Protein and Peptide Letters - Volume 24, Issue 11, 2017

Background: Two-component signaling systems are wildly distributed in most bacteria, which allow bacteria to sense and respond to the stimuli for survival in fluctuating environmental conditions. Histidine kinase response regulator hybrid protein (HK) from G. oxydans, a polypeptide with a response regulator attached to the histidine protein kinase, was identified as a member of two-component signaling system in the bacterium. However, the role of the protein in the bacterium remained unknown. Objectives: The main objective of this study was to screen and identify the proteins in G. oxydans, potentially interactive with HK, to reveal the roles played by HK in the bacterium. Methods: Potential HK interactive proteins were screened by yeast two-hybrid assay. Among those candidate proteins, a specific interaction between HK and a 2-keto-3-deoxy-D-manno-octulosonate- 8-phosphate synthase (KDO8PS) was established by GST pull-down assay in vitro and BiFC assay in vivo. Results: 60 positive clones, representing 27 different prey proteins in G. oxydans, were screened by yeast two-hybrid assay with HK as the bait. Among these interactive proteins, KDO8PS can catalyze the condensation reaction between phosphoenolpyruvate (PEP) and arabinose 5-phosphate (A5P) to produce a precursor essential for LPS biosynthesis in Gram-negative bacteria. After expression and purification, the interaction between HK and KDO8PS was further verified by GST pull-down assay. Subsequent BiFC assay revealed the interaction between these two proteins in vivo. Conclusion: Our results revealed an interaction between a two-component system protein HK with KDO8PS in G. oxydans. As KDO8PS plays a vital role in lipopolysaccharide biosynthesis required for growth in Gram-negative bacteria, and its activity strongly depends on several conserved histidine residues, the interaction described here implied that HK may play certain roles in bacterial growth under stress.

Background: Plant lectins are a group of highly diverse proteins that possess at least one non-catalytic domain that binds reversibly to a specific mono- or oligosaccharide. So far, only seven members in the lectin-arcelin-αAI1 supergene family in legume lectins have been reported to have inhibitory activity of α-amylases. Objective & Methods: A proteinaceous α-amylase inhibitor was isolated and purified using Ammonium sulfate precipitation (ASP), Ion exchange chromatography (IEC) and Reversed phase liquid chromatography (RPLC) from the mature seeds of chickpea. Results & Conclusion: Identification by Ultra-performance liquid chromatography-tandem mass spectrometry (UPLC/MS-MS) indicated that the purified proteinaceous α-amylase inhibitor was a chickpea lectin CAL in GenBank (accession No. AGL46982.1). CAL had 227 aa containing a hemopexin- like repeats domain and was a cytoplasm protein. It had very low (<17%) identity with seven members in the lectin-arcelin-aAI1 supergene family in legumes that have α-amylase inhibitory activity. The purified CAL derived from prokaryotic expression was confirmed to have inhibitory activity against various α-amylases. The inhibitory activity of CAL against various α-amylases was severely affected by temperature, pH, incubation time, substrate concentration and CAL protein concentration. Feeding CAL reduced the weight of potato beetle larvae by 27.21% (P<0.05) and survival rate by 6.67% (P>0.05). Our results indicated that CAL is a new type of lectin with inhibitory activity against α-amylases in legume lectins, which can be used as a candidate in genetic engineering for breeding for pest resistance.

Background: Mastoparan B (MPB) is a venom peptide isolated from Vespa basalis (black-bellied hornet), one of the dangerous vespine wasps found in Taiwan. MPB is a tetradecapeptide (LKLKSIVSWAKKVL), amphiphilic venom peptide, with a molecular mass of 1.6 kDa. MPB belongs to an evolutionarily conserved component of the innate immune response against microbes. In this study, we attempted to modify a reliable oleosin-based fusion expression strategy coupled with the artificial oil body (AOB)-cyanogen bromide (CNBr) platform to produce bioactive MPB. Objectives: The aim of this study was to develop an artificial oil body (AOB)-cyanogen bromide (CNBr) platform to produce the bioactive form of mastoparan B (MPB), which in a manner identical to that of its native counterpart. Methods: The plasmid pET30-His6-rOle(127M→L)-MPB was constructed, and then four different E. coli strains- BL21(DE3), BL21(DE3)pLysS, C41(DE3), and C43(DE3) were tested to identify the most suitable host for the pET30-His6-rOle(127M→L)-MPB fusion protein expression. We optimized the expression conditions by testing different growth temperatures, isopropyl-β-D-thiogalactoside (IPTG) concentrations, and post-induction collection times. Afterwards, the His6-rOle(127M→L)-MPB protein was purified by one-step nickel-chelated affinity chromatography (Ni2+-NTA) under denaturing conditions. The purified His6-rOle(127M→L)-MPB was selectively cleaved by thrombin protease to remove the His6-tag and the leader peptide from the N-terminus. Subsequently, rOle(127M→L)-MPB protein was constituted into AOB and incubated with CNBr for a cleavage reaction, which resulted in the release of the MPB from rOle(127M→L)-MPB protein via AOB. The purified MPB was identified by MALDI-MS and HPLC analysis, and its bioactivity was examined by antimicrobial testing. Results: After a 2-h induction period, the E. coli C43(DE3) was found to be superior to BL21(DE3) and the other protease-deficient strains as an expression host. And, the optimal His6-rOle(127M→L)-MPB expression at 37°C for 2 h after induction with 5 μM IPTG. The purified MPB showed that a single major peak was detected by HPLC/UV detection with a retention time of 22.5 minutes, which was approximately 90% pure. The putative MPB, and over two-third of the peptide sequence was verified by the MALDI-MS analysis. Finally, the purified MPB was examined by a broth dilution-antimicrobial susceptibility test. These results indicated that the purified MPB was bioactive and very effective in anti-bacterial (E. coli J96) activity. Here, we successfully used the oleosin-based fusion expression strategy coupled with the artificial oil body (AOB)-cyanogen bromide (CNBr) platform to produce bioactive MPB peptide which, in a manner identical to that of its native counterpart. Conclusion: In this study, the recombinant oleosin based fusion strategy coupled with AOB-CNBr purification platform open a new avenue for the production of active MPB and facilitate the studies and applications of the peptide in the future for medicinal applications such as hypotension and antibacterial effect.

Background: Nowadays, the biological effects of microgravity have been the subject of various experimental researches. Microgravity has been confirmed to affect biological systems. Furthermore, as a result of improvement in space technology for instance a manned mission to the moon, probabilities for human exposed to microgravity have incremented undoubtedly. Objectives: The purpose of this study was to investigate the probable biological effects of microgravity on the human serum albumin (HSA) structure after 3 and 24 h exposure. It is worth mentioning that this is the first effort to investigate the structural alternations of HSA under simulated microgravity condition in biophysico-chemical terms thru different spectroscopic instruments. Methods: 2D clinostat was utilized for simulating microgravity. The UV-Vis, intrinsic and extrinsic fluorescence, dynamic light scattering (DLS) and circular dichroism (CD) spectra of 3.76 μM HSA in Tris–HCl buffer (pH 7.4, 0.1 M) and 3.76 μM HSA in Tris–HCl buffer (pH 7.4, 0.1 M) kept at simulated microgravity for 3 and 24h were verified. Results: The UV-Visible, near-UV-CD and intrinsic fluorescence spectroscopy represented that microgravity can remarkably change the tertiary structure of HSA. Additionally, the ANS affinity for HSA incremented when the protein was exposed to simulate microgravity compared to unexposed HSA, which may possibly have appeared attributable to expansion of the structure of simulated HSA. Fluorescence quenching by acrylamide demonstrated higher stern-volmer constant for exposed HSA. The results of zeta potential and dynamic light scattering (DLS) experiments depicted that simulated microgravity cause raise in the surface charge and size of HSA. Far-UV CD data demonstrated that simulated microgravity did not perturb the secondary structures of the protein. Conclusion: Collectively, our results suggest that HSA after 24 h exposure to microgravity can exhibit a molten globule (MG) structure. This is the first report to demonstrate the molten globule state formation in microgravity condition. Results from this study could give knowledge to understand the role of gravity on protein folding process. In addition, this finding could help to find out safe limits for astronauts and space travelers and to develop adequate countermeasures against any harmful effects of microgravity.

Background: The chaperone activity of α-crystallin (α-Cry) plays an important role in maintenance of eye lens transparency. Various mutations in the α-Cry genes have been indicated to cause cataract diseases in human. Also, the calcium imbalance has been shown to induce aggregation in α-Cry. We investigated the impact of calcium ion on structure, chaperone activity of the recombinant wild-type and mutant R12C αA-Cry. We suggested that the raise of calcium level in eye lens is an additional contributory factor accelerating the development of cataract diseases in patients with R12C mutation. Objectives: The main objective of this study was to investigate the impact of calcium ion on structure, chaperone activity and amyloidogenic properties of the recombinant wild-type and mutant R12C αA-Cry, in a comparative study. Methods: The mutagenesis was performed on confirmed αA-Cry cDNA in pET-28b (+) which applied as a template to generate R12C mutant, using polymerase chain reaction (PCR) and a Quick Change Lightning Multi Site-Directed Mutagenesis kit (Stratgene). Both wild-type and mutant plasmids were chemically transformed into E.coli BL21 (DE3) and the respective recombinant proteins over-expressed in LB broth. The protein purification was done using Q-Sepharose anion exchange and Sephacryl S-300 gel filtration chromatography. The purified αA-Cry samples were incubated with different concentrations of calcium ion (0-40 mM) at 37 °C for 1 week. The secondary and tertiary structural analyses of each protein were performed by far-UV CD and Try/Trp and ANS fluorescence assessments, respectively. The assessment of chaperone activity was done spectrophotometrically in both thermal and chemical-induced aggregation systems using γ-Cry and bovine pancreatic insulin as the substrate proteins, respectively. Also, the amyloidogenic properties of proteins was investigated by CR absorption and ThT fluorescence measurements. Results: The results of fluorescence and CD assessments suggested the significant secondary and tertiary structural alterations upon R12C mutation. R12C mutant αA-Cry demonstrated preserved secondary and tertiary structures in the presence of calcium. The chaperone activity of wild-type and mutant R12C αA-Cry was reduced in the presence of calcium. Also, the extent of chaperone activity reduction was significantly higher for R12C αA-Cry. Both wild-type and mutant R12C αA-Cry revealed slight amount of aggregation when incubated with different calcium concentrations for 1 week, at 37 °C. However, the susceptibility of both proteins for aggregation was significantly increased in the presence of 40 mM calcium, at the elevated temperature (60 °C). Also, the mutant protein exhibited extensive disulfide bridge cross-linking as indicated by gel electrophoresis. Moreover, the mutant R12C αA-Cry significantly resists against amyloid fibril formation in the presence of calcium ion compared to the wild-type protein as indicated by CR and ThT assessments. Conclusion: Our data suggested that αA-Cry conformational changes occurring upon R12C mutation and further functional damages induced by calcium may play an important role in the pathomechanism of the cataract development by this mutant protein.

Background: Identification of immunogenic antigens is an important step for the vaccine improvement. Previous studies indicated that Actinobacillus pleuropneumoniae PalA is homologous to a Haemophilus influenzae protective antigen Hi-PAL (P6) protein. However, PalA protein adversely affects the Apx toxinbased subunit vaccine. The role of PalA in the inactivated vaccine is not known, and the mechanism involved in the PalA-mediated interference has not been investigated. Objectives: The main objective of this study was to investigate the possible impacts of PalA on the protective immunity of A. pleuropneumoniae inactivated vaccine. Methods: Coding sequence of the mature peptide of PalA was amplified from A. pleuropneumoniae SLW01, and inserted into the prokaryotic expression plasmid pGEX-KG, so as to generate the recombinant PalA (rPalA) protein. The immunogenicity of rPalA was verified in rabbits. For the protection assay, mice were assigned into 4 groups, and were immunized with TSB, rPalA, bacterin (Bac) and bacterin + rPalA (BacPal), respectively. Humoral immune response was evaluated before each immunization and before challenge. Two weeks after three immunizations, mice were infected with virulent A. pleuropneumoniae 4074. The clinical signs, survival rates and lung bacteria loads were determined. Then a passive protection assay was performed using pooled sera from the active immunization assay. Results: rPalA was produced in E. coli and was confirmed to be immune-reactive. rPalA is able to elicit a strong humoral immune response in rabbit. Besides, polyclonal antibodies against rPalA is able to recognize the natural PalA in the outer membrane of A. pleuropneumoniae. The positive immunization assay showed that mice immunized with BacPal produced significantly less antibodies against Apx toxins, relative to that of animals immunized with Bac before challenge (P <0.01). After virulent challenge, all mice in the TSB and rPalA groups died within 48 hpi. The survival rates of the Bac and the BacPal groups were 100% and 75%, respectively. The average bacterial loads of the BacPal group was lower than that of the TSB and rPalA groups (P <0.01), but higher than that of the Bac group (P <0.01). The survival rates of mice received pooled anti-sera against TSB, rPalA, BacPal and Bac, were 0%, 0%, 37.5% and 100% after challenge, respectively. In addition, mice in the BacPal group showed moderate to severe lung damage, whereas mice in the Bac group showed relatively normal lung tissues during the histological examination. Conclusion: Our results demonstrate that A. pleuropneumoniae PalA is an immunogenic but not protective antigen, the existence of PalA suppresses the production of protective antibodies, and thus reduces the protective immunity of inactivated vaccine. Therefore, it should be taken into consideration of these immunogenic but not protective proteins during the development of highly effective vaccines in future.