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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

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

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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]