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Cite this: Chem. Commun., 2014, 50, 4248 Received 30th October 2013, Accepted 28th February 2014

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Circular permutation of chicken interleukin-1 beta enhances its thermostability† Wen-Ting Chen,a Ting Chen,a Chao-Sheng Cheng,a Wen-Yang Huang,a Xinquan Wangb and Hsien-Sheng Yin*a

DOI: 10.1039/c3cc48313d www.rsc.org/chemcomm

Interleukin-1b is a cytokine critically involved in immune and inflammatory responses. To extend its use as a component of avian vaccines, a circularly permuted chicken interleukin-1b was synthesized that maintains its activity after pre-incubation at high temperatures, unlike wild-type chicken interleukin-1b, which is irreversibly inactivated at high temperatures.

Interleukin-1 beta (IL-1b) is a proinflammatory cytokine that plays a crucial role in regulating the production of chemokines, cytokines, adhesion molecules, and acute inflammatory-phase proteins.1 It is expressed as a pre-pro-protein mainly in monocytes and macrophages.2 This pre-pro-protein is activated by caspase 1, and by interacting with the IL-1 receptor, mature IL-1b triggers a cascade of immune responses.2 Increased secretion of active IL-1b usually correlates with the pathogenesis of autoinflammatory diseases.3 When IL-1b is included in a vaccine, it acts as an immunostimulant to enhance the immune response, e.g., after vaccination against influenza virus,4 S. pneumonia,5 and coccidiosis.6 Although human IL-1b has been well characterized in terms of its structure and biology, the same is not true for avian IL-1bs. The primary sequences of avian IL-1bs are B35% identical to that of human IL-1b.7,8 The crystal structure of chicken IL-1b (PDB entry, 2WRY) revealed that it is a typical b-trefoil protein containing 14 b-strands and an a-helix (Fig. S1, ESI†).9 Its interior contains a cavity formed by non-polar residues.9 Although the tertiary structures of chicken and human IL-1b are similar, the residues in the loops involved in receptor binding differ, which leads to different cross-reactivities and immunological responses.7,10 To increase the potential of IL-1b for therapeutic use, e.g., as an adjuvant, we created a circularly permuted chicken IL-1b (CP36) and determined its physicochemical and biological properties.

Circular permutation of a protein rearranges its primary sequence by forming a peptide bond between its native N- and C-termini and creating new ends at another location;11 circular permutation may thus alter the protein’s folding landscape,12 stability,12 and/or bioactivity.13 Circularly permuted IL-1b variants were cloned and expressed (see ESI†). CPred14 and B-factor analyses of the wild-type (WT) chicken IL-1b structure were used to predict potential circular permutation sites. Peptide linkers of various lengths were introduced to connect the original N- and C-termini. Most of the variants were insoluble or expressed in very low yield. The CP36 (which has new N- and C-termini in WT residues, Gln36 and Leu35 in loop 3, respectively, and the linker GT(GGS)8) could be expressed and purified with no aggregation observed. To investigate the structural impact of circular permutation on chicken IL-1b, far-UV circular dichroism (CD) and fluorescence spectra were recorded.15 The CD spectrum of WT chicken IL-1b revealed a strong negative signal at B205 nm (Fig. 1A), indicating the presence of b-sheets (63  4%). In contrast, the spectrum of CP36 indicated that a substantial change in secondary structure occurred upon circular permutation. This spectrum contained a peak at 195 nm and two minima at 209 nm and 222 nm (Fig. 1A), suggestive of a protein containing

a

Institute of Bioinformatics and Structural Biology, National Tsing Hua University, Hsinchu, Taiwan. E-mail: [email protected] b School of Life Sciences, Tsing Hua University, Beijing, China † Electronic supplementary information (ESI) available: Experimental methods and supplementary figures. See DOI: 10.1039/c3cc48313d

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Fig. 1 Far-UV CD spectra and thermal stabilities of WT chicken IL-1b and CP36. (A) The far-UV CD spectra of WT IL-1b and CP36 were recorded between 260 and 195 nm at 25 1C. (B) Thermal denaturation curves recorded at 217 nm from 4 1C to 96 1C in 2 1C increments.

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Table 1 Binding affinities and spectroscopic characteristics of WT chicken IL-1b and CP36. Cm is the concentration of guanidine-HCl at which 50% of a protein is unfolded. lmax and Fmax refer to the wavelength of the maximum intrinsic fluorescence and the intensity of a protein–ANS complex, respectively. AU, arbitrary unit. Kd, dissociation constants

Protein

Cm (M)

lmax (nm)

Fmax (AU)

Kd (nM)

WT chicken IL-1b CP36

1.4 1.7

326.5 358.5

60.11 5.14

0.12 199

mostly a-helices (63  4% (a) and 33  4% (b)). Moreover, the fluorescence emission spectrum of WT IL-1b had a maximum (lmax) at 326.5 nm (excitation at 280 nm), whereas the maximum was shifted to 358.5 nm in the spectrum of CP36 (Fig. S2, ESI† and Table 1), which suggests that the tyrosine residues in CP36 are more exposed to solvent.16 The buried aromatic residues in IL-1b contribute to the formation of a well-packed non-polar core.17 The fluorescence intensity (Fmax) of 1,8-anilinonaphthalenesulfonate (ANS) in a solution of CP36 was greater than that of an ANS/WT IL-1b solution, suggesting a more loosely packed non-polar core in CP36 (Fig. S3, ESI†).16,18 Therefore, circular permutation of WT IL-1b apparently altered its secondary structure characteristics and perturbed the packing of its non-polar core. The buried, conserved aromatic residues in IL-1b are important to the stability of the hydrophobic core and, consequently, to the entire protein.17 We examined the stability of CP36 against guanidine-HCl (a chemical denaturant) and against temperature.15 The guanidine-HCl denaturation curves for CP36 and WT IL-1b were similar (Fig. S4, ESI†), with Cm values—the concentration of guanidine-HCl at which 50% of a protein is unfolded—of 1.7 M and 1.4 M, respectively. Between 0 and 1.0 M guanidine-HCl, the population of CP36 that was denatured appeared to be less than that of WT IL-1b, implying that the structure of CP36 is more resistant to small concentrations of guanidine-HCl than is IL-1b. The WT IL-1b thermal denaturation curve showed a well-defined two-state unfolding transition (Fig. 1B and Fig. S5, ESI,† left panel). Interestingly, the spectrum of CP36 (and therefore its secondary structure) was relatively unchanged by temperature (Fig. 1B and Fig. S5, ESI,† right panel). The main effect of temperature on the spectrum was an apparent and almost linear increase in ellipticity not accompanied by a two-state transition (Fig. 1B). Accordingly, the circular permutation of IL-1b changes the protein’s secondary content and may form extra helical conformations to stabilize it. Consequently, CP36 appeared to contain a loose and solventaccessible non-polar interior, which may be responsible for its enhanced stability and altered secondary structure. To gain insight into the three-dimensional structure of CP36, it was modeled by ab initio modeling at the Robetta server.19 The model revealed 11 b-strands and three a-helices (Fig. 2A). The CP36 and IL-1b structures share a similar core fold (residues 22–126) with a root mean square deviation (rmsd) of 2.14 Å for the Ca atoms of these residues (Fig. 2B). Larger structural deviations were evident for the Ca atoms at the N- and C-termini and for residues sequentially near the linker sequence with a rmsd of 20.83 Å. VOIDOO analysis20 showed that both CP36 and WT IL-1b have pockets formed by non-polar

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Fig. 2 Ribbon diagram for CP36 and superpositioned CP36 and WT IL-1b. (A) A ribbon diagram of the three-dimensional structure of CP36. The a-helices and b-strands are shown as helices and arrows, respectively. The N- and C-termini are labeled. The location of the linker is shown. (B) The X-ray structure of WT chicken IL-1b (red) is superimposed onto that of CP36 (blue). The alignment indicates that the major structural differences occur at the termini and before and after the linker. The loops in WT chicken IL-1b that are directly involved in receptor binding are colored yellow. The N- and C-termini are represented as spheres that, for CP36, are colored blue and cyan, respectively, and for WT IL-1b are colored red and pink.

residues at similar locations (Fig. S6, ESI†), although the pockets are of different sizes. The pocket in the model of CP36 is 454.0 Å3, which is considerably larger than that found in WT IL-1b (50.5 Å3) (Table S1, ESI†) and is consistent with the ANS-binding data. Additionally, the aromatic residues in CP36 were found to be more solvent accessible than those in IL-1b (820.1 Å2 vs. 444.6 Å2, respectively; Table S1, ESI†). This greater degree of solvent accessibility correlates with the results of the intrinsic fluorescence study (Fig. S2, ESI†). With respect to the orientations of WT IL-1b, different orientations were found for Y122, Y159, F157, F165, F168, and F175 in the CP36 model (Fig. S7, ESI†). These residues are probably responsible for the increased size of the pocket in CP36 and the difference in the stabilities of the two proteins. Surface plasmon resonance assays were performed to determine the dissociation constants (Kd) for the chicken IL-1 receptor–IL-1b and chicken IL-1 receptor–CP36 complexes (Fig. 3).21 The Kd values were 0.12 nM and 199 nM for WT chicken IL-1b and CP36, respectively, showing that WT chicken IL-1b binds the receptor more tightly. Conversely, WT human IL-1b did not bind the receptor (Fig. S8, ESI†). Loops 1, 3, 4, 9, and 11 in WT chicken IL-1b are important for its association with the receptor.9 After superpositioning the structures of WT chicken IL-1b and CP36 (Fig. 2B), we found that the positions and orientations of some of these loops (loops 1, 3, and 4) were not the same, and these altered

Fig. 3 Surface plasmon resonance assays to determine the binding affinities of WT chicken IL-1b and CP36 to the chicken IL-1 receptor. Sensorgrams were obtained by injecting various concentrations of (A) WT chicken IL-1b or (B) CP36 over a receptor-immobilized chip. The curves were fitted with a 1 : 1 interaction model (black lines). RU, response unit.

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Fig. 4 Functional assays of IL-1bs. (A) To determine the bioactivities of WT chicken IL-1b, CP36, and WT human IL-1b, each protein was directly injected into the wing vein of adult chickens, after which the plasma cortisol level was measured. Inset: chicken fibroblasts were incubated with WT chicken IL-1b or CP36 to detect K60 mRNA expression. b-Actin mRNA expression served as the internal control. (B) To investigate the impact of temperature on the subsequent ability of WT chicken IL-1b or CP36 to increase the plasma cortisol level, solutions of the proteins were heated to various temperatures for 10 min and cooled to 25 1C. They were then immediately injected into chickens. The residual activity was defined as the ratio of the cortisol level after and before the temperature treatment.

positions/orientation may cause the decreased affinity of CP36 for the receptor. To characterize the bioactivity of CP36, the mRNA expression level of the pro-inflammatory CXC chemokine K60 in chicken fibroblasts was determined (Fig. 4A, inset).9 An obvious agarose-gel band corresponding to K60 mRNA was present in response to CP36 treatment, implying that CP36, like WT chicken IL-1b, triggers an immune response in fibroblasts. Conversely, exposure of chicken fibroblasts to human IL-1b did not induce K60 mRNA expression despite the similar tertiary structures of WT chicken and human IL-1b (Fig. S9, ESI†).9 Additionally, the plasma cortisol levels in chickens were significantly enhanced by intravenous injections of WT chicken IL-1b and CP36, which was not found for human IL-1b (Fig. 4A). Therefore, CP36 can induce immune and inflammatory responses in chickens, which is consistent with the K60 mRNA study. However, the ability of CP36 to increase the plasma cortisol level was Btwofold lower than that of WT chicken IL-1b, which may be a consequence of a decreased binding affinity of CP36 for the chicken receptor. Our CD thermal study revealed that CP36 is more thermally stable than WT IL-1b. We therefore subjected solutions of WT chicken IL-1b and CP36 to various temperatures and subsequently injected the solutions into chickens to measure the serum cortisol level (Fig. 4B). Heat-treated CP36 caused strikingly better immune responses than heat-treated WT chicken IL-1b. When heat-treated at 50 1C, WT IL-1b lost more than 50% of its activity. Conversely, CP36, even after being exposed to 65 1C, retained more than 80% of its activity. These results demonstrate that the heat tolerance of CP36 allows it to retain its bioactivity, thereby enhancing cold chain storage and the vaccine potency.22

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To conclude, we synthesized a circularly permuted IL-1b (CP36) and found that it was also monomeric (Fig. S10, ESI†) but more stable to chemical, thermal and proteolytic denaturation (Fig. S11, ESI†) compared with WT IL-1b. Despite the decreased binding affinity of CP36 for the IL-1 receptor, CP36 still exhibited in vitro and in vivo bioactivities. Importantly, CP36 retained its bioactivity after high-temperature treatment, which may allow CP36 to be used in therapeutic applications such as an avian vaccine adjuvant. The authors are thankful to Ms Ting-Yu Chiang for helpful discussions and data analysis. This work was supported by the National Science Council, Taiwan (grant number, NSC-101-2627B-007-003- and the Toward World-Class University Project).

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Circular permutation of chicken interleukin-1 beta enhances its thermostability.

Interleukin-1β is a cytokine critically involved in immune and inflammatory responses. To extend its use as a component of avian vaccines, a circularl...
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