. Haemostasis 65, 936 (1991).
380
BLOOD COAGULATION FACTORS AND INHIBITORS
[25]
Assay Method
Principle TGase activity was determined by the fluorescence of monodansylcadaverine (DCA) incorporated into N,N-dimethylcasein as described in the method of Lorand and Gotoh. 14
Reagents and Procedure The reaction mixture, containing 20/zl of 50 mM Tris-HC1 buffer, pH 7.5, 20 ~1 of 100 mM CaCI2, 20/zl of 100 mM dithiothreitol (DTT), 100 /xl of 2 mM DCA (Sigma Chemical Co., St. Louis, MO), 200/zl of 0.4% (w/v) dimethylcasein (Sigma), and the sample, in a total of 400 ttl, is incubated at 37° for 30 min. After incubation, 400/zl of 10% trichloroacetic acid is added to terminate the reaction and the mixture is centrifuged at 15,000 rpm for 15 min. The precipitate is washed three times with 1 ml of ethanol/ethyl ether (1 : I, v/v) and dried by Speed-Vac concentrator (Savant Instruments, NY). The dried precipitate is dissolved in 1 ml of 50 mM Tris-HC1, pH 8.0, containing 8 M urea and 0.5% SDS. Fluorescent intensity is then measured with excitation at 355 nm and emission at 525 nm. DCA dissolved in the same solution to give a concentration of 10 /xmol/liter is used as a standard. One unit of the enzyme activity, defined as amine incorporation unit per minute (AIU), is calculated as described. 14 Purification Procedure Japanese horseshoe crab (T. tridentatus) hemocytes are collected as described.15 Briefly, the fresh hemocyte pellet is homogenized three times with 300 ml of 50 mM Tris-acetate buffer, pH 7.5, containing 1 mM EDTA (TAE) using a Physcotron homogenizer (Nihon-Seimitsu Kogyo, Ltd., Tokyo) in a sterilized centrifugation tube; it is then centrifuged at 8000 rpm for 30 min. The lysate (890 ml) thus obtained from 32.4 g of hemocytes (wet weight) is dialyzed at 4 ° for 12 hr against 20 liters of 50 mM TAE buffer. This procedure is very important to stabilize the Limulus TGase. The dialyzed lysate is centrifuged at 8000 rpm for I hr and the supernatant is then applied to a CM-Sepharose CL-6B column (3.5 x 22 cm) equilibrated with 50 mM TAE buffer. The column is washed with 700 ml of the equilibrated buffer and proteins are eluted with TAE buffer containing 2 14 L. Lorand and T. Gotoh, this series, Vol. 19, p. 770. 15 T. Nakamura, T. Morita, and S. Iwanaga, J. Biochem. (Tokyo) 97, 1561 0985).
[25]
HORSESHOE CRAB TRANSGLUTAMINASE
381
M NaCI. The breakthrough fractions containing TGase activity are pooled and dialyzed overnight against 50 mM TAE buffer. The dialyzate is brought to 50% saturation of ammonium sulfate and the resulting precipitate is collected by centrifugation at 8000 rpm for 30 min. The pellet thus obtained is dissolved in 460 ml of 50 mM TAE buffer and dialyzed overnight against same buffer. The dialyzed solution is applied to a DEAE-cellulose (DE52) column (2.0 x 14.5 cm) equilibrated with 50 mM TAE buffer. The column is washed with 300 ml of the equilibration buffer and the elution is carried out with a linear salt gradient with 500 ml each of 50 mM TAE buffer and the same buffer containing 0.3 M NaCl. As shown in Fig. l, TGase is eluted in the fractions with a conductivity of 8 mmho. These fractions (indicated by a bar) are collected, dialyzed overnight against 50 mM TAE buffer, and concentrated by Diaflo ultrafiltation (Amicon, Danvers, MA). The concentrated sample is applied to a Sephacryl S-300 column (3.0 x 94 cm) equilibrated with 50 mM TAE buffer and eluted 3O A
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FIG. 1. Chromatography of Limulus TGase fraction on a DEAE-cellulose column (2.0 x 14.5 cm). The breakthrough fractions obtained from a CM-Sepharose CL-6B column were pooled and fractionated by 50% saturated (NH4)2SO4, and the resulting precipitate was dissolved in 50 mM TAE buffer and dialyzed overnight against the same buffer. The dialyzed solution was then applied to a DEAE-cellulose column equilibrated with 50 mM TAE buffer. The elution was performed with a linear salt gradient from 0 to 0.3 M NaCl in the equilibration buffer. Fractions of 8 ml were collected at 4°, and the fractions indicated by a bar were pooled. The absorbance of the eluate at 280 nm (O) and the TGase activity ( e ) are shown.
382
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BLOOD COAGULATION FACTORS AND INHIBITORS
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FIG. 2. Gel filtration of T G a s e fraction on a Sephacryl S-300 c o l u m n (3.0 × 94 cm). T h e pooled fraction obtained from Fig. 1 was concentrated a n d applied to a Sephacryl S-300 column. T h e elution w a s carried out with 50 m M T A E buffer and fractions o f 3.4 ml were collected at 4 °, a n d the fractions indicated by a bar were pooled. The a b s o r b a n c e o f eluate at 280 n m (O) a n d the T G a s e activity ( 0 ) are shown.
with the same buffer (Fig. 2). The pooled TGase-containing fraction (indicated by a bar) is applied to a DEAE-Cosmogel (7.5 x 50 mm) column (Nakalai Tesque Co.) linked to high-performance liquid chromatography. The column is equilibrated with 50 mM Tris-acetate, pH 7.5, and the elution is performed with a linear gradient from 0 to 0.4 M NaCI in the same buffers for 120 min at a flow rate of 1.0 ml/min, as shown in Fig. 3. The pooled TGase-containing fraction is dialyzed against 50 mM Tris-acetate, pH 7.5, and the dialyzed fraction is finally applied to a zincchelating Sepharose 6B (1.6 × 25 cm) column equilibrated with 50 mM Tris-acetate, pH 7.5. The proteins are eluted with a linear gradient of histidine (0-30 mM) in 50 mM Tris-acetate, pH 7.5. In each of the procedures, the protein concentrations are determined by a dye-binding method using bovine serum albumin as a standard (Bio-Rad, Richmond, CA). Table I shows the summary of the purification of TGase from the hemocyte lysate. The yield of purified TGase is about 1.6 mg from 32.4 g of hemocytes (wet weight, collected from 10 horseshoe crabs) and 156-fold purification is achieved. The purified TGase shows a single band
[9-5]
HORSESHOE CRAB TRANSGLUTAMINASE
383
I I
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FIG. 3. Chromatography of TGase fraction on a DEAE-Cosmogel column (7.5 x 50 mm). The pooled fraction obtained from Fig. 2 was dialyzed against 50 mM TAE buffer and the dialyzed solution was applied to a DEAE-Cosmogel column connected to highperformance liquid chromatography. The elution was performed with a linear salt gradient to 0.4 M NaC1 in 50 mM Tris-acetate, pH 7.5, at a flow rate of 1.0 ml/min. The fractions indicated by a bar were collected manually• (A) Absorbance of eluate at 280 nm; (B) shaded bars indicate the TGase activity. o f 86 k D a u n d e r n o n r e d u c e d S D S - P A G E (Fig. 4), a n d it g a v e h i g h e r m o l e c u l a r m a s s in t h e p r e s e n c e o f 2 - m e r c a p t o e t h a n o l .
Properties T h e e x t i n c t i o n c o e f f i c i e n t o f Limulus T G a s e (1% s o l u t i o n in 50 m M T A E b u f f e r ) is e s t i m a t e d to b e 21.7 at 280 nm. T h e s a m p l e s h o u l d b e s t o r e d at 4 °, n o t in a f r o z e n s t a t e , b e c a u s e f r e e z e - t h a w i n g p r o c e d u r e s u n s t a b i l i z e this e n z y m e . Limulus T G a s e s h o w s C a 2 ÷ - d e p e n d e n t a c t i v i t y like m a m m a l i a n t i s s u e T G a s e a n d 10 m M C a 2÷ is r e q u i r e d f o r m a x i m u m
384
BLOOD COAGULATION FACTORS AND INHIBITORS
[25]
TABLE I Limulus TGAsE
P U R I F I C A T I O N OF
Volume (ml)
Step Hemocyte lysate CM-Sepharose CL-6B Ammonium sulfate precipitation DEAE-cellulose Sephacryl S-300 DEAE-Cosmogel Zinc-chelating Sepharose 6B a
890 1000 225 175 27.0 12.0 13.0
Total protein (rag) 1335 462 191 17.1 9.2 2.7 1.6
Total activity (AIU) ~
Specific activity (AIU/mg)
8055 8425 2690
6.0 18.3 14.0
4541 3109 2305 1528
266 340 851 938
Purification (fold)
Yield (%)
1.0 3.0 2.3
100 105 33.3
44.3 56.6 142 156
56.4 38.6 28.6 19.0
Amine incorporation u n i t )
1
2
94 kDa -.~ 67 kDa
43 kDa
30 kDa - ~
20.1 kDa - I ~ Fro. 4. S D S - P A G E of purified Limulus TGase. The purified Limulus TGase was electrophoresed in the absence (lane 1) and presence (lane 2) of 2-mercaptoethanol. S D S - P A G E was carried out using a 6-15% gradient gel. After electrophoresis, the gel was stained with Coomassie brilliant blue.
[25]
HORSESHOE CRAB TRANSGLUTAMINASE
385
TABLE II EFFECTOF VARIOUSINHIBITORSON ACTIVITY OF Limulus TGASE Inhibitor
Remaining activity (%)
None 100 mM hydroxylamine 1 mM ICH2COOH 1 mM N-ethylmaleimide 1 mM CuCl 2 5 mM EDTA 5 mM diisopropyl fluorophosphate
100 0 6 2 0 0 100
activity. 5 Interestingly, however, dithiothreitol, which enhances tissue TGase activity, shows little effect on Limulus TGase activity. Table II shows the inhibitor spectrum of Limulus TGase. At concentrations of 1 to 5 mM, CuCl2, EDTA, and SH-blocking reagents such as iodoacetic acid and N-ethylmaleimide strongly inhibit the Limulus TGase activity. Furthermore, hydroxylamine shows inhibitory effect, but diisopropyl fluorophosphate (DFP) has no effect. The Limulus TGase activity is also inhibited by high concentrations (>0.5 M) of NaC1. A micromolar amount of GTP is known to inhibit guinea pig liver TGase] 6 but nucleoside 5'-triphosphates such as ATP, CTP, GTP, and UTP have no effect on the Limulus TGase activity up to final concentrations of I mM in the presence of 5 mM CaC12. cDNA and Amino Acid Sequence We have succeeded in cloning the cDNA encoding Limulus TGase and have determined the entire amino acid sequence using recombinant DNA technique. As shown in Fig. 5, Limulus TGase consists of 764 amino acid residues with a calculated molecular mass of 87,1 I0 Da, which is in good agreement with the value estimated on SDS-PAGE. Based on a sequence similarity with other mammalian tissue TGases, Cys-343 is deduced to be a catalytic site. Although there are five potential carbohydrate attachment sites (Asn-X-Ser/Thr) at positions of 12, 196, 206, 578, and 611, no hexosamines are detected by amino acid analysis, indicating that Limulus TGase is a simple protein.
t6 K. E. Achyuthan and C. S. Greenberg, J. Biol. Chem. 262, 1901 (1987).
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The entire sequence of Limulus TGase shows a significant similarity to mammalian TGase family sequences as follows: guinea pig liver TGase (32.7%), ll human factor XIIIa subunit (36.9%), 9 human keratinocyte TGase (39.9%), l° and human erythrocyte band 4.2 (23.7%). 12 However, Limulus TGase has a unique NH2-terminal cationic 60-residue extension with no homology to the mammalian TGases. The function of this extension is not known.
Fie. 5. The composite nucleotide sequence (upper) and the deduced amino acid sequence (lower) of Limulus TGase. A polyadenylation signal (AATAAA) is double underlined. The amino acid residues that have been confirmed by sequencing of purified peptides are single underlined. The amino acid residue suspected to be an active Cys residue is circled.