scholarly journals Generation of “nanometer-size aggregates” using Solubility Controlling Peptide tags and their ability to increase a protein’s immunogenicity in vivo

2019 ◽  
Author(s):  
Nafsoon Rahman ◽  
Mohammad Monirul Islam ◽  
Satoru Unzai ◽  
Shiho Miura ◽  
Yutaka Kuroda
Keyword(s):  
Underlying Mechanism ◽  
Igg Antibody ◽  
Nanometer Size ◽  
C Terminus ◽  
Peptide Tags ◽  
Pancreatic Trypsin Inhibitor ◽  
Model Protein ◽  
Antibody Titers ◽  
Micrometer Size

AbstractSub-visible aggregates of proteins are suspected to cause adverse immune response, and a recent FDA guideline has recommended the monitoring of micrometer-size aggregates (2-10 μm) though recognizing that the underlying mechanism behind aggregation and immunogenicity remains unclear. Here, we report a correlation between the immunogenicity and the size of nanometer-scale aggregates of a small 6.5 kDa model protein, Bovine Pancreatic Trypsin Inhibitor (BPTI) variant. BPTI-19A, a monomeric and non-immunogenic protein, was oligomerized into sub-visible aggregates with hydrodynamic radii (Rh) of 3~4 nm by attaching hydrophobic solubility controlling peptide (SCP) tags to its C-terminus. The results showed that the association of non-immunogenic BPTI into nanometer-size aggregates made it highly immunogenic, as assessed by the IgG antibody titers of the mice’s sera. Overall, the study emphasizes that sub-visible aggregates, as small as a few nanometers, which are presently ignored, are worth monitoring for deciphering the origin of undesired immunogenicity of therapeutic proteins.

scholarly journals A systematic mutational analysis identifies a 5-residue proline tag that enhances the in vivo immunogenicity of a non-immunogenic model protein 240 folds

2020 ◽  
Author(s):  
Nafsoon Rahman ◽  
Mohammad Monirul Islam ◽  
Md Golam Kibria ◽  
Satoru Unzai ◽  
Yutaka Kuroda
Keyword(s):  
Mutational Analysis ◽  
Peptide Vaccines ◽  
Tertiary Structures ◽  
Small Proteins ◽  
C Terminus ◽  
Pancreatic Trypsin Inhibitor ◽  
Model Protein ◽  
Soluble Aggregates ◽  
Igg Level

AbstractSmall proteins are generally non-immunogenic, which can be a major hurdle in developing protein and peptide vaccines or producing antibodies for biopharmaceutical usage. For improving a protein’s immunogenicity, we previously proposed to use short Solubility Controlling Peptide (SCP) tags that oligomerize proteins into soluble aggregates. Here, we systematically analyzed the effect of SCP-tags that do not induce oligomerization on the immunogenicity of a small, non-immunogenic, model protein, Bovine Pancreatic Trypsin Inhibitor (BPTI-19A; 6 kDa). We assessed the effect of the following ten SCP-tags: Six tags made of five consecutive Arg, Lys, His, Asp, Asn, Pro; one made of seven Pro; two tags made of consecutive Arg-lle and Asn-Ile, all attached at the C-terminus of BPTI-19A; and a 5-proline tag attached at the N-terminus. Circular dichroism, fluorescence, dynamic light scattering measurements, and analytical ultra-centrifugation indicated that the addition of the SCP-tags did not change the secondary structure content nor the tertiary structures of the protein nor its monomeric state. On the other hand, the C-terminus 5-proline (C5P) tag unexpectedly increased the immunogenicity (IgG level) of BPTI-19A by up to 240 fold as assessed by ELISA. Additionally, the 5-arginine tag (C5R) increased the titer by up to 73 fold. The titer increase lasted for several weeks, and the effect was cumulative to that of the Freund’s adjuvant, which is commonly used to boost a protein’s immunogenicity. Altogether, SCP-tags that do not oligomerize proteins substantially increased the immunogenicity of a non-immunogenic protein, suggesting that the 5-proline and the 5-arginine SCP-tags may provide a novel tool for facilitating the production of antibodies or improving the effectiveness of protein-based vaccines.

scholarly journals Gp37 Regulates the Pathogenesis of Avian Leukosis Virus Subgroup J via Its C Terminus

2020 ◽  
Vol 94 (11) ◽  
Author(s):  
Tuofan Li ◽  
Xiaohui Yao ◽  
Chunping Li ◽  
Jun Zhang ◽  
Quan Xie ◽  
...  
Keyword(s):  
Myeloid Leukemia ◽  
Antiviral Drug ◽  
Underlying Mechanism ◽  
Vital Role ◽  
Significant Loss ◽  
Viral Shedding ◽  
C Terminus ◽  
New Perspective

ABSTRACT Different from other subgroups of avian leukosis viruses (ALVs), ALV-J is highly pathogenic. It is the main culprit causing myeloid leukemia and hemangioma in chickens. The distinctiveness of the env gene of ALV-J, with low homology to those of other ALVs, is linked to its unique pathogenesis, but the underlying mechanism remains unclear. Previous studies show that env of ALV-J can be grouped into three species based on the tyrosine motifs in the cytoplasmic domain (CTD) of Gp37, i.e., the inhibitory, bifunctional, and active groups. To explore whether the C terminus or the tyrosine motifs in the CTD of Gp37 affect the pathogenicity of ALV-J, a set of ALV-J infectious clones containing different C termini of Gp37 or the mutants at the tyrosine sites were tested in vitro and in vivo. Viral growth kinetics indicated not only that ALV-J with active env is the fastest in replication and ALV-J with inhibitory env is the lowest but also that the tyrosine sites essentially affected the replication of ALV-J. Moreover, in vivo studies demonstrated that chickens infected by ALV-J with active or bifunctional env showed higher viremia, cloacal viral shedding, and viral tissue load than those infected by ALV-J with inhibitory env. Notably, the chickens infected by ALV-J with active or bifunctional env showed significant loss of body weight compared with the control chickens. Taken together, these findings reveal that the C terminus of Gp37 plays a vital role in ALV-J pathogenesis, and change from inhibitory env to bifunctional or active env increases the pathogenesis of ALV-J. IMPORTANCE ALV-J can cause severe immunosuppression and myeloid leukemia in infected chickens. However, no vaccine or antiviral drug is available against ALV-J, and the mechanism for ALV-J pathogenesis needs to be elucidated. It is generally believed that gp85 and LTR of ALV contribute to its pathogenesis. Here, we found that the C terminus and the tyrosine motifs (YxxM, ITIM, and ITAM-like) in the CTD of Gp37 of ALV-J could affect the pathogenicity of ALV-J in vitro and in vivo. The pathogenicity of ALV-J with Gp37 containing ITIM only was significantly less than ALV-J with Gp37 containing both YxxM and ITIM and ALV-J with Gp37 containing both YxxM and ITAM-like. This study highlights the vital role of the C terminus of Gp37 in the pathogenesis of ALV-J and thus provides a new perspective to elucidate the interaction between ALV-J and its host and a molecular basis to develop efficient strategies against ALV-J.

scholarly journals Phosphorylation of α-dystrobrevin is essential for αkap accumulation and acetylcholine receptor stability

2020 ◽  
Vol 295 (31) ◽  
pp. 10677-10688
Author(s):  
Po-Ju Chen ◽  
Diego Zelada ◽  
Dina Cheryne Belhasan ◽  
Mohammed Akaaboune
Keyword(s):  
Acetylcholine Receptor ◽  
Muscle Cells ◽  
Underlying Mechanism ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Tyrosine Residues ◽  
C Terminus ◽  
The Stability ◽  
High Level

The maintenance of a high density of the acetylcholine receptor (AChR) is the hallmark of the neuromuscular junction. Muscle-specific anchoring protein (αkap) encoded within the calcium/calmodulin-dependent protein kinase IIα (CAMK2A) gene is essential for the maintenance of AChR clusters both in vivo and in cultured muscle cells. The underlying mechanism by which αkap is maintained and regulated remains unknown. Here, using human cell lines, fluorescence microscopy, and pulldown and immunoblotting assays, we show that α-dystrobrevin (α-dbn), an intracellular component of the dystrophin glycoprotein complex, directly and robustly promotes the stability of αkap in a concentration-dependent manner. Mechanistically, we found that the phosphorylatable tyrosine residues of α-dbn are essential for the stability of α-dbn itself and its interaction with αkap, with substitution of three tyrosine residues in the α-dbn C terminus with phenylalanine compromising the αkap–α-dbn interaction and significantly reducing both αkap and α-dbn accumulation. Moreover, the αkap–α-dbn interaction was critical for αkap accumulation and stability. We also found that the absence of either αkap or α-dbn markedly reduces AChRα accumulation and that overexpression of α-dbn or αkap in cultured muscle cells promotes the formation of large agrin-induced AChR clusters. Collectively, these results indicate that the stability of αkap and α-dbn complex plays an important role in the maintenance of high-level expression of AChRs.

scholarly journals Silicone Oil-Based Nanoadjuvants as Candidates for a New Formulation of Intranasal Vaccines

Vaccines ◽  
2021 ◽  
Vol 9 (3) ◽  
pp. 234
Author(s):  
Agnieszka Razim ◽  
Marcelina Pyclik ◽  
Katarzyna Pacyga ◽  
Sabina Górska ◽  
Jintao Xu ◽  
...  
Keyword(s):  
Silicone Oil ◽  
Storage Conditions ◽  
Igg Antibody ◽  
Mucosal Surfaces ◽  
Model Protein ◽  
Antibody Titers ◽  
Mucosal Vaccines ◽  
New Formulation ◽  
Factor Α

Many conventional vaccines are administered via a needle injection, while most pathogens primarily invade the host via mucosal surfaces. Moreover, protective IgA antibodies are insufficiently induced by parenteral vaccines. Mucosal immunity induces both local and systemic response to pathogens and typically lasts for long periods of time. Therefore, vaccination via mucosal routes has been increasingly explored. However, mucosal vaccines require potent adjuvants to become efficacious. Despite many efforts to develop safe and robust adjuvants for mucosal vaccines, only a few have been approved for use in human formulations. The aim of our study was to design, develop and characterize new silicone oil-based nanoadjuvant candidates for intranasal vaccines with potential to become mucosal adjuvants. We have developed an array of nanoadjuvant candidates (NACs), based on well-defined ingredients. NAC1, 2 and 3 are based on silicone oil, but differ in the used detergents and organic solvents, which results in variations in their droplet size and zeta potential. NACs’ cytotoxicity, Tumor Necrosis Factor α (TNF-α) induction and their effect on antigen engulfment by immune cells were tested in vitro. Adjuvant properties of NACs were verified by intranasal vaccination of mice together with ovalbumin (OVA). NACs show remarkable stability and do not require any special storage conditions. They exhibit bio-adhesiveness and influence the degree of model protein engulfment by epithelial cells. Moreover, they induce high specific anti-OVA IgG antibody titers after two intranasal administrations. Nanoadjuvant candidates composed of silicone oil and cationic detergents are stable, exhibit remarkable adjuvant properties and can be used as adjuvants for intranasal immunization.

scholarly journals A Developmentally Regulated Chaperone Complex for the Endoplasmic Reticulum of Male Haploid Germ Cells

2007 ◽  
Vol 18 (8) ◽  
pp. 2795-2804 ◽  
Author(s):  
Marcel van Lith ◽  
Anna-Riikka Karala ◽  
Dave Bown ◽  
John A. Gatehouse ◽  
Lloyd W. Ruddock ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Germ Cells ◽  
Specific Protein ◽  
Developmentally Regulated ◽  
C Terminus ◽  
Pancreatic Trypsin Inhibitor ◽  
Disulfide Isomerases ◽  
Chaperone Complex

Glycoprotein folding is mediated by lectin-like chaperones and protein disulfide isomerases (PDIs) in the endoplasmic reticulum. Calnexin and the PDI homologue ERp57 work together to help fold nascent polypeptides with glycans located toward the N-terminus of a protein, whereas PDI and BiP may engage proteins that lack glycans or have sugars toward the C-terminus. In this study, we show that the PDI homologue PDILT is expressed exclusively in postmeiotic male germ cells, in contrast to the ubiquitous expression of many other PDI family members in the testis. PDILT is induced during puberty and represents the first example of a PDI family member under developmental control. We find that PDILT is not active as an oxido-reductase, but interacts with the model peptide Δ-somatostatin and nonnative bovine pancreatic trypsin inhibitor in vitro, indicative of chaperone activity. In vivo, PDILT forms a tissue-specific chaperone complex with the calnexin homologue calmegin. The identification of a redox-inactive chaperone partnership defines a new system of testis-specific protein folding with implications for male fertility.

SAC3B is a target of CML19, the centrin 2 of Arabidopsis thaliana

2020 ◽  
Vol 477 (1) ◽  
pp. 173-189 ◽  
Author(s):  
Marco Pedretti ◽  
Carolina Conter ◽  
Paola Dominici ◽  
Alessandra Astegno
Keyword(s):  
Excision Repair ◽  
Complex Structure ◽  
Binding Motif ◽  
C Terminus ◽  
Repair Protein ◽  
Nucleotide Excision ◽  
Ef Hand ◽  
Molecular Determinants ◽  
Structure In Solution

Arabidopsis centrin 2, also known as calmodulin-like protein 19 (CML19), is a member of the EF-hand superfamily of calcium (Ca2+)-binding proteins. In addition to the notion that CML19 interacts with the nucleotide excision repair protein RAD4, CML19 was suggested to be a component of the transcription export complex 2 (TREX-2) by interacting with SAC3B. However, the molecular determinants of this interaction have remained largely unknown. Herein, we identified a CML19-binding site within the C-terminus of SAC3B and characterized the binding properties of the corresponding 26-residue peptide (SAC3Bp), which exhibits the hydrophobic triad centrin-binding motif in a reversed orientation (I8W4W1). Using a combination of spectroscopic and calorimetric experiments, we shed light on the SAC3Bp–CML19 complex structure in solution. We demonstrated that the peptide interacts not only with Ca2+-saturated CML19, but also with apo-CML19 to form a protein–peptide complex with a 1 : 1 stoichiometry. Both interactions involve hydrophobic and electrostatic contributions and include the burial of Trp residues of SAC3Bp. However, the peptide likely assumes different conformations upon binding to apo-CML19 or Ca2+-CML19. Importantly, the peptide dramatically increases the affinity for Ca2+ of CML19, especially of the C-lobe, suggesting that in vivo the protein would be Ca2+-saturated and bound to SAC3B even at resting Ca2+-levels. Our results, providing direct evidence that Arabidopsis SAC3B is a CML19 target and proposing that CML19 can bind to SAC3B through its C-lobe independent of a Ca2+ stimulus, support a functional role for these proteins in TREX-2 complex and mRNA export.

The Inducing Roles of the New Isolated Bursal Hexapeptide and Pentapeptide on the Immune Response of AIV Vaccine in Mice

2019 ◽  
Vol 26 (7) ◽  
pp. 542-549 ◽  
Author(s):  
Shan Shan Hao ◽  
Man Man Zong ◽  
Ze Zhang ◽  
Jia Xi Cai ◽  
Yang Zheng ◽  
...  
Keyword(s):  
Immune Response ◽  
Dendritic Cells ◽  
Amino Acid ◽  
T Cell ◽  
Antigen Presentation ◽  
Amino Acid Sequences ◽  
Cell Subpopulation ◽  
Igg Antibody ◽  
Antibody Titers ◽  
Specific Igg

Background: Bursa of Fabricius is the acknowledged central humoral immune organ. The bursal-derived peptides play the important roles on the immature B cell development and antibody production. Objective: Here we explored the functions of the new isolated bursal hexapeptide and pentapeptide on the humoral, cellular immune response and antigen presentation to Avian Influenza Virus (AIV) vaccine in mice immunization. Methods: The bursa extract samples were purified following RP HPLC method, and were analyzed with MS/MS to identify the amino acid sequences. Mice were twice subcutaneously injected with AIV inactivated vaccine plus with two new isolated bursal peptides at three dosages, respectively. On two weeks after the second immunization, sera samples were collected from the immunized mice to measure AIV-specific IgG antibody levels and HI antibody titers. Also, on 7th day after the second immunization, lymphocytes were isolated from the immunized mice to detect T cell subtype and lymphocyte viabilities, and the expressions of co-stimulatory molecule on dendritic cells in the immunized mice. Results: Two new bursal hexapeptide and pentapeptide with amino acid sequences KGNRVY and MPPTH were isolated, respectively. Our investigation proved the strong regulatory roles of bursal hexapeptide on AIV-specific IgG levels and HI antibody titers, and lymphocyte viabilities, and the significant increased T cells subpopulation and expressions of MHCII molecule on dendritic cells in the immunized mice. Moreover, our findings verified the significantly enhanced AIV-specific IgG antibody and HI titers, and the strong increased T cell subpopulation and expressions of CD40 molecule on dendritic cells in the mice immunized with AIV vaccine and bursal pentapeptide. Conclusion: We isolated and identified two new hexapeptide and pentapeptide from bursa, and proved that these two bursal peptides effectively induced the AIV-specific antibody, T cell and antigen presentation immune responses, which provided an experimental basis for the further clinical application of the bursal derived active peptide on the vaccine improvement.

scholarly journals A Peptide Found in Human Serum, Derived from the C-Terminus of Albumin, Shows Antifungal Activity In Vitro and In Vivo

2020 ◽  
Vol 8 (10) ◽  
pp. 1627
Author(s):  
Tecla Ciociola ◽  
Pier Paolo Zanello ◽  
Tiziana D’Adda ◽  
Serena Galati ◽  
Stefania Conti ◽  
...  
Keyword(s):  
Antifungal Activity ◽  
Human Serum ◽  
Fungicidal Activity ◽  
Galleria Mellonella ◽  
Serum Proteins ◽  
Morphological Alterations ◽  
C Terminus ◽  
Different Sources

The growing problem of antimicrobial resistance highlights the need for alternative strategies to combat infections. From this perspective, there is a considerable interest in natural molecules obtained from different sources, which are shown to be active against microorganisms, either alone or in association with conventional drugs. In this paper, peptides with the same sequence of fragments, found in human serum, derived from physiological proteins, were evaluated for their antifungal activity. A 13-residue peptide, representing the 597–609 fragment within the albumin C-terminus, was proved to exert a fungicidal activity in vitro against pathogenic yeasts and a therapeutic effect in vivo in the experimental model of candidal infection in Galleria mellonella. Studies by confocal microscopy and transmission and scanning electron microscopy demonstrated that the peptide penetrates and accumulates in Candida albicans cells, causing gross morphological alterations in cellular structure. These findings add albumin to the group of proteins, which already includes hemoglobin and antibodies, that could give rise to cryptic antimicrobial fragments, and could suggest their role in anti-infective homeostasis. The study of bioactive fragments from serum proteins could open interesting perspectives for the development of new antimicrobial molecules derived by natural sources.

scholarly journals Regulation of Swarming Motility and flhDCSm Expression by RssAB Signaling in Serratia marcescens

2008 ◽  
Vol 190 (7) ◽  
pp. 2496-2504 ◽  
Author(s):  
Po-Chi Soo ◽  
Yu-Tze Horng ◽  
Jun-Rong Wei ◽  
Jwu-Ching Shu ◽  
Chia-Chen Lu ◽  
...  
Keyword(s):  
Serratia Marcescens ◽  
Binding Site ◽  
Promoter Region ◽  
Transcriptional Start Site ◽  
Direct Interaction ◽  
Underlying Mechanism ◽  
Inhibitory Effect ◽  
Start Codon ◽  
Mobility Shift

ABSTRACT Serratia marcescens cells swarm at 30°C but not at 37°C, and the underlying mechanism is not characterized. Our previous studies had shown that a temperature upshift from 30 to 37°C reduced the expression levels of flhDCSm and hagSm in S. marcescens CH-1. Mutation in rssA or rssB, cognate genes that comprise a two-component system, also resulted in precocious swarming phenotypes at 37°C. To further characterize the underlying mechanism, in the present study, we report that expression of flhDCSm and synthesis of flagella are significantly increased in the rssA mutant strain at 37°C. Primer extension analysis for determination of the transcriptional start site(s) of flhDCSm revealed two transcriptional start sites, P1 and P2, in S. marcescens CH-1. Characterization of the phosphorylated RssB (RssB∼P) binding site by an electrophoretic mobility shift assay showed direct interaction of RssB∼P, but not unphosphorylated RssB [RssB(D51E)], with the P2 promoter region. A DNase I footprinting assay using a capillary electrophoresis approach further determined that the RssB∼P binding site is located between base pair positions −341 and −364 from the translation start codon ATG in the flhDCSm promoter region. The binding site overlaps with the P2 “−35” promoter region. A modified chromatin immunoprecipitation assay was subsequently performed to confirm that RssB∼P binds to the flhDCSm promoter region in vivo. In conclusion, our results indicated that activated RssA-RssB signaling directly inhibits flhDCSm promoter activity at 37°C. This inhibitory effect was comparatively alleviated at 30°C. This finding might explain, at least in part, the phenomenon of inhibition of S. marcescens swarming at 37°C.

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