http://www.abbs.info e-mail:[email protected] ISSN 0582-9879 ACTA BIOCHIMICA et BIOPHYSICA SINICA 2002, 34(4): 494-497 CN 31-1300/Q |
Short Communication |
Conformation
nearby Trp Residues of APIA and APIB Modulates the Inhibitory Specificity of
the Protease
(
Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological
Sciences,
the
Chinese Academy of Science, Shanghai 200031,
China )
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.
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 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.
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.
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]