A one-process production of completely biotinylated proteins in a T7 expression system.

Streptavidin is a tetrameric protein with high specificity and affinity for biotin. The interaction between avidin and biotin has become a valuable tool in nanotechnology. In recent years, the site-specific biotin modification of proteins using biotin ligases, such as BirA, has attracted attention. This study established an in vivo method for achieving the complete biotinylation of target proteins using a single plasmid co-expressing BirA and its target proteins. Specifically, a biotin-modified protein was produced in Escherichia coli strain BL21(DE3) using a single plasmid containing genes encoding both BirA and a protein fused to BirA's substrate sequence, Avitag. This approach simplifies the production of biotinylated proteins in E. coli and allows the creation of various biotinylated protein types through gene replacement. Furthermore, the biotin modification rate of the obtained target protein could be evaluated using Native-PAGE without performing complicated isolation operations of biotinylated proteins. In Native-PAGE, biotin-modified proteins and unmodified proteins were confirmed as clearly different bands, and it was possible to easily derive the modification rate from the respective band intensities.

Development of a Transcriptional Activator-Like Effector Protein to Accurately Discriminate Single Nucleotide Difference.

Transcriptional activator-like effector (TALE), a DNA-binding protein, is widely used in genome editing. However, the recognition of the target sequence by the TALE is adversely affected by the number of mismatches. Therefore, the association constant of DNA-TALE complex formation can be controlled by appropriately introducing a mismatch into the TALE recognition sequence. This study aimed to construct a TALE that can distinguish a single nucleotide difference. Our results show that a single mismatch present in repeats 2 or 3 of TALE did not interfere with the complex formation with DNA, whereas continuous mismatches present in repeats 2 and 3 significantly reduced association with the target DNA. Based on these findings, we constructed a detection system of the one nucleotide difference in gene with high accuracy and constructed a TALE-nuclease (TALEN) that selectively cleaves DNA with a single mismatch.

Production of pentaglycine-fused proteins using Escherichia coli expression system without in vitro peptidase treatment.

Conjugation of functional molecules to peptides is necessary for protein analysis and applications. Transpeptidase sortase A catalyzes the ligation reaction between the amino acid sequence LPXTG and polyglycine and allows for peptide sequence-specific molecular modifications. In this study, the preparation of pentaglycine-fused green fluorescent protein (G5-GFP) via methionine truncation mediated by Escherichia coli endogenous methionyl aminopeptidase was investigated. Some expression vectors of GFP presenting MetGly5 at the N-terminal were constructed, and N-terminal sequence analyses of the protein expressed in E. coli were performed. When the first codon of the GFP-encoding sequence was AUG, a mixture of GFP without pentaglycine and G5-GFP was obtained. In contrast, when the first codon AUG was replaced with a codon encoding alanine, G5-GFP was obtained uniformly. These results showed that the location of AUG in the expression vector had a significant influence on the preparation of polyglycine-fused proteins. The obtained findings are useful for the preparation of polyglycine-fused substrates using E. coli.

Dimerization of ER-resident molecular chaperones mediated by ERp29.

The lectin chaperones calnexin (CNX) and calreticulin (CRT) localized in the endoplasmic reticulum play important roles in glycoprotein quality control. Although the interaction between these lectin chaperones and ERp57 is well known, it has been recently reported that endoplasmic reticulum protein 29 (ERp29), a member of PDI family, interacts with CNX and CRT. The biochemical function of ERp29 is unclear because it exhibits no ERp57-like redox activity. In this study, we addressed the possibility that ER chaperones CNX and CRT are connected via ERp29, based on our observation that ERp29 exists as a dimer. As a result, we showed that CNX dimerizes through ERp29. These results endorse the hypothesis that ERp29 serves as a bridge that links two molecules of CNX. Also, we showed that similar complexes such as CNX-CRT were formed via ERp29.

Repressor-Like On-Off Regulation of Protein Expression by the DNA-Binding Transcription Activator-Like Effector in T7 Promoter-Based Cell-Free Protein Synthesis.

The aim of this study was to develop a transcription activator-like effector (TALE)-based technology to regulate protein synthesis in cell-free systems. We attempted to regulate the T7 promoter system, which has no natural mechanism of expression control, and sought to arbitrarily induce protein expression through the formation and dissociation of TALE and target DNA complexes. Protein synthesis was performed in a cell-free system in the presence of TALE, which recognized and bound to a sequence upstream of the T7 promoter, and protein expression was suppressed by approximately 80 % compared to in the absence of TALE. This suggests that masking part of the promoter region strongly suppresses protein synthesis. Additionally, competitive inhibition of TALE binding to the target DNA template led to protein synthesis levels that were equivalent to the levels in the absence of TALE. Our results demonstrate that DNA recognition by TALE can regulate the expression of the T7 promoter system.

Monitoring of Glycoprotein Quality Control System with a Series of Chemically Synthesized Homogeneous Native and Misfolded Glycoproteins.

The glycoprotein quality control (GQC) system in the endoplasmic reticulum (ER) effectively uses chaperone-type enzymes and lectins such as UDP-glucose:glycoprotein glucosyltransferase (UGGT), calnexin (CNX), calreticulin (CRT), protein disulfide bond isomerases (ERp57 or PDIs), and glucosidases to generate native-folded glycoproteins from nascent glycopolypeptides. However, the individual processes of the GQC system at the molecular level are still unclear. We chemically synthesized a series of several homogeneous glycoproteins bearing M9-high-mannose type oligosaccharides (M9-glycan), such as erythropoietin (EPO), interferon-ß (IFN-ß), and interleukin 8 (IL8) and their misfolded counterparts, and used these glycoprotein probes to better understand the GQC process. The analyses by high performance liquid chromatography and mass spectrometer clearly showed refolding processes from synthetic misfolded glycoproteins to native form through folding intermediates, allowing for the relationship between the amount of glucosylation and the refolding of the glycoprotein to be estimated. The experiment using these probes demonstrated that GQC system isolated from rat liver acts in a catalytic cycle regulated by the fast crosstalk of glucosylation/deglucosylation in order to accelerate refolding of misfolded glycoproteins.

ATP-independent inhibition of amyloid beta fibrillation by the endoplasmic reticulum resident molecular chaperone GRP78.

Neuronal cell death induced by an accumulation of amyloid beta (Aß) peptides, which are pathogenic molecules for Alzheimer's disease, is closely related with endoplasmic reticulum (ER) stress. In the ER stress condition, part of the ER resident chaperones is known to be translocated to another cellular location, such as the cell surface. The ER chaperone 78-kDa glucose-regulated protein (GRP78), which shows ATP-dependent chaperone activity, also shows translocation to the cell surface. In this study, we examined the influence of GRP78 on Aß fibrillation in the presence or absence of ATP. We revealed that a small amount of GRP78 effectively inhibited fibrillation of Aß fragments. Intriguingly, the fibrillation inhibition by GRP78 was confirmed in the absence of ATP, suggesting GRP78 exhibited ATP-independent interaction with Aß fragments.

PDI family protein ERp29 recognizes P-domain of molecular chaperone calnexin.

Endoplasmic reticulum (ER) resident lectin chaperone calnexin (CNX) and calreticulin (CRT) assist folding of nascent glycoproteins. Their association with ERp57, a member of PDI family proteins (PDIs) which promote disulfide bond formation of unfolded proteins, has been well documented. Recent studies have provided evidence that other PDIs may also interact with CNX and CRT. Accordingly, it seems possible that the ER provides a repertoire of CNX/CRT-PDI complexes, in order to facilitate refolding of various glycoproteins. In this study, we examined the ability of PDIs to interact with CNX. Among them ERp29 was shown to interact with CNX, similarly to ERp57. Judging from the dissociation constant, its ability to interact with CNX was similar to that of ERp57. Results of further analyses by using a CNX mutant imply that ERp29 and ERp57 recognize the same domain of CNX, whereas the mode of interaction with CNX might be somewhat different between them.

Enzymatic Installation of Functional Molecules on Amyloid-Based Polymers.

We produced a functional polymer whose framework comprised transthyretin (TTR) amyloid fibrils. In order to immobilize functional molecules onto the amyloid fibrils, transpeptidase sortase A (srtA), which catalyzes the covalent binding of LPXTG with polyglycine, was employed. After the preparation of the amyloid fibril of LPETGG-tagged TTR, immobilization of Gly5-fused GFP on the amyloid fibrils by srtA-mediated transpeptidation was carried out. SrtA recognized the amyloid fibrils consisting of an LPETGG-tagged TTR variant (L55P) as a good substrate, resulting in successful preparation of a GFP-immobilized amyloid. Intriguingly, the replacement of GFP with Gly5-fused luciferase was confirmed when the GFP-immobilized amyloids were mixed with Gly5-luciferase in the presence of srtA. Thus, it was found that functional molecules covalently immobilized on amyloid could be detached and substituted with other tagged molecules by using srtA.

Glycoprotein quality control-related proteins effectively inhibit fibrillation of amyloid beta 1-42.

Formation of amyloid beta (Aß) aggregates is a risk factor for Alzheimer's disease. Accumulation of Aß aggregates on the cell surface causes oxidative stress, and ultimately results in cell death. Consequently, inhibition of aggregate formation is predicted to be beneficial. Recently, translocation of glycoprotein quality control-related (GPQC) proteins such as chaperones and protein disulfide-isomerase (PDI) family members was reported under oxidative stress conditions. Therefore, it is possible that GPQC proteins contact Aß peptides on the cell membrane during stress conditions. Here, we examined the effect of ER resident proteins on Aß aggregation. Our results show that minimal expression of GPQC proteins enables Aß to effectively avoid aggregation. Moreover, further analyses show that Aß structure remains in the monomer state in the presence of ER proteins. Thus, our findings show that GPQC proteins have strong affinity for Aß monomers, and suggests that the interaction between them repeatedly associates and dissociates in a short period of time.

Hydrophobic Tagged Dihydrofolate Reductase for Creating Misfolded Glycoprotein Mimetics.

In the endoplasmic reticulum (ER), nascent glycoproteins that have not acquired the native conformation are either repaired or sorted for degradation by specific quality-control systems composed by various proteins. Among them, UDP-glucose:glycoprotein glucosyltransferase (UGGT) serves as a folding sensor in the ER. However, the molecular mechanism of its recognition remains obscure. This study used pseudo-misfolded glycoproteins, comprising a modified dihydrofolate reductase with artificial pyrene-cysteine moiety on the protein surface (pDHFR) and Man9 GlcNAc2 -methotrexate (M9-MTX). All five M9-MTX/pDHFR complexes, with a pyrene group at different positions, were found to be good substrates of UGGT, irrespective of the site of pyrene modification. These results suggest UGGT's mode of substrate recognition is fuzzy, thus allowing various glycoproteins to be accommodated in the folding cycle.

Glycan specificity of a testis-specific lectin chaperone calmegin and effects of hydrophobic interactions.

BACKGROUND: Testis-specific chaperone calmegin is required for the generation of normal spermatozoa. Calmegin is known to be a homologue of endoplasmic reticulum (ER) residing lectin chaperone calnexin. Although functional similarity between calnexin and calmegin has been predicted, detailed information concerned with substrate recognition by calmegin, such as glycan specificity, chaperone function and binding affinity, are obscure. METHODS: In this study, biochemical properties of calmegin and calnexin were compared using synthetic glycans and glycosylated or non-glycosylated proteins as substrates. RESULTS: Whereas their amino acid sequences are quite similar to each other, a certain difference in secondary structures was indicated by circular dichroism (CD) spectrum. While both of them inhibited protein heat-aggregation to a similar extent, calnexin exhibited a higher ability to facilitate protein folding. Similarly to calnexin, calmegin preferentially recognizes monoglucosylated glycans such as Glc1Man9GlcNAc2 (G1M9). While the surface hydrophobicity of calmegin was higher than that of calnexin, calnexin showed stronger binding to substrate. We reasoned that lectin activity, in addition to hydrophobic interaction, contributes to this strong affinity between calnexin and substrate. CONCLUSIONS: Although their similarity in carbohydrate binding specificities is high, there seems to be some differences in the mode of substrate recognition between calmegin and calnexin. GENERAL SIGNIFICANCE: Properties of calmegin as a lectin-chaperone were revealed in comparison with calnexin.

PDI family protein ERp29 forms 1:1 complex with lectin chaperone calreticulin.

Lectin chaperone calreticulin is well known to interact with ERp57 which is one of PDI family proteins. The interaction of ERp57 with calreticulin is believed to assist disulfide bond formation of nascent glycoprotein in the ER. Various kinds of PDI family proteins are present in the ER, however, their precise roles have been unclear. In this study, interaction assay between PDI family proteins and calreticulin by SPR analysis was performed. Our analysis revealed for the first time formation of a 1:1 complex between ERp29 and calreticulin. The dissociation constant of interaction between ERp29 and calreticulin was shown to be almost identical to ERp57-calreticulin interaction. We speculate that the recognition site of ERp29 within calreticulin is different from that of ERp57.

Folding of synthetic homogeneous glycoproteins in the presence of a glycoprotein folding sensor enzyme.

UDP-glucose:glycoprotein glucosyltransferase (UGGT) plays a key role in recognizing folded and misfolded glycoproteins in the glycoprotein quality control system of the endoplasmic reticulum. UGGT detects misfolded glycoproteins and re-glucosylates them as a tag for misfolded glycoproteins. A flexible model to reproduce in vitro folding of a glycoprotein in the presence of UGGT in a mixture containing correctly folded, folding intermediates, and misfolded glycoproteins is described. The data demonstrates that UGGT can re-glucosylate all intermediates in the in vitro folding experiments, thus indicating that UGGT inspects not only final folded products, but also the glycoprotein folding intermediates.

Human prefoldin inhibits amyloid-ß (Aß) fibrillation and contributes to formation of nontoxic Aß aggregates.

Amyloid-ß (Aß) peptides represent key players in the pathogenesis of Alzheimer's disease (AD), and mounting evidence indicates that soluble Aß oligomers mediate the toxicity. Prefoldin (PFD) is a molecular chaperone that prevents aggregation of misfolded proteins. Here we investigated the role of PFD in Aß aggregation. First, we demonstrated that PFD is expressed in mouse brain by Western blotting and immunohistochemistry and found that PFD is upregulated in AD model APP23 transgenic mice. Then we investigated the effect of recombinant human PFD (hPFD) on Aß(1-42) aggregation in vitro and found that hPFD inhibited Aß fibrillation and induced formation of soluble Aß oligomers. Interestingly, cell viability measurements using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay showed that Aß oligomers formed by hPFD were 30-40% less toxic to cultured rat pheochromocytoma (PC12) cells or primary cortical neurons from embryonic C57BL/6CrSlc mice than previously reported Aß oligomers (formed by archaeal PFD) and Aß fibrils (p < 0.001). Thioflavin T measurements and immunoblotting indicated different structural properties for the different Aß oligomers. Our findings show a relation between cytotoxicity of Aß oligomers and structure and suggest a possible protective role of PFD in AD.

Formation of non-toxic Aß fibrils by small heat shock protein under heat-stress conditions.

Small heat shock protein (sHsp) is a molecular chaperone with a conserved alpha-crystallin domain that can prevent protein aggregation. It has been shown that sHsps exist as oligomers (12-40 mer) and their dissociation into small dimers or oligomers is functionally important. Since several sHsps are upregulated and co-localized with amyloid-ß (Aß) in senile plaques of patients with Alzheimer's disease (AD), sHsps are thought to be involved in AD. Previous studies have also shown that sHsp can prevent Aß aggregation in vitro. However, it remains unclear how the quaternary structure of sHsp influences Aß aggregation. In this study, we report for the first time the effect of the quaternary structure of sHsp on Aß aggregation using sHsp from the fission yeast Schizosaccharomyces pombe (SpHsp16.0) showing a clear temperature-dependent structural transition between an oligomer (30 °C) and dimer (50 °C) state. Aß aggregation was inhibited by the oligomeric form of SpHsp16.0. In contrast, amyloid fibrils were formed in the presence of dimeric SpHsp16.0. Interestingly, these amyloid fibrils consisted of both Aß and SpHsp16.0 and showed a low ThT intensity and low cytotoxicity due to their low binding affinity to the cell surface. These results suggest the formation of novel fibrillar Aß amyloid with different characteristics from that of the authentic Aß amyloid fibrils formed in the absence of sHsp. Our results also suggest the potential protective role of sHsp in AD under stress conditions.

One-pot synthesis of fibrillar-shaped functional nanomaterial using microbial transglutaminase.

Recently, functional nanowire production using amyloids as a scaffold for protein immobilization has attracted attention. However, protein immobilization on amyloid fibrils often caused protein inactivation. In this study, we investigated protein immobilization using enzymatic peptide ligation to suppress protein inactivation during immobilization. We attempted to immobilize functional molecules such as green fluorescent protein (GFP) and Nanoluc to a transthyretin (TTR) amyloid using microbial transglutaminase (MTG), which links the glutamine side chain to the primary amine. Linkage between amyloid fibrils and functional molecules was achieved through the MTG substrate sequence, and the functional molecules-loaded nanowires were successfully fabricated. We also found that the synthetic process from amyloidization to functional molecules immobilization could be achieved in a single-step procedure.All rights reserved.

Chemical synthesis of intentionally misfolded homogeneous glycoprotein: a unique approach for the study of glycoprotein quality control.

Biosynthesis of glycoproteins in the endoplasmic reticulum employs a quality control system, which discriminates and excludes misfolded malfunctional glycoproteins from a correctly folded one. As chemical tools to study the glycoprotein quality control system, we systematically synthesized misfolded homogeneous glycoproteins bearing a high-mannose type oligosaccharide via oxidative misfolding of a chemically synthesized homogeneous glycopeptide. The endoplasmic reticulum folding sensor enzyme, UDP-glucose:glycoprotein glucosyltransferase (UGGT), recognizes a specific folding intermediate, which exhibits a molten globule-like hydrophobic nature.

Biophysical properties of UDP-glucose:glycoprotein glucosyltransferase, a folding sensor enzyme in the ER, delineated by synthetic probes.

UDP-glucose:glycoprotein glucosyltransferase plays a key role in glycoprotein quality control in the endoplasmic reticulum, by virtue of its ability to discriminate folding states. Although lines of evidence have clarified the ability of UGGT to recognize a partially unfolded protein, its mechanistic rationale has been obscure. In this study, the substrate recognition mechanism of UGGT was studied using synthetic substrate of UGGT. Although UGGT has high extent of surface hydrophobicity, it clearly lacks property of typical molecular chaperones. Furthermore, it was revealed that the addition of the substrate caused secondary structure change of UGGT in a dose-dependent manner, resulting that the K(d) value of the UGGT-substrate interaction was estimated from theoretical formula based on 1:1 complexation between UGGT and the acceptor substrate. Moreover, the kinetic analysis of glucosyltransferase activity of UGGT elucidated Michaelis constant K(m) correctly.

Cell interaction study of amyloid by using luminescent conjugated polythiophene: implication that amyloid cytotoxicity is correlated with prolonged cellular binding.

Needles and noodles: Studying amyloid toxicity is important for understanding protein misfolding diseases. Using a luminescent conjugated polythiophene, we found that cell binding of nontoxic filamentous amyloids of insulin and ß2-microglobulin was less efficient than that of toxic fibrillar amyloids; this suggests a correlation between amyloid toxicity and cell binding.