Biomolecules Department, National Institute of Bioscience and Human Technology, 11 Higashi, Tsukuba, Ibaraki 305-8566, Japan
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Abstract |
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Keywords: Chemical synthesis/chitin-binding activity/hevein domain/non-natural amino acid residue/site-specific mutagenesis
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Introduction |
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Materials and methods |
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Solid-phase peptide synthesis, cleavage from resin, deprotection, separation by reversed-phase HPLC (RP-HPLC) and oxidative refolding of the reduced peptide were performed as described previously (Muraki et al., 1998). The samples were prepared as peptide amides. Molecular weights (MW) of the purified samples were measured with MALDI-TOF/MS Voyager (PerSeptive Biosystems).
-Cyano-4-hydroxycinnamic acid was used as the matrix, UDA-isolectin VI(UDA VI) (Does et al., 1999
), bovine insulin chain A (oxidized, purity 96%) and WIN2-N was obtained as described (Muraki et al., 1998
). All chemical reagents were of the purest grade available. A polyacrylamide gradient gel (1525%), SDSTrisTricine buffer and peptide MW markers were purchased from Daiich Kagaku Yakuhin.
Assay of chitin binding activity
A chitin bead slurry was purchased from New England Biolabs (Beverly, MA). The affinity for chitin was evaluated basically according to the methods of Oita et al. (1996) and Nielsen et al. (1997) as described (Muraki et al., 1998). A 50 µg amount of sample as determined with a BCA protein assay kit (Pierce) was used for each assay. The amount of unbound sample after each elution step was determined from the peak area in RP-HPLC analysis. The amount of sample bound to chitin was calculated by subtracting the amount of unbound sample from the total amount of sample. Three replicate assays were performed with each sample at room temperature (25 ± 2°C).
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Results and discussion |
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The amino acid sequences of UDA VI (hinge region excluded), WIN2-N, CPB20-N and Ac-AMP2 were aligned in Fig. 1a. The RP-HPLC profiles of CBP20-N after oxidative refolding (Fig. 2a
) resembled that of WIN2-N (Muraki et al., 1998
) as expected from the striking similarity (40/43 identical) in amino acid sequence. Specifically, a pair of peaks (peaks 1 and 2) of the refolded product were observed after the oxidation of reduced peptide. Judging from the MW of peak 1 (4439) and peak 2 (4421), peaks 1 and 2 were identified as CBP20-N with an N-terminal glutamine residue ([Gln1]-CBP20-N) (calculated MW: 4443) and CBP20-N with an N-terminal pyroglutamate residue ([Pgl1]-CBP20-N) (calculated MW: 4426), respectively. The partial conversion of Gln1 into Pgl1 catalyzed by weak acids including acetic acid (AcOH) and the resulting similar separation of peaks in the RP-HPLC profile have been reported (Dimarchi et al., 1982
, Or
owska et al., 1987
). Since we used pure N
-Fmoc-N
-tritylglutamine in the last coupling step, the formation of Pgl1 should be ascribed to the deprotection and purification steps using 1050% AcOH.
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All purified samples were homogeneous as judged by both MALDI-TOF/MS and RP-HPLC analysis.
Mutational effect on the affinity for chitin
Recently, we have determined the X-ray structure of UDA VI(GlcNAc)3 complex at 1.9 Å resolution (Harata and Muraki, 2000). The structure of the ligand-binding region of the N-terminal domain of UDA VI (UDA VI-N) is shown in Figure 1b
. As expected from the amino acid sequence homology, the overall three-dimensional structures of all HDs revealed so far are very similar to each other (Martins et al., 1996
; Asensio et al., 1998
). The spatial arrangement of the side chains of three aromatic residues which are involved in proteincarbohydrate interaction was also well conserved (Martins et al., 1996
). Therefore, it is reaonable to discuss the mutational effect on the affinity for chitin in this study by referring to the X-ray structure of the UDA VI(GlcNAc)3 complex.
The affinity of HD samples for chitin was examined in 10 mM TrisHCl (pH 8.0) and 0.5 M AcOH (pH 2.5). In Figure 3
, the results of the assay are summarized. UDA VI (sample a) and insulin A-chain (sample b) were used as the positive and the negative control of the experiment, respectively. The behavior of intact HDs, WIN2-N (sample c) and [Pgl1]-CBP20-N (sample d) was similar to that of UDA VI under either pH conditions, whereas the affinity of a C-terminal truncated HD, Ac-AMP2 (sample f), was much less than that of UDA VI under acidic conditions. In the X-ray structure of the UDA VI-(GlcNAc)3 complex, several possible hydrogen bonds were observed between the main chain of the C-terminal region (residues 3643) of the first domain and that of the core region including the ligand binding site (not shown). Therefore, the C-terminal region in intact HDs may contribute to strengthening the affinity by stabilizing the binding conformation of the domain. The stronger affinity of [Pgl1]-CBP20-N than Ac-AMP2 for chitin in this study was consistent with the larger association constant of hevein (Asension et al., 1995) than Ac-AMP2 (Verheyden et al., 1995
) with (GlcNAc)3, which were determined by NMR experiments.
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The involvement of three conserved aromatic residues in the ligand binding of Ac-AMP2 has been suggested by NMR studies (Verheyden et al., 1995). Our previous study demonstrated that the replacement of Tyr73 in WGA which corresponds sterically to Tyr27 in Ac-AMP2 by Phe did not have much effect on the affinity for (GlcNAc)3 (Nagahora et al., 1995
). In the present study, the site-specific mutant concerning Phe18, Tyr20 and Tyr27 of Ac-AMP2 were examined. At pH 8.0, all Ac-AMP2 samples possessing three aromatic residues at position 18, 20 and 27 (samples f, g, h, i, j, k, l, o and p) were completely bound to chitin, whereas a significant amount of unbound sample was observed with the mutants with replacement of any one aromatic residue of Ac-AMP2 by Ala (samples m, q and r). This suggested that the complete set of the three aromatic residues in the ligand binding site was important for the full expression of the affinity for chitin. As judged from the percentage of the remaining samples at pH 8.0, the extent of reduction of the affinity by the mutation to Ala decreased in the order Tyr20
Tyr27>Phe18.
The stacking interaction between the aromatic side-chain group of protein and the apolar face of the carbohydrate moiety frequently occurs in the ligand recognition of carbohydrate binding proteins (Vyas, 1991; Elgavish and Shaanan, 1997
). The reduction in affinity for another water-insoluble ß-1,4-linked polysaccharide, crystalline cellulose, by the mutation of Tyr492 in the ligand binding site to Ala has been reported with the cellulose binding domain of Trichoderma reesei cellobiohydrase I (Reinikainen et al., 1992
). In the UDA VI(GlcNAc)3 complex, Trp21 and Trp23, which correspond to Phe18 and Tyr20 in Ac-AMP2, stacked with the apolar face of NAG C and NAG B in parallel, respectively (Figure 1b
). Phe18 and Tyr20 in Ac-AMP2 were first replaced with two other natural aromatic amino acid residues. Tyr18 mutant (sample g) and more significantly Trp18 mutant (sample j) showed an enhanced affinity compared with wild-type Ac-AMP2 (sample f) under acidic conditions, whereas Phe20 mutant (sample o) and Trp20 mutant (sample p) exhibited similar and lower affinity, respectively.
The mutants containing a non-natural amino acid residue at position 18 were then further examined to probe another possibility of the affinity enhancement for chitin. Cha18 mutant (sample n) exhibited less affinity than Ala18 mutant (sample m), indicating that the side-chain benzene ring of Phe18 was important not simply as hydrophobic six-membered carbon-atom ring but as a planar aromatic ring. The pNO2-Phe18 mutant (sample h) and F5-Phe18 mutant (sample i) displayed similar and reduced affinity compared with wild-type Ac-AMP2, showing the weak and detrimental effect of these electron-withdrawing substituent groups, respectively. In contrast, the replacement of Phe18 with 1-Nal18 or 2-Nal18 enhanced the affinity. The increased extent by the replacement with 2-Nal18 (sample 1) was larger than that with 1-Nal18 (sample k) and comparable to that with Trp18 mutant (sample j).
Our recent study suggested that CH interactions (Nishio et al., 1995
) involving a stacking aromatic residue, Tyr63, played an important role of the synergism between apolar and polar interactions in the recognition of a carbohydrate ligand by human lysozyme (Muraki et al., 2000
). Although the parallelism between the side chain of Trp21 and the ring of NAG C is not perfect in the UDA VI(GlcNAc)3 complex, six possible CH
interactions less than 3.2 Å, specifically H5 (in NAG C)CD1 (in Trp21), 3.0; H5CD2, 2.9; H5NE1, 3.2; H5CE2, 3.1; H5CG, 2.8; and H61CE3, 3.2 Å, were observed by generating hydrogen atoms on the NAG C residue using the `h-build' protocol in X-PLOR (Brünger, 1992
). The difference in the size or the substituent group of stacking aromatic rings should affect the strength of CH
interactions by increasing or decreasing the ability of the aromatic ring as a hydrogen-bond acceptor. Therefore, the results of the present study may suggest that such an interaction is a major determinant of the affinity of Ac-AMP2 for chitin again. Although the detailed mechanism of the enhancement of affinity caused by the mutation of Phe18 to the residue with a larger aromatic ring remains to be clarified, the present study demonstrated the possibility of improving the binding strength of an HD to chitin by protein engineering. Further investigations to reveal the structure and function relationships of HD are in progress.
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Notes |
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Acknowledgments |
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References |
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Received November 11, 1999; revised February 3, 2000; accepted February 29, 2000.