Construction and validation of a synthetic phage-displayed nanobody library.

Nanobodies derived from camelids and sharks offer unique advantages in therapeutic applications due to their ability to bind to epitopes that were previously inaccessible. Traditional methods of nanobody development face challenges such as ethical concerns and antigen toxicity. Our study presents a synthetic, phagedisplayed nanobody library using trinucleotide-directed mutagenesis technology, which allows precise amino acid composition in complementarity-determining regions (CDRs), with a focus on CDR3 diversity. This approach avoids common problems such as frameshift mutations and stop codon insertions associated with other synthetic antibody library construction methods. By analyzing FDA-approved nanobodies and Protein Data Bank sequences, we designed sub-libraries with different CDR3 lengths and introduced amino acid substitutions to improve solubility. The validation of our library through the successful isolation of nanobodies against targets such as PD-1, ATXN1 and STAT3 demonstrates a versatile and ethical platform for the development of high specificity and affinity nanobodies and represents a significant advance in biotechnology.

Wound healing induced by new synthetic peptide, A7-1, in C57BL/6 mouse model.

The effects of the novel synthetic peptide, A7-1, on wound healing and skin grafts were evaluated in a C57BL/6 mouse model. Two 15-mm wide circular skin excisions were made on the backs of mice and to each excision, 100 µM A7-1 or normal saline was applied daily. The treatments were applied and sutured for skin graft analysis. Digital photos were acquired on days 4, 7, 11, and 14 and fluorescein angiography was conducted. Wound sizes were verified using stereoscopic microscopy. Histological analysis was performed via hematoxylin and eosin staining and Masson's trichrome staining. Western blotting was performed using vascular endothelial growth factor. Using a stereoscopic microscope, significantly faster wound healing (17.3%) and skin graft healing (16.5%) were observed in the A7-1 treatment group in comparison to that of the control. The angiogenesis was significantly faster in fluorescein angiography examination in wound healing (11%) and skin grafts (15%). However, the average completion of epithelization (overall time for wound healing), did not show any significant differences. In comparison to the control, the new protein, A7-1, led to significantly faster wound healing in the initial angiogenesis.

Characterization of Synthetic Peptides by Mass Spectrometry.

In the quality control of synthetic peptides, mass spectroscopy (MS) serves as an optimal method for evaluating authenticity and integrity. Typically, the sequence of a synthetic peptide is already established, thereby directing the focus of analysis towards validating its identity and purity. This chapter outlines straightforward methodologies for conducting MS analyses specifically tailored for synthetic peptides.

Non-invasive urine-based ELISA using a recombinant Leishmania protein to diagnose tegumentary leishmaniasis.

The diagnosis of tegumentary leishmaniasis (TL) is hampered by variable sensitivity and/or specificity of the tests. Serological assays are suitable to diagnose visceral leishmaniasis (VL); however, they present low performance for the detection of TL cases. Additionally, blood collection to obtain patient serum represents a challenge, as it is an invasive and uncomfortable procedure, requiring laboratorial infrastructure and trained professionals. In this context, the present study proposed to evaluate patient urine to detect TL, given that this analyte has proven to be effective in ELISA experiments for the detection of VL cases. For this, a Leishmania protein called LiHyV, two specific B-cell epitopes derived from protein amino acid sequence, and a Leishmania antigenic extract (SLA) were used as antigens. A total of 215 paired urine and serum samples were evaluated, and results showed that, when serum was employed as an analyte, rLiHyV, Peptide1, Peptide2, and SLA presented a sensitivity of 85 %, 29 %, 58 %, and 31 %, respectively, and a specificity of 97.5 %, 98 %, 100 %, and 97.5 %, respectively, in the diagnosis of TL. When urine was used, rLiHyV, Peptide1, Peptide2, and SLA presented a sensitivity of 95 %, 74 %, 67 %, and 52 %, respectively, and a specificity of 100 %, 99 %, 98 %, and 86 %, respectively. In conclusion, preliminary data suggest that urine could be considered as an alternative biological sample for the detection of TL cases.

DAP1-2: a synthetic peptide targeting IL-1R1 receptor effectively suppresses IL-1ß in vitro.

The pathological manifestation of the inflammatory process primarily stems from the heightened release of pro-inflammatory cytokines, with IL-1ß standing out as a pivotal cytokine. The excessive presence of IL-1ß disrupts immune signaling, thereby assuming a pathogenic and exacerbating role in the pathophysiology of numerous inflammatory diseases. Regulating IL-1ß levels becomes crucial, and the IL-1Ra molecule serves this purpose by binding to the IL-1R1 receptor, thereby impeding the binding of IL-1ß. Several pharmaceuticals have entered the market, aiming to neutralize IL-1ß's biological function through diverse mechanisms. However, the existing IL-1ß inhibitors are recombinant proteins, characterized by a high production cost and limited stability. Therefore, this study aimed to predict a peptide, named DAP1-2, based on the IL-1Ra molecule. DAP1-2 was designed to attenuate responses triggered by IL-1ß by blocking the IL-1R1 receptor. The selection of amino acids from the IL-1Ra molecule (PDB: I1RA) that interact with the three domains of the IL-1R1 receptor was performed using Swiss PDB Viewer. After prediction, chemical synthesis was made using the Fmoc-Synthesis technique. The efficacy of DAP1-2 was assessed using RAW 264.7 cells, which were exposed to LPS (5 µg/mL) for 24 h to induce IL-1ß expression and treated with the peptides in different concentrations. IL-1ß levels were assessed using ELISA, and the gene expression of IL-1ß was measured by RT-qPCR, additionally to the viability test. Results revealed a significant reduction in IL-1ß levels and gene expression in cells stimulated by LPS and treated with DAP1-2 in different concentrations. Furthermore, the MTT assay confirmed the nontoxic nature of the peptides on the cell lineage. This alternative approach shows promise as an IL-1 inhibitor, due to the stability, ease of production, and cost-effectiveness provided by the use of synthetic peptides.

First Generic Teriparatide: Structural and Biological Sameness to Its Reference Medicinal Product.

Teriparatide is an anabolic peptide drug indicated for the treatment of osteoporosis. Recombinant teriparatide was first approved in 2002 and has since been followed by patent-free alternatives under biosimilar or hybrid regulatory application. The aim of this study is to demonstrate the essential similarity between synthetic teriparatide BGW and the reference medicinal product (RMP), and thus to ensure the development of the first generic teriparatide drug. Hence, an extensive side-by-side comparative exercise, focusing on structural and biological activity, was performed using a wide range of state-of-the-art orthogonal methods. Nuclear magnetic resonance (NMR), ion mobility-mass spectrometry (IM-MS), UV, circular dichroism (CD) and Fourier transform infrared (FTIR) demonstrated the structural similarity between teriparatide BGW and the RMP. Comparative cell-based bioassays showed that the synthetic and recombinant peptides have identical behaviors. Teriparatide BGW, as a generic drug, provides an available treatment option for patients with osteoporosis and offers clinical benefits identical to those provided by the RMP.

The bright side of chemistry: Exploring synthetic peptide-based anticancer vaccines.

The present review focuses on synthetic peptide-based vaccine strategies in the context of anticancer intervention, paying attention to critical aspects such as peptide epitope selection, adjuvant integration, and nuanced classification of synthetic peptide cancer vaccines. Within this discussion, we delve into the diverse array of synthetic peptide-based anticancer vaccines, each derived from tumor-associated antigens (TAAs), including melanoma antigen recognized by T cells 1 (Melan-A or MART-1), mucin 1 (MUC1), human epidermal growth factor receptor 2 (HER-2), tumor protein 53 (p53), human telomerase reverse transcriptase (hTERT), survivin, folate receptor (FR), cancer-testis antigen 1 (NY-ESO-1), and prostate-specific antigen (PSA). We also describe the synthetic peptide-based vaccines developed for cancers triggered by oncovirus, such as human papillomavirus (HPV), and hepatitis C virus (HCV). Additionally, the potential synergy of peptide-based vaccines with common therapeutics in cancer was considered. The last part of our discussion deals with the realm of the peptide-based vaccines delivery, highlighting its role in translating the most promising candidates into effective clinical strategies. Although this discussion does not cover all the ongoing peptide vaccine investigations, it aims at offering valuable insights into the chemical modifications and the structural complexities of anticancer peptide-based vaccines.

Enantiomeric purity of synthetic therapeutic peptides: A review.

Synthetic therapeutic peptides are a complex and popular class of pharmaceuticals. In recent years, peptides with proven therapeutic activity have gained significant interest in the market. The determination of synthetic peptide enantiomeric purity plays a critical role in the evaluation of the quality of the medicine. Since racemization is one of the most common side reactions occurring in AAs or peptides, enantiomeric impurities such as D-isomers can form during the peptide synthesis or can be introduced from the starting materials (e.g., AAs). The therapeutic effect of a synthetic or semi-synthetic bioactive peptide molecule depends on its AA enantiomeric purity and secondary/tertiary structure. Therefore, the enantiomeric purity determination for synthetic peptides is supportive for interpreting unwanted therapeutic effects and determining the quality of synthetic peptide therapeutics. However, enantiomeric purity analysis encounters formidable analytical challenges during chromatographic separation, as D/L isomers have identical physical-chemical properties except stereochemical configuration. To ensure peptides AA stereochemical configuration whether in the free or bound state, sensitive and reproducible quantitative analytical method is mandatory. In this regard, numerous analytical techniques were emerged for the quantification of D-isomeric impurities in synthetic peptides, but still, very few reports are available in the literature. Thus, the purpose of this paper is to provide an overview of the importance, regulatory requirements, and various analytical methods used for peptide enantiomeric purity determination. In addition, we discussed the available literature in terms of enantiomeric impurity detection, common hydrolysis procedural aspects, and different analytical strategies used for sample preparation.

A Review on the use of Synthetic and Recombinant Antigens for the Immunodiagnosis of Tegumentary Leishmaniasis.

Improving the diagnostic technology used to detect tegumentary leishmaniasis (TL) is essential in view of it being a widespread, often neglected tropical disease, with cases reported from the Southern United States to Northern Argentina. Recombinant proteins, recombinant multiepitope proteins, and synthetic peptides have been extensively researched and used in disease diagnosis. One of the benefits of applying these antigens is a measurable increase in sensitivity and specificity, which improves test accuracy. The present review aims to describe the use of these antigens and their diagnostic effectiveness. With that in mind, a bibliographic survey was conducted on the PudMed platform using the search terms "tegumentary leishmaniasis" AND "diagno", revealing that recombinant proteins have been described and evaluated for their value in TL diagnosis since the 1990s. However, there was a spike in the number of publications using all of the antigens between 2013 and 2022, confirming an expansion in research efforts to improve diagnosis. Moreover, all of the studies involving different antigens had promising results, including improved sensitivity and specificity. These data recognize the importance of doing research with new technologies focused on developing quick, more effective diagnostic kits as early diagnosis facilitates treatment.

Potential role of the cell-penetrating peptide-conjugated soluble N-ethylmaleimide-sensitive factor attachment protein receptor motif of vesicle-associated membrane protein 2-patterned peptide in novel cosmeceutical skin product development.

AIM: This study aimed to investigate and verify the effect of cell-penetrating peptide (CPP)-conjugated soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) motif of vesicle-associated membrane protein 2 (VAMP2)-patterned peptide (INCI name: Acetyl sh-Oligopeptide-26 sh-Oligopeptide-27 SP, trade name: M.Biome-BT) on improving skin function in vitro. METHODS: The cytotoxicity of CPP-conjugated SNARE motif of VAMP2-patterned peptide (CVP) was investigated using the 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT) assay against B16-F10 cells and human dermal fibroblasts (HDFs) and a reconstructed skin irritation test. The anti-wrinkle activity of M.Biome-BT was determined by assessing the release of norepinephrine and dopamine in PC-12 cells via ELISA. The skin-whitening effects of CVP were assessed in B16-F10 cells by measuring the intra- and extracellular melanin contents and expression levels of melanin production-related genes, such as microphthalmia-associated transcription factor (MITF), tyrosinase (TYR), tyrosinase-related protein-1 (TRP-1), and TRP-2. RESULTS: CVP is not cytotoxic to B16-F10 cells and HDFs, and no skin irritation was observed. CVP treatment considerably diminished K+ -induced norepinephrine and dopamine secretion compared with the non-treated control group (62% and 40%, respectively). Additionally, the inhibition ability of CVP on norepinephrine and dopamine release was comparable to that of botulinum neurotoxin type A (BoNT/A). CVP also increased intracellular melanin content in a dose-dependent manner, whereas extracellular melanin content decreased (76%-85%). However, CVP treatment did not affect the mRNA expression of MITF, TYR, TRP-1, and TRP-2. These results suggest that CVP does not inhibit melanin production; however, it may induce a whitening effect by inhibiting melanin transport. CONCLUSIONS: Taken together, our findings indicate that CVP could be used as an active and safe cosmeceutical ingredient for antiaging applications.

A methodological approach by capillary electrophoresis coupled to mass spectrometry via electrospray interface for the characterization of short synthetic peptides towards the conception of self-assembled nanotheranostic agents.

Nanostructures formed by the self-assembling peptide building blocks are attractive materials for the design of theranostic objects due to their intrinsic biocompatibility, accessible surface chemistry as well as cavitary morphology. Short peptide synthesis and modification are straightforward and give access to a great diversity of sequences, making them very versatile building blocks allowing for the design of thoroughly controlled self-assembled nanostructures. In this work, we developed a new CE-DAD-ESI-MS method to characterize short synthetic amphiphilic peptides in terms of exact sequence and purity level in the low 0.1 mg.mL-1 range, without sample treatment. This study was conducted using a model sequence, described to have pH sensitive self-assembling property. Peptide samples obtained from different synthesis processes (batch or flow, purified or not) were thus separated by capillary zone electrophoresis (CZE). The associated dual UV and MS detection mode allowed to evidence the exact sequence together with the presence of impurities, identified as truncated or non-deprotected sequences, and to quantify their relative proportion in the peptide mixture. Our results demonstrate that the developed CE-DAD-ESI-MS method could be directly applied to the characterization of crude synthetic peptide products, in parallel with the optimization of peptide synthetic pathway to obtain controlled sequences with high synthetic yield and purity, which is crucial for further design of robust peptide based self-assembled nanoarchitectures.

Biomedical applications of synthetic peptides derived from venom of animal origin: A systematic review.

Development of therapeutic agents that have fewer adverse effects and have higher efficacy for diseases, such as cancer, metabolic disorders, neurological diseases, infections, cardiovascular diseases, and respiratory diseases, are required. Recent studies have focused on identifying novel sources for pharmaceutical molecules to develop therapies against these diseases. Among the sources for potentially new therapies, animal venom-derived molecules have generated much interest. Various animal venom-derived proteins and peptides have been isolated, identified, synthesized, and tested to develop drugs. Venom-derived peptides have several biomedical properties, such as proapoptotic, cell migration, and autophagy regulation activities in cancer cell models; induction of vasodilation by nitric oxide and regulation of angiotensin II; modification of insulin response by controlling calcium and potassium channels; regulation of pain receptor activity; modulation of immune cell activity; alteration of motor neuron activity; degradation or inhibition of ß-amyloid plaque formation; antibacterial, antifungal, antiviral, and antiprotozoal activities; increase in sperm motility and potentiation of erectile function; reduction of intraocular pressure; anticoagulation, fibrinolytic, and antithrombotic activities; etc. This systematic review compiles these biomedical properties and potential biomedical applications of synthesized animal venom-derived peptides reported in the latest research. In addition, the limitations and areas of opportunity in this research field are discussed so that new studies can be developed based on the data presented.

Structural Shifts of the Parvovirus B19 Capsid Receptor-binding Domain: A Peptide Study.

BACKGROUND: Binding appropriate cellular receptors is a crucial step of a lifecycle for any virus. Structure of receptor-binding domain for a viral surface protein has to be determined before the start of future drug design projects. OBJECTIVE: Investigation of pH-induced changes in the secondary structure for a capsid peptide with loss of function mutation can shed some light on the mechanism of entrance. METHODS: Spectroscopic methods were accompanied by electrophoresis, ultrafiltration, and computational biochemistry. RESULTS: In this study, we showed that a peptide from the receptor-binding domain of Parvovirus B19 VP1 capsid (residues 13-31) is beta-structural at pH=7.4 in 0.01 M phosphate buffer, but alpha- helical at pH=5.0, according to the circular dichroism (CD) spectroscopy results. Results of infra- red (IR) spectroscopy showed that the same peptide exists in both alpha-helical and beta-structural conformations in partial dehydration conditions both at pH=7.4 and pH=5.0. In contrast, the peptide with Y20W mutation, which is known to block the internalization of the virus, forms mostly alpha-helical conformation in partial dehydration conditions at pH=7.4. According to our hypothesis, an intermolecular antiparallel beta structure formed by the wild-type peptide in its tetramers at pH=7.4 is the prototype of the similar intermolecular antiparallel beta structure formed by the corresponding part of Parvovirus B19 receptor-binding domain with its cellular receptor (AXL). CONCLUSION: Loss of function Y20W substitution in VP1 capsid protein prevents the shift into the beta-structural state by way of alpha helix stabilization and the decrease of its ability to turn into the disordered state.

Scorpion venom heat-resistant peptide alleviates mitochondrial dynamics imbalance induced by PM2.5 exposure by downregulating the PGC-1α/SIRT3 signaling pathway.

Background: Epidemiological inquiry reveals that neuroinflammation and mitochondrial dysfunction caused by PM2.5 exposure are associated with Alzheimer's disease. Nevertheless, the molecular mechanisms of mitochondrial dynamics and neuroinflammation induced by PM2.5 exposure remain elusive. In this study, our objective was to explore the impact of PM2.5 on mitochondrial dynamics and neuroinflammation, while also examining the reparative potential of scorpion venom heat-resistant synthetic peptide (SVHRSP). Methods: Western blot and quantitative reverse transcription polymerase chain reaction (RT-qPCR) were employed to ascertain the protein and gene levels of IL-1ß, IL-6, and TNF-α in BV2 cells. The concentration of IL-6 in the supernatant of the BV2 cell culture was measured by enzyme-linked immunosorbent assay. For the assessment of mitochondrial homeostasis, western blot, RT-qPCR, and cellular immunohistochemistry methods were utilized to investigate the protein and gene levels of DRP1 and MFN-2 in HT22 cells. In the context of signal pathway analyses, western blot, RT-qPCR, and immunofluorescence techniques were employed to detect the protein and gene expressions of PGC-1α and SIRT3 in HT22 cells, respectively. Following the transfection with siPGC-1αRNA, downstream proteins of PGC-1α/SIRT3 pathway in HT22 cells were investigated by Western blot and RT-qPCR. Results: The experimental findings demonstrated that exposure to PM2.5 exacerbated neuroinflammation, resulting in elevated levels of IL-1ß, IL-6, and TNF-α. Furthermore, it perturbed mitochondrial dynamics, as evidenced by increased DRP1 expression and decreased MFN-2 expression. Additionally, dysfunction was observed in the PGC-1α/SIRT3 signal pathway. However, intervention with SVHRSP ameliorated the cellular damage induced by PM2.5 exposure. Conclusions: SVHRSP alleviated neuroinflammation and mitochondrial dynamics imbalance induced by PM2.5 exposure by downregulating the PGC-1α/SIRT3 signaling pathway.

United we stand, divided we fall: in-depth proteomic evaluation of the synergistic effect of Mo-CBP3-PepI and Ciprofloxacin against Staphylococcus aureus biofilms.

Staphylococcus aureus forms biofilms, a structure that protects bacterial cells, conferring more resistance to difficult treatment. Synthetic peptides surge as an alternative to overcome the biofilm of multidrug-resistant pathogens. Mo-CBP3-PepI, when combined with Ciprofloxacin, reduced preformed S. aureus biofilm by 50% at low concentrations (0.2 and 6.2 µg. mL-1, respectively). The goal of this study was to evaluate the proteomic profile of biofilms after treatment with the Mo-CBP3-PepI combined with ciprofloxacin. Here, proteomic analysis confirmed with more depth previously described mechanisms and revealed changes in the accumulation of proteins related to DNA and protein metabolism, cell wall biosynthesis, redox metabolism, quorum sensing, and biofilm formation. Some proteins related to DNA and protein metabolism were reduced, while other proteins, like redox system proteins, disappeared in Ciprofloxacin+Mo-CBP3-PepI treatment. Our results indicated a synergistic effect of these two molecules with several mechanisms against S. aureus biofilm and opened new doors for combined treatments with other drugs.

Advances in Synthetic Grafts in Spinal Fusion Surgery.

Degenerative spine disease is increasing in prevalence as the global population ages, indicating a need for targeted therapies and continued innovations. While autograft and allograft have historically demonstrated robust results in spine fusion surgery, they have significant limitations and associated complications such as infection, donor site morbidity and pain, and neurovascular injury. Synthetic grafts may provide similar success while mitigating negative outcomes. A narrative literature review was performed to review available synthetic materials that aim to optimize spinal fusion. The authors specifically address the evolution of synthetics and comment on future trends. Novel synthetic materials currently in use include ceramics, synthetic polymers and peptides, bioactive glasses, and peptide amphiphiles, and the authors focus on their success in both human and animal models, physical properties, advantages, and disadvantages. Advantages include properties of osteoinduction, osteoconduction, and osteogenesis, whereas disadvantages encompass a lack of these properties or growth factor-induced complications. Typically, the use of synthetic materials results in fewer complications and lower costs. While the development and tuning of synthetic materials are ongoing, there are many beneficial alternatives to autografts and allografts with promising fusion results.

SARS-CoV-2 inhibitory activity of a short peptide derived from internal fusion peptide of S2 subunit of spike glycoprotein.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has posed a great concern in human population. To fight coronavirus emergence, we have dissected the conserved amino acid region of the internal fusion peptide in the S2 subunit of Spike glycoprotein of SARS-CoV-2 to design new inhibitory peptides. Among the 11 overlapping peptides (9-23-mer), PN19, a 19-mer peptide, exhibited a powerful inhibitory activity against different SARS-CoV-2 clinical isolate variants in absence of cytotoxicity. The PN19 inhibitory activity was found to be dependent on conservation of the central Phe and C-terminal Tyr residues in the peptide sequence. Circular dichroism spectra of the active peptide exhibited an alpha-helix propensity, confirmed by secondary structure prediction analysis. The PN19 inhibitory activity, exerted in the first step of virus infection, was reduced after peptide adsorption treatment with virus-cell substrate during fusion interaction. Additionally, PN19 inhibitory activity was reduced by adding S2 membrane-proximal region derived peptides. PN19 showed binding ability to the S2 membrane proximal region derived peptides, confirmed by molecular modelling, playing a role in the mechanism of action. Collectively, these results confirm that the internal fusion peptide region is a good candidate on which develop peptidomimetic anti SARS-CoV-2 antivirals.

A Porcine DNMT1 Variant: Molecular Cloning and Generation of Specific Polyclonal Antibody.

DNA methyltransferase 1 (DNMT1), the first-identified DNA methyltransferase in mammals, has been well studied in the control of embryo development and somatic homeostasis in mice and humans. Accumulating reports have demonstrated that DNMT1 plays an important role in the regulation of differentiation and the activation of immune cells. However, little is known about the effects of porcine DNMT1 on such functional regulation, especially the regulation of the biological functions of immune cells. In this study, we report the cloning of DNMT1 (4833 bp in length) from porcine alveolar macrophages (PAMs). According to the sequence of the cloned DNMT1 gene, the deduced protein sequence contains a total of 1611 amino acids with a 2 amino acid insertion, a 1 amino acid deletion, and 12 single amino acid mutations in comparison to the reported DNMT1 protein. A polyclonal antibody based on a synthetic peptide was generated to study the expression of the porcine DNMT1. The polyclonal antibody only recognized the cloned porcine DNMT1 and not the previously reported protein due to a single amino acid difference in the antigenic peptide region. However, the polyclonal antibody recognized the endogenous DNMT1 in several porcine cells (PAM, PK15, ST, and PIEC) and the cells of other species (HEK-293T, Marc-145, MDBK, and MDCK cells). Moreover, our results demonstrated that all the detected tissues of piglet express DNMT1, which is the same as that in porcine alveolar macrophages. In summary, we have identified a porcine DNMT1 variant with sequence and expression analyses.

ADY tripeptide is a minimum sequence for Tyrosylprotein sulfotransferases 1 and 2 substrate recognition.

Tyrosylprotein sulfotransferases (TPSTs) catalyze the transfer of a sulphonate moiety from 3'-Phosphoadenosine 5'-Phosphosulfate (PAPS) to the hydroxyl group of a tyrosine residue in substrate proteins. The positively charged substrate binding region of TPST homodimer interacts with acidic residues located in N-terminal region from the sulfated tyrosine in substrates. However, the sequence pattern in TPST substrate recognition remains unclear. Therefore, we aimed to determine the minimum recognition chain length required for tyrosine sulfation. We prepared His-tagged polypeptide, His-TPST143-370 and His-TPST243-377, form 43-370 of TPST1 and 43-377 of TPST2. Next, we prepared a series of synthesized ADYAE peptides and used a combination of reverse phase high-performance liquid chromatography (RP-HPLC) and mass spectrometric analysis to show that the tripeptide amino acid sequence, ADY, was sulfated by TPST1 and TPST2. Furthermore, we found that the acidic residue, located two residues C-terminal region from the tyrosine residue, may be involved in the TPST-induced sulfation regulation. The results in our study propose that proteins with the ADY sequence may be useful for searching the novel TPST tyrosine sulfated substrates.

Two MP2CL5 Antigen Vaccines from Naegleria fowleri Stimulate the Immune Response against Meningitis in the BALB/c Model.

Naegleria fowleri is an etiological agent that generates primary amoebic meningoencephalitis; unfortunately, no effective treatment or vaccine is available. The objective of this work was to determine the immunoprotective response of two vaccine antigens, as follows: (i) the polypeptide band of 19 kDa or (ii) a predicted immunogenic peptide from the membrane protein MP2CL5 (Smp145). Both antigens were administered intranasally in mice using cholera toxin (CT) as an adjuvant. The survival rate and immune response of immunized mice with both antigens and challenged with N. fowleri trophozoites were measured in the nose-associated lymphoid tissue (NALT) and nasal passages (NPs) by flow cytometry and enzyme-linked immunosorbent assay (ELISA). We also determined the immunolocalization of both antigens in N. fowleri trophozoites by confocal microscopy. Immunization with the polypeptide band of 19 kDa alone or coadministered with CT was able to confer 80% and 100% of protection, respectively. The immunization with both antigens (alone or coadministered with CT) showed an increase in T and B lymphocytes. In addition, there was an increase in the expression of integrin α4ß1 and IgA in the nasal cavity of protected mice, and the IgA, IgG, and IgM levels were increased in serum and nasal washes. The immunolocalization of both antigens in N. fowleri trophozoites was observed in the plasma membrane, specifically in pseudopod-like structures. The MP2CL5 antigens evaluated in this work were capable of conferring protection which would lead us to consider them as potential candidates for vaccines against meningitis caused by N. fowleri.