Deletion Of Peptide Research Articles

The Ile35 Residue of the ALS-Associated Mutant SOD1 Plays a Crucial Role in the Intracellular Aggregation of the Molecule.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease with an unknown pathogenesis. It has been reported that mutations in the gene for Cu/Zn superoxide dismutase (SOD1) cause familial ALS. Mutant SOD1 undergoes aggregation and forms amyloid more easily, and SOD1-immunopositive inclusions have been observed in the spinal cords of ALS patients. Because of this, SOD1 aggregation is thought to be related to the pathogenesis of ALS. Some core regions of amyloid have been identified, but the issue of whether these regions form aggregates in living cells remains unclear, and the mechanism responsible for intracellular SOD1 aggregation also remains unclear. The findings reported in this study indicate that the aggregation of the ALS-linked mutant SOD1-EGFP was significantly enhanced when the BioID2 gene was fused to the N-terminus of the mutant SOD1-EGFP plasmid for cellular expression. Expression of a series of BioID2-(C-terminal deletion peptides of SOD1)-EGFP permitted us to identify 1-35 as a minimal N-terminal sequence and Ile35 as an essential amino acid residue that contributes to the intracellular aggregation of SOD1. The findings also showed that an additional substitution of Ile35 with Ser into the ALS mutant SOD1 resulted in the significant suppression of aggregate formation. The fact that no Ile35 mutations have been reported to date in ALS patients indicates that all ALS mutant SOD1s contain Ile35. Taken together, we propose that Ile35 plays a pivotal role in the aggregation of the ALS-linked SOD1 and that this study will contribute to our understanding of the mechanism responsible for SOD1 aggregation.

Assessing the immunogenicity risk of salmon calcitonin peptide impurities using in silico and in vitro methods.

Advances in synthetic peptide synthesis have enabled rapid and cost-effective peptide drug manufacturing. For this reason, peptide drugs that were first produced using recombinant DNA (rDNA) technology are now being produced using solid- and liquid-phase peptide synthesis. While peptide synthesis has some advantages over rDNA expression methods, new peptide-related impurities that differ from the active pharmaceutical ingredient (API) may be generated during synthesis. These impurity byproducts of the original peptide sequence feature amino acid insertions, deletions, and side-chain modifications that may alter the immunogenicity risk profile of the drug product. Impurities resulting from synthesis have become the special focus of regulatory review and approval for human use, as outlined in the FDA's Center for Drug Evaluation and Research guidance document, "ANDAs for Certain Highly Purified Synthetic Peptide Drug Products That Refer to Listed Drugs of rDNA Origin," published in 2021. This case study illustrates how in silico and in vitro methods can be applied to assess the immunogenicity risk of impurities that may be present in synthetic generic versions of the salmon calcitonin (SCT) drug product. Sponsors of generic drug abbreviated new drug applications (ANDAs) should consider careful control of these impurities (for example, keeping the concentration of the immunogenic impurities below the cut-off recommended by FDA regulators). Twenty example SCT impurities were analyzed using in silico tools and assessed as having slightly more or less immunogenic risk potential relative to the SCT API peptide. Class II human leukocyte antigen (HLA)-binding assays provided independent confirmation that a 9-mer sequence present in the C-terminus of SCT binds promiscuously to multiple HLA DR alleles, while T-cell assays confirmed the expected T-cell responses to SCT and selected impurities. In silico analysis combined with in vitro assays that directly compare the API to each individual impurity peptide may be a useful approach for assessing the potential immunogenic risk posed by peptide impurities that are present in generic drug products.

ESM1 facilitates the EGFR/HER3-triggered epithelial-to-mesenchymal transition and progression of gastric cancer via modulating interplay between Akt and angiopoietin-2 signaling.

Gastric cancer (GC) poses global challenges due to its difficult early diagnosis and drug resistance, necessitating the identification of early detection markers and understanding of oncogenic pathways for effective GC therapy. Endothelial cell-specific molecule 1 (ESM1), a secreted glycoprotein, is elevated in various cancers, but its role in GC remains controversial. In our study, ESM1 was elevated in GC tissues, and its concentration was correlated with progression and poorer patient prognosis in independent cohorts. Functionally, ESM1 expression promoted proliferation, anoikis resistance, and motility of GC cells, as well as tumor growth in PDOs and in GC xenograft models. Mechanistically, ESM1 expression triggered the epithelial-to-mesenchymal transition (EMT) of GC cells by enhancing epidermal growth factor receptor (EGFR)/human EGFR 3 (HER3) association and activating the EGFR/HER3-Akt pathway. Additionally, angiopoietin-2 (ANGPT2) was found to be highly correlated with ESM1 and interplayed with Akt to induce the EMT and cancer progression. Use of a signal peptide deletion mutant (ESM1-19del) showed that the secreted form of ESM1 is crucial for its protumorigenic effects by activating the EGFR/HER3-Akt/ANGPT2 pathway to promote the EMT. Patients with high levels of both ESM1 and ANGPT2 had the poorest prognoses. Furthermore, therapeutic peptides successfully inhibited ESM1's induction of the aforementioned signals and motility of GC cells. ESM1's oncogenic role in GC involves activating the EGFR/HER3-Akt/ANGPT2 pathway, presenting a potential therapeutic target for GC.

Removal of N-Terminal Peptide Impacts Structural Aspects of an IgE-Reactive Recombinant Der p 5

(1) Background: Modification of the structural elements of allergens is widely used in the field of allergies. The goal of the present research was to express, purify, and characterize the shortened recombinant group 5 allergen of Dermatophagoides pteronyssinus (rDer p 5). (2) Methods: rDer p 5 storage stability and aggregation capacity were explored through in silico analysis, dynamic light scattering (DLS), and SDS-PAGE. Serum IgE reactivity and cytokine amount were investigated in sera or cell culture supernatants through ELISA, MULTIPLEX®, and Western blot analysis using sera from sensitized humans from Brazil, Colombia, and Ecuador. (3) Results: Dimeric rDer p 5 was detected through native PAGE, and this result was confirmed by data from DLS. The protein was thermically stable, as it did not degrade at 4 °C for 21 days. The shortened rDer p 5 was classified as a major IgE allergen in Brazil and Colombia, but minor in Ecuador. IL-13, IL-10, IL-1β, and IL-6 were significantly elevated in the sera of rDer p 5-reactive patients. The same cytokines plus IL-5 were more secreted by human cells upon rDer p 5 stimulation. (4) Conclusions: N-terminal peptide deletion led to a higher rDer p 5 folding stability, which, even though dimeric, was an IgE-reactive protein. Therefore, rDer p 5 could be used for molecular diagnostic applications or as backbone for hypoallergen design.

Structural displacement model of chitooligosaccharide transport through chitoporin

VhChiP is a chitooligosaccharide-specific porin identified in the outer membrane of Vibrio campbellii type strain American Type Culture Collection BAA 1116. VhChiP contains three identical subunits, and in each subunit, the 19-amino acid N-terminal segment serves as a molecular plug (the "N-plug") that controls the closed/open dynamics of the neighboring pores. In this study, the crystal structures of VhChiP lacking the N-plug were determined in the absence and presence of chitohexaose. Binding studies of sugar-ligand interactions by single-channel recordings and isothermal microcalorimetry experiments suggested that the deletion of the N-plug peptide significantly weakened the sugar-binding affinity due to the loss of hydrogen bonds around the central affinity sites. Steered molecular dynamic simulations revealed that the movement of the sugar chain along the sugar passage triggered the ejection of the N-plug, while the H-bonds transiently formed between the reducing end GlcNAc units of the sugar chain with the N-plug peptide may help to facilitate sugar translocation. The findings enable us to propose the structural displacement model, which enables us to understand the molecular basis of chitooligosaccharide uptake by marine Vibrio bacteria.

Dual single guide RNAs-mediating deletion of mature myostatin peptide results in concomitant muscle fibre hyperplasia and adipocyte hypotrophy in pigs

Myostatin (MSTN) is a major gene target for skeletal muscle overgrowth in animals. We hypothesized that deletion of the entire mature peptide encoded by MSTN in pigs would knock out its bioactive form and accordingly stimulate skeletal muscle overgrowth. Thus, we engineered two pairs of single-guide RNAs (sgRNAs) to target exons 1 and 3 of MSTN in primary fetal fibroblasts of Taoyuan black pigs. We found that sgRNAs targeting exon 3, which encodes the mature peptide, had higher biallelic null mutation efficiency than those targeting exon 1. Somatic cell nuclear transfer was conducted using the exon 3 mutation cells as donor cells to generate five cloned MSTN null piglets (MSTN−/−). Growth testing revealed that both the growth rate and average daily weight gain of MST−/− pigs were greater than those of wild-type (MSTN+/+) pigs. Slaughter data demonstrated that the lean ratio of MSTN−/− pigs was 11.3% higher (P < 0.01) while the back-fat thickness was 17.33% lower (P < 0.01) than those of MSTN+/+ pigs. Haematoxylin–eosin staining indicated that the increased leanness of MSTN−/− pigs resulted from muscle fibre hyperplasia rather than hypertrophy.HE staining showed markedly decreased adipocyte size in MSTN−/− pigs. We also critically examined the off-target and random integration by resequencing, which showed that the founder MSTN−/− pigs contained no non-target mutations or exogenous plasmid elements. This study is the first to report the successful knock out of the mature MSTN peptide using dual sgRNA-mediated deletion, leading to the most prominent alteration of meat production traits in pigs published thus far. This new strategy is expected to have a wide impact on genetic improvements in food animals.

Regulation of Cell Membrane Permeability Enhanced the Non-Classical Secretion of γ-Cyclodextrin Glycosyltransferase in Bacillus subtilis

γ-Cyclodextrin glycosyltransferase (γ-CGTase, EC 2.4.1.19) is an essential enzyme required in the production of γ-cyclodextrin, which shows huge prospects in the food, medicine, materials, and chemical industries. In this study, γ-CGTase from Bacillus sp. G-825-6 STB17 was successfully cloned and expressed in Bacillus subtilis WB600. The final extracellular activity of γ-CGTase can reach 45.34 U/mL with the deletion of the signal peptide, which was about 11.3 times of the initial level of γ-CGTase secreted by the general pathway. By monitoring the whole cultivation process, secretion was divided into two stages, which were dominated by cell membrane changes and apoptosis. The measurement of lactate dehydrogenase and the results of fluorescence microscopy demonstrated that the cell membrane permeability changed significantly in the middle stage of fermentation, proving that it played a crucial role in the non-classical secretion of γ-CGTase. Furthermore, the addition of Triton X-100, a non-ionic surfactant, remarkably enhanced the secretion level of γ-CGTase by 21.5%, which was caused by the increase in cell membrane permeability. This work is the first to obtain the extracellular expression of CGTase via the non-classical secretion pathway in B. subtilis and provides a new strategy to enhance extracellular expression by regulating the cell membranes.

Effects of a Shift of the Signal Peptide Cleavage Site in Signal Peptide Variant on the Synthesis and Secretion of SARS-CoV-2 Spike Protein.

The COVID-19 pandemic is caused by SARS-CoV-2; the spike protein is a key structural protein that mediates infection of the host by SARS-CoV-2. In this study, we aimed to evaluate the effects of signal peptide on the secretion and release of SARS-CoV-2 spike protein. Therefore, we constructed a signal peptide deletion mutant and three signal peptide site-directed mutants. The (H) region and (C) region in the signal peptide of L5F-S13I mutant have changed significantly, compared with wild type, L5F and S13I. We demonstrated the effects of signal peptide on the secretion and synthesis of RBD protein, finding that mutation of S13 to I13 on the signal peptide is more conducive to the secretion of RBD protein, which was mainly due to the shift of the signal peptide cleavage site in the mutant S13I. Here, we not only investigated the structure of the N-terminal signal peptide of the SARS-CoV-2 spike protein but also considered possible secretory pathways. We suggest that the development of drugs that target the signal peptide of the SARS-CoV-2 spike protein may have potential to treat COVID-19 in the future.

Inhibition of CSPG receptor PTPσ promotes migration of newly born neuroblasts, axonal sprouting, and recovery from stroke.

In addition to neuroprotective strategies, neuroregenerative processes could provide targets for stroke recovery. However, the upregulation of inhibitory chondroitin sulfate proteoglycans (CSPGs) impedes innate regenerative efforts. Here, we examine the regulatory role of PTPσ (a major proteoglycan receptor) in dampening post-stroke recovery. Use of a receptor modulatory peptide (ISP) or Ptprs gene deletion leads to increased neurite outgrowth and enhanced NSCs migration upon inhibitory CSPG substrates. Post-stroke ISP treatment results in increased axonal sprouting as well as neuroblast migration deeply into the lesion scar with a transcriptional signature reflective of repair. Lastly, peptide treatment post-stroke (initiated acutely or more chronically at 7days) results in improved behavioral recovery in both motor and cognitive functions. Therefore, we propose that CSPGs induced by stroke play a predominant role in the regulation of neural repair and that blocking CSPG signaling pathways will lead to enhanced neurorepair and functional recovery in stroke.

Efficient synthesis of Aib8 -Arg34 -GLP-1 (7-37) by liquid-phase fragment condensation.

Glucagon-like peptide-1 (GLP-1) is a potential therapeutic agent for treating Type 2 diabetes, owing to its glucose-dependent capability to stimulate insulin secretion. Semaglutide is currently the best GLP-1 analogue; however, the Aib8 -Arg34 -GLP-1 (7-37) of semaglutide contains an unnatural amino acid at the eighth position (Aib: 2-aminoisobutyric acid), which hinders its fermentation process. Aib8 -Arg34 -GLP-1 (7-37) is mainly synthesised by solid-phase synthesis. However, solid-phase synthesis of Aib8 -Arg34 -GLP-1 (7-37) has many shortcomings: (i) The synthesis requires many organic solvents, (ii) the existence of deletion peptides impedes the subsequent purification process, (iii) the yield is low (approximately 16%), and (iv) it is not suitable for large-scale synthesis. However, the synthesis of Aib8 -Arg34 -GLP-1 (7-37) by liquid-phase fragment condensation of expressed and synthetic peptides (Arg34 -GLP-1 (9-37) and Boc-His (Boc)-Aib) has many advantages: (i) The synthesis process only requires a few organic reagents, (ii) the yield is high (approximately 60%), (iii) the purification conditions are simple and Aib8 -Arg34 -GLP-1 (7-37) with a purity of over 98% is obtained through one-step reverse-phase purification, and (iv) the raw materials are inexpensive and large-scale synthesis is possible. In conclusion, here, we developed an efficient method for synthesising Aib8 -Arg34 -GLP-1 (7-37).

Enhancing Extracellular Pullulanase Production in Bacillus subtilis Through dltB Disruption and Signal Peptide Optimization.

Bacillus subtilis has many attributes that make it a popular host for recombinant protein production. Although its protein production ability has been enhanced through protease gene disruption, residual proteases like quality control HtrA and HtrB can limit protein yield by degrading inadequately folded proteins present during overexpression. In this study, two strategies were employed to increase production of industrial enzyme pullulanase: enhancing extracellular pullulanase folding and optimizing its signal peptide. The hypothesis was that disruption of dltB gene of expression host B. subtilis WS9 would enhance recombinant extracellular folding by increasing cation binding to the cell's outer envelope. Consistent with this hypothesis, disrupting dltB enhanced pullulanase production by 49% in shake-flask cultures. The disruption also enhanced extracellular α-CGTase and β-CGTase production by 25% and 35%, respectively. Then, more effective signal peptide for pullulanase production was identified through high-throughput screening of 173 unique B. subtilis signal peptides. Replacing the native signal peptide of pullulanase with that encoded by ywtF increased extracellular pullulanase activity by an additional 12%. Three-liter fermenter scale-up production yielded the highest extracellular pullulanase activity reported to date: 8037.91 U·mL-1. This study highlights the usefulness of dltB deletion and signal peptide optimization in enhancing extracellular protein production.

CD9 inhibition reveals a functional connection of extracellular vesicle secretion with mitophagy in melanoma cells.

Tetraspanins are often used as Extracellular Vesicle (EV) detection markers because of their abundance on these secreted vesicles. However, data on their function on EV biogenesis are controversial and compensatory mechanisms often occur upon gene deletion. To overcome this handicap, we have compared the effects of tetraspanin CD9 gene deletion with those elicited by cytopermeable peptides with blocking properties against tetraspanin CD9. Both CD9 peptide or gene deletion reduced the number of early endosomes. CD9 peptide induced an increase in lysosome numbers, while CD9 deletion augmented the number of MVB and EV secretion, probably because of compensatory CD63 expression upregulation. In vivo, CD9 peptide delayed primary tumour cell growth and reduced metastasis size. These effects on cell proliferation were shown to be concomitant with an impairment in mitochondrial quality control. CD9 KO cells were able to compensate the mitochondrial malfunction by increasing total mitochondrial mass reducing mitophagy. Our data thus provide the first evidence for a functional connection of tetraspanin CD9 with mitophagy in melanoma cells.

Functional Characterization of a New GH107 Endo-α-(1,4)-Fucoidanase from the Marine Bacterium Formosa haliotis.

Fucoidans from brown macroalgae are sulfated fucose-rich polysaccharides, that have several beneficial biological activities, including anti-inflammatory and anti-tumor effects. Controlled enzymatic depolymerization of the fucoidan backbone can help produce homogeneous, defined fucoidan products for structure-function research and pharmaceutical uses. However, only a few endo-fucoidanases have been described. This article reports the genome-based discovery, recombinant expression in Escherichia coli, stabilization, and functional characterization of a new bacterial endo-α-(1,4)-fucoidanase, Fhf1, from Formosa haliotis. Fhf1 catalyzes the cleavage of α-(1,4)-glycosidic linkages in fucoidans built of alternating α-(1,3)-/α-(1,4)-linked l-fucopyranosyl sulfated at C2. The native Fhf1 is 1120 amino acids long and belongs to glycoside hydrolase (GH) family 107. Deletion of the signal peptide and a 470 amino acid long C-terminal stretch led to the recombinant expression of a robust, minimized enzyme, Fhf1Δ470 (71 kDa). Fhf1Δ470 has optimal activity at pH 8, 37–40 °C, can tolerate up to 500 mM NaCl, and requires the presence of divalent cations, either Ca2+, Mn2+, Zn2+ or Ni2+, for maximal activity. This new enzyme has the potential to serve the need for controlled enzymatic fucoidan depolymerization to produce bioactive sulfated fucoidan oligomers.

The tree of life of polyamine oxidases

Polyamine oxidases (PAOs) are characterized by a broad variability in catalytic properties and subcellular localization, and impact key cellular processes in diverse organisms. In the present study, a comprehensive phylogenetic analysis was performed to understand the evolution of PAOs across the three domains of life and particularly within eukaryotes. Phylogenetic trees show that PAO-like sequences of bacteria, archaea, and eukaryotes form three distinct clades, with the exception of a few procaryotes that probably acquired a PAO gene through horizontal transfer from a eukaryotic donor. Results strongly support a common origin for archaeal PAO-like proteins and eukaryotic PAOs, as well as a shared origin between PAOs and monoamine oxidases. Within eukaryotes, four main lineages were identified that likely originated from an ancestral eukaryotic PAO before the split of the main superphyla, followed by specific gene losses in each superphylum. Plant PAOs show the highest diversity within eukaryotes and belong to three distinct clades that underwent to multiple events of gene duplication and gene loss. Peptide deletion along the evolution of plant PAOs of Clade I accounted for further diversification of function and subcellular localization. This study provides a reference for future structure–function studies and emphasizes the importance of extending comparisons among PAO subfamilies across multiple eukaryotic superphyla.

Screening of a genome-reduced Corynebacterium glutamicum strain library for improved heterologous cutinase secretion.

The construction of microbial platform organisms by means of genome reduction is an ongoing topic in biotechnology. In this study, we investigated whether the deletion of single or multiple gene clusters has a positive effect on the secretion of cutinase from Fusarium solani pisi in the industrial workhorse Corynebacterium glutamicum. A total of 22 genome-reduced strain variants were compared applying two Sec signal peptides from Bacillus subtilis. High-throughput phenotyping using robotics-integrated microbioreactor technology with automated harvesting revealed distinct cutinase secretion performance for a specific combination of signal peptide and genomic deletions. The biomass-specific cutinase yield for strain GRS41_51_NprE was increased by~200%, although the growth rate was reduced by~60%. Importantly, the causative deletions of genomic clusters cg2801-cg2828 and rrnC-cg3298 could not have been inferred a priori. Strikingly, bioreactor fed-batch cultivations at controlled growth rates resulted in a complete reversal of the screening results, with the cutinase yield for strain GRS41_51_NprE dropping by~25% compared to the reference strain. Thus, the choice of bioprocess conditions may turn a 'high-performance' strain from batch screening into a 'low-performance' strain in fed-batch cultivation. In conclusion, future studies are needed in order to understand metabolic adaptations of C.glutamicum to both genomic deletions and different bioprocess conditions.

Oncogenic fusion protein FGFR2-PPHLN1: Requirements for biological activation, and efficacy of inhibitors.

Aim of studyChromosomal translocations such as t(10;12)(q26,q12) are associated with intrahepatic cholangiocarcinoma, a universally fatal category of liver cancer. This translocation creates the oncogenic fusion protein of Fibroblast Growth Factor Receptor 2 joined to Periphilin 1. The aims of this study were to identify significant features required for biological activation, analyze the activation of downstream signaling pathways, and examine the efficacy of the TKIs BGJ398 and TAS-120, and of the MEK inhibitor Trametinib.MethodsThese studies examined FGFR2-PPHLN1 proteins containing a kinase-dead, kinase-activated, or WT kinase domain in comparison with analogous FGFR2 proteins. Biological activity was assayed using soft agar colony formation in epithelial RIE-1 cells and focus assays in NIH3T3 cells. The MAPK/ERK, JAK/STAT3 and PI3K/AKT signaling pathways were examined for activation. Membrane association was analyzed by indirect immunofluorescence comparing proteins altered by deletion of the signal peptide, or by addition of a myristylation signal.ResultsBiological activity of FGFR2-PPHLN1 required an active FGFR2-derived tyrosine kinase domain, and a dimerization domain contributed by PPHLN1. Strong activation of canonical MAPK/ERK, JAK/STAT3 and PI3K/AKT signaling pathways was observed. The efficacy of the tyrosine kinase inhibitors BGJ398 and TAS-120 was examined individually and combinatorially with the MEK inhibitor Trametinib; heterogeneous responses were observed in a mutation-specific manner. A requirement for membrane localization of the fusion protein was also demonstrated.Concluding statementOur study collectively demonstrates the potent transforming potential of FGFR2-PPHLN1 in driving cellular proliferation. We discuss the importance of sequencing-based, mutation-specific personalized therapeutics in treating FGFR2 fusion-positive intrahepatic cholangiocarcinoma.

Dedicated to a peptide chemist of few words and immense impact: Louis A. Carpino

Dedicated to a peptide chemist of few words and immense impact: Louis A. Carpino

Absence of α-calcitonin gene-related peptide modulates bone remodeling properties of murine osteoblasts and osteoclasts in an age-dependent way

Mice with an overall deletion of the sensory neuropeptide α-calcitonin gene-related peptide (α-CGRP) develop an age-dependent osteopenic bone phenotype. Underlying molecular mechanisms of how αCGRP affects bone cell metabolism are not well understood. This study aims to characterize differences in metabolic parameters of osteoblast-like cells (OB) and differentiated bone marrow-derived macrophages (BMM)/osteoclast (OC) cultures isolated from 3 month (3 m) and 9 month old (9 m) α-CGRP-deficient mice (−/−) and age-matched WT controls. All WT bone cell cultures endogenously produced and secreted α-CGRP. We found higher BMM but reduced OB numbers and reduced OB vitality after isolation from 9 m compared to 3 m mice, independent of genotype. Absence of α-CGRP reduced cell spreading, increased apoptosis rate throughout osteogenic differentiation, and reduced ALP activity during late osteogenic differentiation in 9 m OB−/− cultures, whereas minor effects were found in 3 m OB−/− cultures. Cathepsin K activity was reduced in 3 m OC−/− cultures. On the contrary, 9 m OC−/− cells demonstrated increased proliferation and caspase3/7 activity. The absence of α-CGRP influenced bone formation and resorption rate differently in bone cells from 3 and 9 m old mice. In summary we suggest, that an increase of dysfunctional mature osteoblasts might occur during aging and contribute to the development of the osteopenic bone phenotype in mature adult (9 m) α-CGRP-deficient mice.

The PAR2 signal peptide prevents premature receptor cleavage and activation.

Unlike closely related GPCRs, protease-activated receptors (PAR1, PAR2, PAR3, and PAR4) have a predicted signal peptide at their N-terminus, which is encoded by a separate exon, suggesting that the signal peptides of PARs may serve an important and unique function, specific for PARs. In this report, we show that the PAR2 signal peptide, when fused to the N-terminus of IgG-Fc, effectively induced IgG-Fc secretion into culture medium, thus behaving like a classical signal peptide. The presence of PAR2 signal peptide has a strong effect on PAR2 cell surface expression, as deletion of the signal peptide (PAR2ΔSP) led to dramatic reduction of the cell surface expression and decreased responses to trypsin or the synthetic peptide ligand (SLIGKV). However, further deletion of the tethered ligand region (SLIGKV) at the N-terminus rescued the cell surface receptor expression and the response to the synthetic peptide ligand, suggesting that the signal peptide of PAR2 may be involved in preventing PAR2 from intracellular protease activation before reaching the cell surface. Supporting this hypothesis, an Arg36Ala mutation on PAR2ΔSP, which disabled the trypsin activation site, increased the receptor cell surface expression and the response to ligand stimulation. Similar effects were observed when PAR2ΔSP expressing cells were treated with protease inhibitors. Our findings indicated that there is a role of the PAR2 signal peptide in preventing the premature activation of PAR2 from intracellular protease cleavage before reaching the cells surface. The same mechanism may also apply to PAR1, PAR3, and PAR4.

Identification of the Epitope Recognized by the Anti-Red Sea Bream Iridovirus (RSIV) Monoclonal Antibody M10 Using a Phage Display RSIV Peptide Library

Red sea bream iridoviral disease (RSIVD) spreads readily throughout the marine environment because of its wide variety of host species. Use of a rapid diagnostic method, such as the immunofluorescence antibody test, helps to control the disease, so the anti-RSIV monoclonal antibody M10 (mAb M10) has been developed in Japan. In the present study, we carried out epitope mapping using the phage display method to identify the antigen of mAb M10. A phage display RSIV peptide library was constructed to cover the entire genome of RSIV, then phage clones recognized by the antibody were selected by biopanning. The selected clones harbored partial fragments of the laminin-type epidermal growth factor-like domain (LEGFD) gene. N-terminal and C-terminal deletion peptides were then prepared from the amino acid sequence deduced from the smallest fragment to precisely determine the epitope. Finally, seven amino acids, EYDCPEY, located in the extracellular domain of the LEGFD protein were determined to be the epitope. Identical residues of the epitope were also identified from the LEGFD protein in other megalocytiviruses including the infectious spleen and kidney necrosis virus and turbot reddish body iridovirus. mAb M10 is considered to be widely available for the diagnostics of megalocytivirus infections.