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https://www.abbs.info ISSN 0582-9879 |
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Short Communication |
Conformation
nearby Trp Residues of APIA and APIB Modulates the Inhibitory Specificity of
the Protease
LI
Jiong, CHI Cheng-Wu, RUAN Kang-Cheng*
(
Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological
Sciences,
the
Chinese Academy of Science, Shanghai 200031,
China )
Abstract The relationship between the micro-environment
of the two tryptophan residues and the inhibitory specificity of arrowhead
protease inhibitors A and B (APIA and APIB) was studied by mutagenesis and
fluorescence spectroscopy. The environment of the two Trp residues at positions
93 and 122 in APIB is more hydrophobic than in APIA. Study after substitution
of Trp with Ala revealed that the environment of Trp122 is more
hydrophobic than that of Trp93. Substitution of Leu82 and Arg87 in
APIB with Ser and Leu respectively made the tryptophan fluorescence of APIB to
be like that of APIA and the
inhibitory specificity to be closer to APIA, indicating that the inhibitory
specificity of the enzyme may be
modulated by the conformation around the tryptophan residues.
Key
words arrowhead protease
inhibitors；site-directed mutagenesis；fluorescence emission spectra； conformation；inhibitory
specificity
The
arrowhead proteinase inhibitors A and B (APIA and APIB) are the major inhibitor
components purified from the tubes of arrowhead (Sagittaria sagittifolia,
Linn.)[1]. Our previous study revealed that the inhibitors are
double-headed and multifunctional, capable of inhibiting many different serine
proteinase including trypsin, chymotrypsin as well as tissue kallikrein[1].
They are both composed of 179 amino acid residues with three disulfide-bridges[2,
3]. As their structures are quite unique, sharing no apparent homologous
sequence with other inhibitors, and absent of a domain boundary usually found
in other double-headed inhibitors, they should belong to a new inhibitor family[4].
Although APIA and APIB share 91% homology in primary structure, and have
identical reactive sites (Lys44 and Arg76)[5],
their specificity is quite different. APIA inhibits equimolar amount of trypsin
and chymotrypsin simultaneously, whereas APIB inhibits two molecules of
trypsin, and cannot inhibit chymotrypsin as strongly as APIA does[1, 5].
Our earlier mutagenesis work has revealed that the residues in position 82 and
87 (Ser and Leu in APIA；
Leu and Arg in APIB) were important for the inhibitory specificity[5].
However, the molecular conformation and its relationship with site-directed
mutation and inhibitory specificity have not been studied yet. There are only
two Trp residues in both APIA and APIB at positions 93 and 122[3],
which should allow to explore conformational changes by fluorescence
spectroscopy. Therefore, the conformation around tryptophan residues in APIA
and APIB as well as in the related mutants was monitored by this method.
1
Materials and Methods
1.1 Materials All of the restriction
enzymes, T4 DNA ligase were purchased from Gibco BRL. The DNA extraction kit
was from Promega. The Sequenase Version 2.0 DNA sequencing system was from
United States Biochemical (USB), [a–32P]
ATP (3×106
Ci/mol) from Amersham. The APIA and APIB were prepared according to the
previously described[1]. Bovine trypsin and chymotrypsin were purchased
from Sigma Chemical. Immobilized trypsin was prepared according to the
described[5]. Tosylarginine methyl ester (TAME) and benzoyl-tyrosin
ethyl ester (BTEE) were from Shanghai Dongfeng Biochemical Reagent Factory. All
other reagents were of analytical grade. PCR primers and mutated primers were
synthesized with an Applied Biosystems 380A DNA synthesizer. Escherichia
coli strain TG1 was given by Dr. WANG En-Duo. Saccharomyces cerevisiae
strain S-78 and yeast secretion expression vector, pVT102U/α,
were gifts from Dr. ZHANG You-Shang.
1.2 Site-directed mutagenesis and
polymerase chain reaction
The megaprimer method was used for site-directed mutation with two PCR
steps to amplify the mutated genes[6]. The primers for PCR and
site-directed mutation are shown in Table 1. The forward primer 1 and the
reversed primer 2 corresponded to the N-terminal and C-terminal sequence of
APIB respectively (in order to make the reading frame of the inhibitor
compatible with the expression vector, pVT102U/a,
in the primer an extra nucleotide T was inserted between the EcoRI site
and the first codon GAT). The first PCR step was used to amplify three
megaprimers corresponding to the gene fragments of residues 1―90,
90―179 and 118―179
with a wild type APIB gene as template (Primer 3 and 4 were used as forward
primer to pair with primer 2, respectively, while primer 5 as reversed primer
to pair with primer 1). These amplified megaprimers were then used to pair with
primer 1 or 2 to amplify the mutated genes of APIB by the second PCR step,
respectively.
1.3 Gene expression of mutated
inhibitors in the yeast secretion system The genes encoding the mutated inhibitor cleaved
with EcoRI/HindIII were ligated with the expression vector,
pVT102U/a,
through the XbaI/EcoRI linker as previously described[5].
The ligated mixture was used to transform E.coli strain DH5a.
The recombinant plasmid was confirmed by DNA sequence determination and used to
transform S. cerevisiae
strain S-78. The transformant was grown overnight in 3 ml of synthetic selected
YSD medium, and then transferred to 50 ml of YPD medium for further culture at
30 ℃ for 3 to 4 days. The mutated gene of
inhibitor fused with the gene encoding the leading peptide of α-mating
factor in the expression plasmid was expressed and processed by the KEX2
proteinase inherent in yeast cells, and then directly secreted into the culture
supernatant. The supernatant was collected, and the pH was adjusted to 8.0 with
Tris base, and then purified by using affinity chromatography with immobilized
trypsin as previously described[5].
1.4 Determination of inhibitory
activities
Theassay of trypsin inhibitory activity was performed in 3 ml of 20
mmol/L Tris-HCl, pH 7.8, 10 mmol/L CaCl2, containing 5 mg
trypsin and various amounts of the wild type or mutated inhibitor using 0.5
mmol/L TAME as a substrate. The residual trypsin activity was measured at 247
nm[5]. The chymotrypsin inhibitory activity was performed in 3 ml of
50 mmol/L Tris-HCl, pH 8.0, 10 mmol/L CaCl2, containing 5 mg
chymotrypsin using 0.5 mmol/L BTEE as a substrate[5].
1.5 Fluorescence emission spectra
measurement The
fluorescence emission spectra were measured with Hitachi F-4010 fluorescent
spectrophotometer equipped with a constant-temperature cell holder.
2
Results and Discussion
Fig.1
shows the fluorescence emission spectra of wild type APIB, W93A-APIB
and W122A-APIB excita-ted with 295 nm wavelength, in which only the
Trp residues in APIB and its mutants were excita-ted[7]. The maximum
emission wavelength lmax
of the wild type APIB was 326.5 nm. Fig.1 also reveals that the spectrum of W93A-APIB
(lmax
was 324 nm) has a blue shift compared to that of W122A-APIB (lmax
was 330 nm). This implies that both Trp122
and Trp93 are buried inside of the molecule without exposure to the
solvent, and that the environment around Trp122 is more hydrophobic
than that of Trp93[7―9].
The difference in spectrum also implies that Trp93 and Trp122
in APIB are not located in a same region. The spectrum of APIB represents the
total contribution of these two Trp residues and it seems reasonable that it
lies between that of W122A-APIB and W93A-APIB. The
inhibitory activity assay indicated that the Trp mutations did not raise any
change in the inhibitory activities against trypsin and chymotrypsin (data not
shown), suggesting that the conformation of W93A-APIB and W122A-APIB
have no obvious change caused by the mutation compared with that wild type
APIB.
Fig.1 Intrinsic fluorescence emission spectra
of APIB and related mutants
APIB and its mutants concentration, 10 mmol/L
in 50 mmol/L pH 8.0 Tris-HCl buffer；
excitation wavelength, 295 nm；
temperature, 25 ℃.
The measurements were carried out using a Hitachi F-4010 fluorescent
spectrophotometer.
Fig.2
shows the fluorescence emission spectra of wild APIA and APIB excited with 295
nm. The spectrum of APIB has a significant spectral blue shift relative to the
spectrum of APIA (lmax
was 338.0 nm for APIA and 326.5 nm for APIB, respectively). This indicates that
the average environment of Trp residues in APIB is more hydrophobic than that
in APIA[7], which might result in different specificity of these two
inhibitors against trypsin and chymotrypsin.
Fig.2 Intrinsic fluorescence emission spectra
of APIA, APIB and L82S-R87L-APIB
All the experimental conditions including
the protein concentrations were same as in Fig.1. The dotted curve represents
the spectrum of L82S-R87L-APIB normalized to the APIA
spectrum.
The
fluorescence emission of the mutant L82S-R87L-APIB is
also shown in Fig.2. The normalized spectrum of L82S-R87L-APIB
(dotted curve in Fig.2) almost totally overlaps with that of APIA. This fact
indicates that the residues 82 and 87 significantly influence the conformation
around the Trp residues. Concomitantly, substitution of 82 (Leu) and 87 (Arg)
of APIB with those Ser and Leu caused the change of the inhibitory activity.
Fig.3 (A) and (B) show the inhibitory activity of APIA, APIB and L82S-R87L-APIB
toward trypsin and chymotrypsin, respectively. The inhibitory activity of the
mutant toward trypsin was decreased, and on the other hand, its inhibitory
activity toward chymotrypsin was obviously increased compared with that of
APIB. Its inhibition curves toward trypsin and chymotrypsin totally overlapped
that of APIA (Fig.3). These results and the results of our previous study
suggest that the residues in 82 and 87 are important determinants of the
inhibitory specificity of the two inhibitors. The parallel change in tryptophan
fluorescence and inhibitory activity of this mutant leads to the conclusion
that the inhibitory specificity is related to the conformation around
tryptophan residues of the inhibitors. It seems likely that a hydrophobic
environment around tryptophan favors trypsin inhibition. There are three
possible explanations for this. (1) The inhibitory specificity is related to
the local conformation around the residues 82 and 87, Trp93 Trp122
are directly involved in this region conformation. Therefore the fluorescence
of these two tryptophan residues can reflect the conformation and its change in
this region. (2) The tryptophan residues are not located near residue 82 and
87, but substitution of residue 82 and 87 which are responsible for specificity
induces a change of the over-all conformation of the molecule, resulting in a
change affecting the environment of tryptophan residues. (3) The inhibitory
specificity is directly related to the conformation around the tryptophan
residues which was influenced by the substitution of residues 82 and 87. It
seems that the first explanation is more reasonable as the residues 82 and 87
are close to Trp93 in primary structure. However it is certainly
that the different specificity of APIA and APIB toward trypsin and chymotrypsin
related to the conformation around tryptophan residues. To get further
information about this relationship, the other methods, such as NMR or X-ray
crystallography combined with more site-directed mutations should be carried
out.
Fig.3 Inhibitory activities of APIA, APIB and
the mutant L82S-R87L-APIB
(A) Inhibitory activities against trypsin；
the measurement condition：
0.5 mmol/L TAME in 20 mmol/L pH 7.8 Tris buffer；
trypsin 5 mg；
reaction time, 5 min；
temperature, 37 ℃；
detected wavelength, 247 nm.(B) Inhibitory activities against chymotrypsin；
the measurement condition：
0.5 mmol/L BTEE in 50 mmol/L pH 8.0 Tris buffer；
chymotrypsin 6 mg；
reaction time, 5 min；
temperature, 25 ℃； detected
wavelength, 256 nm.
In
conclusion, the two Trp residues in APIB and APIA are all located in
hydrophobic regions, and the environment of Trp122 is more
hydrophobic than that of Trp93 in APIB. The tryptophan environments
are different in APIB and APIA. The inhibitory specificity of the inhibitors
for trypsin and chymotrypsin seems to be related to the conformation around the
tryptophan residues, which is significantly affected by residue 82 and 87.
References
1
Zhang XY, Chi CW. Studies on multifunctional crystalline proteinase
inhibitors from arrowhead. Sci Sin, 1979, 22：
1443―1454
2
Luo MJ, Lu WY, Chi CW. Clarification of an uncertain intron within the
cDNA sequences of arrowhead proteinase inhibitors A and B. J Biochem,
1997, 121：
991―995
3
Yang HL, Luo RS, Wang LX, Zhu DX, Chi CW. Primary structure and
disulfide bridge location of arrowhead double-headed proteinase inhibitors. J
Biochem, 1992, 111：
537―545
4
Laskowski M Jr, Kato I. Protein inhibitors of proteinases. Annu Rev
Biochem, 1980, 49：
593―626
5
Xie ZW, Luo MJ, Xu WF, Chi CW. Two reactive site locations and
structure-function study of the arrowhead proteinase inhibitors, A and B, using
mutagenesis. Biochemistry, 1997, 36：
5846―5852.
6
Sarkar G, Sommer SS. The “megaprimer”
method of site-directed mutagenesis. Biotechniques, 1990, 8：
404―407
7
Lakowicz RJ. Protein fluorescence. Principles of Fluorescence
Spectroscopy, 1983, New York：
Plenum Press, 341―381
8
Kronman MJ, Holmes LG. The fluorescence of native, denatured, and reduced
denatured protein. Photochem Photobiol, 1971, 14：
113―134
9
Eftink MR, Ghiron CA. Exposure of tryptophanyl residues in proteins.
Quantitative determination by fluorescence quenching studies. Biochemistry,
1976, 15：
672―680
Received：January
11, 2002 Accepted：February
25, 2002
This
work was supported by a grant from the National Natural Science Foundation of
China, No.30070164
*Corresponding
author：
Tel, 86-21-64740532; Fax, 86-21-64338357；
e-mail, [email protected]