A small chimerically bifunctional monomeric protein: Tapes japonica lysozyme

Article abstract of DOI:10.1007/s00018-003-3082-z

The lysozyme of the marine bilave Tapes japonica (13.8 kDa) is a novel protein. The protein has 46% homology with the destabilase from medicinal leech that has isopeptidase activity. Based on these data, we confirmed hydrolysis activity of T. japonica lysozyme against three substrates: L-γ-Glu-pNA, D-γ-Glu-pNA, and ε-(γ-Glu)-L-Lys. The optimal pH of chitinase and isopeptidase activity was 5.0 and 7.0, respectively. The isopeptidase activity was inhibited with serine protease inhibitor, but the lytic and chitinase activities were not. Moreover, only isopeptidase activity is decreased by lyophilization, but lytic and chitinase activities were not. We conclude that T. japonica lysozyme expresses isopeptidase and chitinase activity at different active sites.

Full text of DOI:10.1007/s00018-003-3082-z

CMLS, Cell. Mol. Life Sci. 60 (2003) 1944–1951
420-682X/03/091944-08
DOI 10.1007/s00018-003-3082-z
Birkhäuser Verlag, Basel, 2003
1
CMLS Cellular and Molecular Life Sciences
©
Research Article
A small chimerically bifunctional monomeric protein:
Tapes japonica lysozyme
K. Takeshita ,Y. Hashimoto , T. Ueda and T. Imotoa,b,*
a
a
a
a
Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka, 812-8582 (Japan), Fax: +81 92 642 6667;
e-mail: Imoto@phar.kyushu-u.ac.jp
b
Department of Applied Microbial Technology, Faculty of Engineering, Sojo University, Ikeda 4-22-1,
Kumamoto 860-0082 (Japan)
Received 25 February 2003; received after revision 29 May 2003; accepted 12 June 2003
Abstract. The lysozyme of the marine bilave Tapes isopeptidase activity was 5.0 and 7.0, respectively. The
japonica (13.8 kDa) is a novel protein. The protein has isopeptidase activity was inhibited with serine protease in-
4
6% homology with the destabilase from medicinal leech hibitor, but the lytic and chitinase activities were not.
that has isopeptidase activity. Based on these data, we con- Moreover, only isopeptidase activity is decreased by
firmed hydrolysis activity of T. japonica lysozyme lyophilization, but lytic and chitinase activities were not.
against three substrates: L-g-Glu-pNA, D-g-Glu-pNA, We conclude that T. japonica lysozyme expresses isopep-
and e-(g-Glu)-L-Lys. The optimal pH of chitinase and tidase and chitinase activity at different active sites.
Key words. Lysozyme; Tapes japonica; chitinase; isopeptidase; invertebrate lysozyme; bifunctional.
Lysozymes (EC 3.2.1.17) are widely distributed in the an- nose (NAM) and 2-acetamido-2-deoxy-D-glucopyranose
imal and plant kingdom [1]. Several types of lysozyme (NAG) on peptidoglycan in Gram-positive cells.
have been described: chicken (c), goose (g), bacteria, Lysozyme belongs to the b-glycosidase group, and most
plant, phage, and invertebrate (i) types [1–6]. In particu- lysozymes hydrolyze chitin (an NAG polymer). Novel
lar, Jollès and Jollès first proposed the invertebrate type types of lysozyme have been reported, but the ability of
lysozyme in 1975 [5], and several further studies have these newly identified lysozymes to hydrolyze the b-1-4-
been made on i-type lysozymes [7–10]. The major func- linked glycosidic bond has not yet been demonstrated.
tions of lysozymes are lysing bacterial cells by hydrolyz- Generally, these enzymes have been classified as
ing the beta-1-4-linked glycoside bond of the peptidogly- lysozyme-like enzymes [7, 15]. Recently, the lyso-
can on the bacterial cell wall. Thus, the biochemical zyme from the marine bivalve Tapes japonica has
function of this enzyme seems to be self-defense against been recognized as an i-type lysozyme [8], but little is
bacterial infection [11, 12]. Additionally, it acts as a di- known about the structure and function of i-type
gestive enzyme in ruminant stomachs [13, 14].
lysozymes.
Lytic enzymes, which belong to the lysozyme family, have Lysozyme from the marine bivalve T. japonica is com-
been classified on the basis of organism, activity, and posed of 123 amino acids (13.8 kDa) [8]. The lytic activ-
structure [6, 11]. The lytic function of lysozyme is to ity of T. japonica lysozyme against Micrococcus luteus is
hydrolyze the beta-1-4-linked glycosidic bond between 2- 425%, the chitinase activity is 85%, and the binding
acetamido-3-O-(1-carboxyethyl)-2-deoxy-D-glucopyra- ability to (NAG) is 22% compared to hen egg lysozyme.
3
The primary sequence of T. japonica lysozyme indicates
*
Corresponding author.
46% identity to the destabilase from medicinal leech [16].

CMLS, Cell. Mol. Life Sci. Vol. 60, 2003
Research Article
1945
The destabilase from medicinal leech is an enzyme that centration was 0.83 mg/ml (59.8 mM). The reaction mix-
hydrolyses e-(g-Glu)-Lys cross-linkage between Glu and ture of enzyme and substrate was incubated for 0 – 8 h at
Lys in stabilized fibrin. Destabilase has been studied in 37°C. The progress of the reaction was calculated with
the context of thrombosis [16–18]. Similar isopeptide the molar extinction coefficient of p-nitroanilide
–
1
–1
bonds are also found on peptidoglycan in bacterial cells. (9694 M cm ) [20]. A reaction mixture without enzyme
Peptidoglycans are composed of straight-chain beta-1-4- was used as a negative control. The kinetic parameters of
linked NAM and NAG that are annularly linked with pep- the hydrolysis of L-g-Glu-pNA or D-g-Glu-pNA, cat-
tide chains. These peptide chains link lactic acid residues alyzed by T. japonica lysozyme, were calculated with an
of NAM, and there are isopeptide bonds between D-glu- Eadie-Hofstee plot. The measurement of pH dependence
tamic acid and lysine residues. Therefore, we investigated for isopeptidase activity was performed in buffers of pH
whether T. japonica lysozyme can hydrolyze an isopeptide from 3 to 9. The protein and substrate concentrations were
bond, and were surprised to find that this small enzyme 0.83 mg/ml (59.8 mM) and 0.25 mg/ml (1.75 mM), re-
exhibits two enzyme activities, chitinase and isopeptidase spectively. We determined the isopeptidase activity for
activity, at different sites. This study may contribute to un- e-(g-Glu)-L-Lys using an amino acid analyzer. The protein
derstanding enzymatic functions of i-type lysozyme.
and substrate concentrations were 0.83 mg/ml (59.8 mM)
and 0.25 mg/ml (1.75 mM), respectively. Isopeptidase ac-
tivity was inhibited with 4-(2-aminoethyl) benzensulfonyl
fluoride hydrochloride (AEBSF; Nakarai). After adding
Materials and methods
1
mM of the inhibitor, we determined isopeptidase, chiti-
Purification of marine bivalve (T. japonica) lysozyme nase, and lysozyme activities.
and estimation of protein concentration
Marine bivalves (T. japonica) were gathered on the shores Affinity column – chitin-coated celite
near Fukuoka (Japan) and stored at –80°C until use. The chitin-coated celite was prepared as described previ-
T. japonica lysozyme was purified following a previous ously [21]. The Chitin-coated celite column (50¥70 mm)
method described [8]. Its molecular weight was deter- was equilibrated with 0.1 M Tris-HCl containing 1.0 M
mined using SDS-PAGE and matrix-assisted laser NaCl (pH 8.5). The purified enzyme was applied to the
desorption/ionization-time-of-flight mass spectrometry column and washed with equilibration buffer and eluted
(
MALDI-TOFMS). Protein quantity was estimated using with 1.0 M acetic acid (0 °C).
–1
-–1
the molecular extinction coefficients of 37,600 (M cm )
for hen lysozyme and 27,000 (M cm ) for T. japonica Instrumentation
–
1
–1
lysozyme.
Circular dichroism (CD) spectrometry measurement
was performed on a spectropolarimeter J-720W (Jasco).
Lysozyme activity assay
The protein concentration was 10 mM in 50 mM MOPS
We measured the activity of lysozyme toward M. luteus buffer (pH 7). MALDI-TOFMS measurement was per-
(
Sigma) cells (0.35 mg/ml) in 50 mM KH PO -NaOH formed on a PerSeptive Voyager (Jasco) using a sinapic
2 4
buffer (pH 7). Fifty microliters of lysozyme solution acid matrix.
0.5 mg/ml) was added to 2 ml of M. luteus solution at
0 °C. The reaction was followed by the decrease in tur-
(
3
bidity at 450 nm (Hitachi-2000S).
Results
Chitinase assay
Purification and analysis of T. japonica lysozyme
Chitinase activity was measured using reducing sugar pro- T. japonica lysozyme was isolated from the homogenate
duced from glycol-chitin by T. japonica lysozyme [19]. of the marine bivalve (1.223 g, without shells) and
The enzyme and substrate concentrations were 52 mg/ml purified by cation exchange and gel filtration chromatog-
and 0.67 mg/ml, respectively.
raphy following the method described previously [8] (data
not shown). Purified T. japonica lysozyme showed one
band with an Mr14 kDa on SDS-PAGE under non-
Isopeptidase assay
The isopeptidase activity of T. japonica lysozyme was reduced conditions (fig.1A) and an Mr of 13.8 kDa by
determined by the production of p-nitroanilide from L- MALDI-MS (fig.1B).
g-glutamine-p-nitroanilide (L-g-Glu-pNA) with the ab-
sorbance at 405 nm [20]. The substrate (L-g-Glu-pNA) Isopeptidase activity of T. japonica lysozyme
was dissolved in 0.05 M 3-morpholinopropanesulfonic Originally, we measured isopeptidase activity using L-g-
acid (MOPS) buffer, pH 7.0, containing 0.01 M NaCl Glu-pNA and D-g-Glu-pNA. These substrates are analo-
and was prepared to a final concentration of gous substrates for isopeptide bonds in D-dimer and
0
.25–0.005 mg/ml (1.75 mM–3.5 mM); the protein con- peptideoglycan, respectively, for bacterial cells. As shown

1946
K. Takeshita et al.
Isopeptidase activity in Tapes japonica lysozyme
A
A
1
34
8
2
4
3
0
2
1
8
7
.2
kDa
1
2
B
Figure 1. Identification of the molecular weight of T. japonica lysozyme. (A) SDS-PAGE (15% polyacrylamide gel) analysis of
T. japonica lysozyme under non-reduced conditions. The molecular weights of the marker bands are 200, 116, 66, 42, 30, and 17 kDa, re-
spectively. (B) MALDI-TOFMS of T. japonica lysozyme indicated at 13,806 Da. The theoretical value of T. japonica lysozyme is
13.825 kDa. There are two extra peaks at one-half (6907.82 Da) and double (27,643.3 Da) the molecular-weight peak.
–
4
–1
in figure 2, T. japonica lysozyme showed distinct activi- Glu-pNA: K = 83.6 mM, k = 0.54¥10 s ). T. japon-
m
cat
ties against L- or D-g-Glu-pNA. The optimum pH was ica lysozyme showed a distinct activity for e-(g-Glu)-L-
–8 (fig. 3). On the other hand, the optimum pH for Lys, an analogous specific substrate (fig. 4).
7
chitinase activity was acidic. Therefore, the results of pH
dependence for chitinase and isopeptidase activity showed Inhibition assay
that each enzyme activity is independent. Kinetic para- Since the isopeptidase activity by T. japonica lyso-
meters of isopeptidase activity by T. japonica lysozyme zyme was inhibited by AEBSF, it was shown to be a
were determined by the method of Eadie and Hofstee (L- serine protease type (table 1). However, T. japonica
–
4
–1
g-Glu-pNA: K = 21.4 mM, k = 0.2 ¥10 s ; D-g- lysozyme retained lytic and chitinase activity after the
m
cat

CMLS, Cell. Mol. Life Sci. Vol. 60, 2003
Research Article
1947
A
B
Figure 2. Assay of isopeptidase activity for T. japonica lysozyme against L- (A) or D-g-Glu-pNA (B). T. japonica lysozyme concentration
was 0.83 mg/ml (59.8 mM). Substrate (L-g-Glu-pNA and D-g-Glu-pNA) concentrations were 0.25–0.005 mg/ml (1.75 mM–3.5 mM),
◆
, 0.25 mg/ml; ◆, 0.1 mg/ml; ◆, 0.05 mg/ml; , 0.01 mg/ml, ◆, 0.005 mg/ml. Buffer only was employed for the negative control reaction.
ꢀ
Reactions were conducted in 0.05 M MOPS buffer (pH 7.0) at 37°C for 8 h. Isopeptide bonds in D-dimer and peptidoglycan are indicated
by arrow heads. (A)
Figure 3. The pH dependence of T. japonica lysozyme activity Figure 4. Isopeptidase activity assay with T. japonica lysozyme
against L- and D-g-Glu-pNA. The activity toward L- (◆) or D-g-Glu- against e-(g-Glu)-L-Lys. Analysis of isopeptidase activity was per-
pNA (◆) was examined at pH 3.0–9.0 at 37 °C. The activity is ex- formed with amino acid analysis. Values of activity indicate reduc-
pressed by a rise of OD 405 nm at 4 or 2 h for the L or D substrate, tion in the amount of e-(g-Glu)-L-Lys.
respectively. The enzyme and substrate concentrations were
0
.83 mg/ml (59.8 mM) and 0.25 mg/ml (1.75 mM), respectively. The
optimal pH of isopeptidase activity was 7.0–8.0, and that of chiti-
nase activity was 4.0–5.0. The result of chitinase activity (◆) is from
Ito et al. [8].

1948
K. Takeshita et al.
Isopeptidase activity in Tapes japonica lysozyme
Table 1. Inhibition with serine protease inhibitor (AEBSF).
Isopeptidase activity
Lytic activity
(DOD450/min)
M. luteus
Chitinase activity
(DOD420 after 1 h)
glycol chitin
(DOD405 after 4 h)
L-g-Glu-pNA
D-g-Glu-pNA
AEBSF (–)
AEBSF (+)
0.15
0.008
0.31
0.007
0.33
0.36
86.2
82.3
Isopeptidase activity toward L-g-Glu-pNA or D-g-Glu-pNA was measured at pH 7.0 and 37 °C. Lytic activity toward M. luteus was mea-
sured at pH 7.0 and 30°C. Chitinase activity toward glycol chitin was measured at pH 5.5 and 40 °C.
treatment with AEBSF. This result suggested that T. japon-
ica lysozyme has different sites for the two activities.
Assay of each enzyme activity for fractions eluted
from an affinity column
T. japonica lysozyme has chitinase activity and thus the
ability to bind chitin. The purified enzyme was applied to
a chitin-coated celite column, washed with 0.1 M Tris-
HCl containing 1.0 M NaCl and eluted by 1.0 M acetic
acid (fig. 5). Then, fractions of 5 ml were collected, de-
salted, and concentrated tenfold. The concentration of
protein in each fraction was determined with the Bicin-
Figure 5. Profile of eluted protein from affinity column. The puri- choninate (BCA) method, and lytic activity and isopepti-
fied protein was loaded on an affinity column (chitin-coated celite).
After washing with 150 ml of equilibration buffer, the column was
eluted with 1 M acetic acid. Amount of protein is shown by the solid
line. Lytic activity (◆) and isopeptidase activity (◆) of each fractions (0.35 mg/ml) suspension in 50 mM KH PO -NaOH buffer
dase activity were measured. Lytic activity was assayed
using 20 ml of each fraction and 2 ml of M. luteus
2
4
is shown. Isopeptidase activity is indicated by ∆OD405 after 2 h.
(pH 7). Isopeptidase activity was assayed using 200 ml of
each fraction further concentrated five times and L-g-Glu-
pNA [0.25 mg/ml (1.75 mM)] solution in 0.05 M MOPS
containing 0.01 M NaCl (pH 7.0). As a result, both activ-
ities were eluted simultaneously. This result confirmed
that Tapes japonica lysozyme is a bifunctional enzyme
possessing both lytic and isopeptidase activities.
Influence of lyophilization
Among three enzyme (lytic, chitinase, and isopeptidase
activities) only the isopeptidase activity was largely de-
graded by lyophilization. The activity for L-g-Glu-pNA
was less than 55% and for D-g-Glu-pNA less than 45%,
while the lysozyme and chitinase activities were not
affected. To investigate the influence of lyophilization, we
measured CD spectra, but did not observe any significant
change in the spectra (fig. 6).
Discussion
As previously reported by Ito et al. [8], T. japonica
lysozyme possesses chitinase activity. The fact that T.
japonica lysozyme hydrolyzed e-(g-Glu)-L-Lys showed
that this lysozyme is also an isopeptidase. Isopeptidase ac-
Figure 6. Secondary-structure analysis of T. japonica lysozyme
using CD spectra. Solid line intact protein; dashed line, lyophilized
protein.
–1
–1
tivity for L-g-Glu-pNA ( k /K = 0.9 s M ) of T. japonica
cat
m
–1
–1
lysozyme was less than that of destabilase (1.6 s M ) [20].

CMLS, Cell. Mol. Life Sci. Vol. 60, 2003
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1949
Originally, isopeptidase activity was very low even in We showed that isopeptidase activity was decreased by
destabilase and we worried that an impurity enzyme might lyophilization, but lytic and chitinase activities were not.
be evoking this activity in our enzyme preparation. How- The result of CD spectra showed that little change was
ever, we could confirm that isopeptidase activity was ab- seen in the secondary structure by lyophilization. From
sorbed on a chitin column and eluted simultaneously with these data, we presume that the active sites in the protein
lytic activity. This confirmed that T. japonica lysozyme are separate. Detailed investigation of the influence of
possesses both chitinase and isopeptidase activity. The freeze-dry conditions using nuclear magnetic resonance
isopeptidase activity of T. japonica lysozyme was inhib- and X-ray crystal structure analyses are underway. Since
ited by AEBSF, but lytic and chitinase activities were not. this lysozyme has isopeptidase activity against D-g-Glu-
The optimal pH for isopeptidase activity was 7.0 and that pNA, we considered that it might enhance the lysozyme
for chitinase activity was 5.0, clearly indicating that these activity in harmony with chitinase activity. However, since
activities are exhibited by independent active sites. Desta- AEBSF only inhibited the isopeptidase activity, this
bilase and chlamysin, which have 46% and 55% homol- activity seems unlikely to be related to lytic activity.
ogy, respectively, to T. japonica lysozyme have given no The isopeptidase activity might be efficiently utilized by
indication so far of chitinase activity [7, 15]. On the basis T. japonica to digest bacteria in the late stage.
of sequence homologies and characteristic activities, T. We feel that the genomic analysis of our enzyme is im-
japonica lysozyme, destabilase, chlamysin and Mytilus portant as undertaken by Bachali et al. [9] and Nilsen and
lysozyme are homologous enzymes and could have a Myrnes [10]. We will attempt this, and so far have only
chitin-binding site.
analyzed and published the cDNA sequence of T. japon-
Figure 7. Multisequence alignment of T. japonica lysozyme and c-type lysozymes or its homologues. The amino acid sequence of two c-
type lysozymes and three T. japonica lysozyme homologues were aligned using Clustal W [22]. Conserved amino acids are indicated by
asterisks. Possible active residues are shadowed. (A) Amino acid sequences of T. japonica lysozyme [8], its homologues (destabilase 2: Gen-
bank accession U24122, chlamysin; EMBL database accession number AJ250028; Mytilus edulis: AF334662; M. galloprovincialis:
AF334665), and c-type lysozyme (EC 3.2.1.17) were aligned on the basis of regions of lytic activity. (B) Amino acid sequences of T. japon-
ica lysozyme and its homologues were aligned on the basis of predicted catalytic residues (serine, histidine) as related to regions of isopep-
tidase activity [23].

1950
K. Takeshita et al.
Isopeptidase activity in Tapes japonica lysozyme
ica lysozyme (AB091383). On the basis of studies by Ackowledgements. The authors thank KN-international (USA) for
improvement of our English. This work was supported in part by
grants from the Japan Foundation for Applied Enzymology and the
Foundation for Advanced Medicine (Japan).
Bachali et al. [9] and Nilsen and Myrnes [10], the catalytic
aspartate (D) in the c-type lysozymes is not conserved in
i-type lysozyme. However, we have predicted that the
catalytic aspartate in T. japonica lysozyme is D30
given the sequence homology with c-type lysozymes
and pH dependence of chitinase activity (pK about 2.8).
Moreover, this aspartate is conserved in i-type lyso-
zymes. We think that the cause of the considerable
difference in the position of the catalytic aspartate in i-
type and c-type lysozymes is the existence of a cysteine
within the homologous catalytic domain in i-type
lysozymes.
It is interesting that the T. japonica lysozyme, a small
protein, has both chitinase and isopeptidase activity, since
bifunctional enzymes are usually large or complex pro-
teins. Since the T. japonica lysozyme is a small,
monomeric, and bifunctional enzyme, detailed analysis
of its structure would be interesting (these studies are
underway). Comparing the amino acid sequence of our
enzyme with those of chicken and human lysozyme, we
predicted that the region responsible for chitinase activity
might be residues 18–46 of the T. japonica lysozyme
1
Prager E. M. and Jollès P. (1996) Animal lysozymes c and g: an
overview. In: Lysozyme: Model Enzymes in Biochemistry and
Biology, pp. 9–31, Jollès P. (ed.), Birkhäuser, Basel
2 Fastrez J. (1996) Phage lysozymes. In: Lysozyme: Model En-
zymes in Biochemistry and Biology, pp. 35–64, Jollès P. (ed.),
Birkhäuser, Basel
3
Beintema J. J. and Terwisscha van Scheltinga A. C. (1996) Plant
lysozymes. In: Lysozyme: Model Enzymes in Biochemistry and
Biology, pp. 75–86, Jollès P. (ed.), Birkhäuser, Basel
Höltje J.-V. (1996) Bacterial lysozymes. In: Lysozyme: Model
Enzymes in Biochemistry and Biology, pp. 65–74, Jollès P.
4
(ed.), Birkhäuser, Basel
5 Jollès J. and Jollès P. (1975) The lysozyme from Asterias rubens.
Eur. J. Biochem. 54: 19–23
6
Jollès J. and Jollès P. (1984) What’s new in lysozyme research?
Always a model system, today as yesterday. Mol. Cell. Bio-
chem. 63: 65–189
7 Nilsen I. W., Overbo K., Sandsdalen E., Sandaker E., Sletten K.
and Myrnes B. (1999) Protein purification and gene isolation
of chlamysin, a cold-active lysozyme-like enzyme with anti-
bacterial activity. FEBS Lett. 464:153–158
8
ItoY., Yoshikawa A., Hotani T., Fukuda S., Sugimura K., Imoto
T. (1999) Amino acid sequences of lysozymes newly purified
from invertebrates imply wide distribution of a novel class in the
lysozyme family. Eur. J. Biochem. 259: 456 – 461
Bachali S., Jager M., Hassanin A., Schoentgen F., Jollès P.,
Fiala-Medioni A. et al. (2002) Phylogenetic analysis of in-
vertebrate lysozyme and the evolution of lysozyme function. J.
Mol. Evol 54: 652–664
(fig. 7). As catalytic residues for isopeptidase activity,
H92, the only histidine residue present, and the conserved
serine residue (S62) between destabilase 1 and 2 se-
quences in the second hydrophilic region were predicted
by Zavalova et al. [23]. When we compare T. japonica
lysozyme with destabilase, the H92 and S62 of destabilase
are conserved in T. japonica lysozyme (H94 and S66).
However, the serine is not conserved in the lysozyme se-
quence from Mytilus edulis and Mytilus galloprovincialis.
Moreover, the lysozyme from M. edulis lacked iso-
peptidase activity [9]. Accordingly, we considered that
catalytic residues for isopeptidase activity in T. japonica
lysozyme are S66 and H94. Therefore, the region respon-
sible for isopeptidase activity might be the residues
9
1
0 Nilsen I. W and Myrnes B. (2001) The gene of chlamysin, a
marine invertebratetype lysozyme, is organized similar to ver-
tebrate but different from invertebrate chicken-type lysozyme
genes. Gene 269: 27–32
1
1 Jollès P. (ed.) (1996) Lysozyme: Model Enzymes in Biochem-
istry and Biology, Birkhäuser, Basel
1
2 Imoto T., Johnson L. N., North A. C. T., Phillips D. C. and
Rupley J. A. (1972) Vertebrate lysozyme. In: The Enzymes,
pp. 665–836, Boyer P. D., (ed.), Academic Press, New York
3 Dobson D. E., Prager E. M. and Wilson A. C. (1984) Stomach
lysozymes of ruminats. J. Biol. Chem. 259: 11607–11616
4 Irwin D. M. (1996) Molecular evolution of ruminant lysozyme.
In: Lysozyme: Model Enzymes in Biochemistry and Biology.
pp 347–361, Jollès P. (ed.), Birkhäuser, Basel
5 Zavalova L. L., Baskova I. P., Lukyanov S. A., Sass A.V.,
Snezhkov E. V., Akopov S. B. et al. (2000) Destabilase from
the medicinal leech is a representative of a novel family of
lysozyme. Biochim. Biophys. 1478: 69–77
1
1
65–95. We suggest that the region for chitinase activity
lies at the N-terminal end and that for isopeptidase activ-
ity at the C-terminal end of the T. japonica lysozyme
sequence.
We must search for catalytic residues in detail using mu-
tants of T. japonica lysozyme. We have already con-
structed an expression system for recombinant T. japonica
lysozyme, and the bifunctional property has also been
confirmed by gene engineering.
1
1
6 Fradkov A., Berezhnoy S., Barsova E., Zavalova L., Lukyanov
S., Baskova, I. et al. (1996) Enzyme from the medicinal leech
(
Hirudo medicinalis) that specifically splits endo-e(-g -Glu)-Lys
isopeptide bonds: cDNA cloning and protein primary structure.
Thus, this enzyme is an interesting and unique lysozyme
because it is a helix protein, judging from CD spectra. We
assume that other i-type lysozymes have the same char-
acteristics as T. japonica lysozyme.
FEBS Lett. 390: 145–148
1
7 Baskova I., Zavalova L., Berezhnoy S., Avdonin P., Afanasjeva
G., Popov E. et al. (2000) Inhibition of induced and spontaneous
platelet aggregation by destabilase from medicinal leech.
Platelets. 112: 83–86
Finally, thrombosis or fibrinogenolysis exhibited by 18 Baskova I. P. and Nikonov G. I. (1991) Destabilase, the novel
e-(g-Glu)-Lys isopeptidase with thrombolytic activity. Blood
destabilase has drawn great attention [17, 18]. T. japonica
lysozyme is a small and very stable protein with isopep-
tidase activity, and we expect that it will have numerous
applications.
Coagul. Fibrin. 21: 167–172
1
9 Yamada H. and Imoto T. (1981) A convenient synthesis of
glycolchitin, a substrate of lysozyme. Carbohydr. Res. 92:
160–162

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2
2
2
0 Baskova I. P., Nikonov G. I., Zavalova L. L. and Larionova N. I.
sequence alignment through sequence weighting, position-
specific gap penalties and weight matrix choice. Nucleic Acids
Res. 22: 4673–4680
(
1990) Kinetics of the hydrolysis of L-g-Glu-pNA by destabi-
lase, an enzyme from the leech Hirudo medicinalis. Biochem-
istry (USSR) 55: 495 – 499
23 Zavalova L., Lukyanov S., Baskova I., Snezhkov E., Akopov
S., Berezhnoy S. et al. (1996) Genes from the medicinal
leech (Hirudo medicinalis) coding for unusual enzymes
that specifically cleave endo-e(g-Glu)-Lys isopeptidase
bonds and help to dissolve blood clots. Mol. Gen. Genet. 253:
20–25
1 Yamada H., Fukumura T., Ito Y. and Imoto T. (1985) Chitin-
coating celite as an affinity adsorbent for high-performance
liquid chromatography of lysozyme. Anal. Biochem. 146:
7
1–74
2 Thompson J. D., Higgins D. G. and Gibson T. J. (1994)
CLUSTAL W: improving the sensitivity of progressive multiple