Substrate profiling of human transglutaminase 1 using cDNA display and next-generation sequencing.

Human transglutaminase 1 (TG1) modulates skin development, while its involvement in diseases remains poorly understood, necessitating comprehensive exploration of its substrate interactions. To study the substrate profile of TG1, an in vitro selection system based on cDNA display technology was used to screen two peptide libraries with mutations at varying distance from the reactive glutamine. Next-generation sequencing and bioinformatics analysis of the selected DNA pools revealed a detailed TG1 substrate profile, indicating preferred and non-preferred amino acid sequences. The peptide sequence, AEQHKLPSKWPF, was identified showing high reactivity and specificity to TG1. The position weight matrix calculated from the per amino acid enrichment factors was employed to search human proteins using an in-house algorithm, revealing six known TG1 substrate proteins with high scores, alongside a list of candidate substrates currently under investigation. Our findings are expected to assist in future medical diagnoses and development of treatments for skin disorders.

Dentinogenic ghost cell tumor treated with a combination of marsupialization and radical resection: a case report and review of the literature.

BACKGROUND: Dentinogenic ghost cell tumor is a rare benign tumor that accounts for less than 3% of all cases and consists of the stellate reticulum, which is made up of enamel epithelioid and basaloid cells. Although DGCT is a benign tumor, the local infiltration of the odontogenic epithelium or recurrences have been reported, and its detailed pathology and treatments remain unclear. CASE PRESENTATION: This report describes the case of a 60-year-old Japanese male diagnosed with a maxillary dentinogenic ghost cell tumor. Images showed well-circumscribed, multilocular cystic lesions with a calcified substance in the interior. Marsupialization was performed along with biopsy to prevent the expansion of the lesion, and a partial maxillectomy was performed 2 years after the initial examination. Histopathological findings showed ameloblastomatous proliferation containing clusters of ghost cells and dentinoid materials, resulting in the diagnosis of dentinogenic ghost cell tumor. This article also reviews recently reported cases of dentinogenic ghost cell tumor. CONCLUSION: It is important to perform marsupialization, proper resection, and postoperative follow-up because of possible recurrence.

A novel agonist with homobivalent single-domain antibodies that bind the FGF receptor 1 domain III functions as an FGF2 ligand.

Fibroblast growth factor (FGF) is a multifunctional protein that exhibits a wide range of biological effects. Most commonly, it acts as a mitogen, but it also has regulatory, morphological, and endocrine effects. The four receptor subtypes of FGF are activated by more than 20 different FGF ligands. FGF2, one of the FGF ligands, is an essential factor for cell culture in stem cells for regenerative medicine; however, recombinant FGF2 is extremely unstable. Here, we successfully generated homobivalent agonistic single-domain antibodies (variable domain of heavy chain of heavy chain antibodies referred to as VHHs) that bind to domain III and induce activation of the FGF receptor 1 and thus transduce intracellular signaling. This agonistic VHH has similar biological activity (EC50) as the natural FGF2 ligand. Furthermore, we determined that the agonistic VHH could support the proliferation of human-induced pluripotent stem cells (PSCs) and human mesenchymal stem cells, which are PSCs for regenerative medicine. In addition, the agonistic VHH could maintain the ability of mesenchymal stem cells to differentiate into adipocytes or osteocytes, indicating that it could maintain the properties of PSCs. These results suggest that the VHH agonist may function as an FGF2 mimetic in cell preparation of stem cells for regenerative medicine with better cost effectiveness.

Comprehensive analysis of transglutaminase substrate preference by cDNA display coupled with next-generation sequencing and bioinformatics.

cDNA display is an in vitro display technology based on a covalent linkage between a protein and its corresponding mRNA/cDNA, widely used for the selection of proteins and peptides from large libraries (1012) in a high throughput manner, based on their binding affinity. Here, we developed a platform using cDNA display and next-generation sequencing (NGS) for rapid and comprehensive substrate profiling of transglutaminase 2 (TG2), an enzyme crosslinking glutamine and lysine residues in proteins. After screening and selection of the control peptide library randomized at the reactive glutamine, a combinatorial library of displayed peptides randomized at positions - 1, + 1, + 2, and + 3 from the reactive glutamine was screened followed by NGS and bioinformatic analysis, which indicated a strong preference of TG2 towards peptides with glutamine at position - 1 (Gln-Gln motif), and isoleucine or valine at position + 3. The highly enriched peptides indeed contained the indicated sequence and showed a higher reactivity as TG2 substrates than the peptide previously selected by phage display, thus representing the novel candidate peptide probes for TG2 research. Furthermore, the obtained information on substrate profiling can be used to identify potential TG2 protein targets. This platform will be further used for the substrate profiling of other TG isozymes, as well as for the selection and evolution of larger biomolecules.

Construction of a Humanized Artificial VHH Library Reproducing Structural Features of Camelid VHHs for Therapeutics.

A variable domain of heavy chain antibody (VHH) has different binding properties than conventional antibodies. Conventional antibodies prefer binding to the convex portion of the antigen, whereas VHHs prefer epitopes, such as crevices and clefts on the antigen. Therefore, developing candidates with the binding characteristics of camelid VHHs is important. Thus, To this end, a synthetic VHH library that reproduces the structural properties of camelid VHHs was constructed. First, the characteristics of VHHs were classified according to the paratope formation based on crystal structure analyses of the complex structures of VHHs and antigens. Then, we classified 330 complementarity-determining region 3 (CDR3) structures of VHHs from the Protein Data Bank (PDB) into three loop structures: Upright, Half-Roll, and Roll. Moreover, these structures depended on the number of amino acid residues within CDR3. Furthermore, in the Upright loops, several amino acid residues in the FR2 are involved in the paratope formation, along with CDR3, suggesting that the FR2 design in the synthetic library is important. A humanized synthetic VHH library, comprising two sub-libraries, Upright and Roll, was constructed and named PharmaLogical. A validation study confirmed that our PharmaLogical library reproduces VHHs with the characteristics of the paratope formation of the camelid VHHs, and shows good performance in VHH screening.

Nasal delivery of single-domain antibody improves symptoms of SARS-CoV-2 infection in an animal model.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes the disease COVID-19 can lead to serious symptoms, such as severe pneumonia, in the elderly and those with underlying medical conditions. While vaccines are now available, they do not work for everyone and therapeutic drugs are still needed, particularly for treating life-threatening conditions. Here, we showed nasal delivery of a new, unmodified camelid single-domain antibody (VHH), termed K-874A, effectively inhibited SARS-CoV-2 titers in infected lungs of Syrian hamsters without causing weight loss and cytokine induction. In vitro studies demonstrated that K-874A neutralized SARS-CoV-2 in both VeroE6/TMPRSS2 and human lung-derived alveolar organoid cells. Unlike other drug candidates, K-874A blocks viral membrane fusion rather than viral attachment. Cryo-electron microscopy revealed K-874A bound between the receptor binding domain and N-terminal domain of the virus S protein. Further, infected cells treated with K-874A produced fewer virus progeny that were less infective. We propose that direct administration of K-874A to the lung could be a new treatment for preventing the reinfection of amplified virus in COVID-19 patients.

Interleukin-17A Peptide Aptamers with an Unexpected Binding Moiety Selected by cDNA Display under Heterogenous Conditions.

Peptide-based drugs are an attractive new modality of therapeutics, and in vitro selection from a large-scale library is a powerful way to identify new lead sequences. In conventional screenings, peptide specificity and stability in physiological heterogenous environments are not evaluated, which sometimes makes subsequent optimization difficult. Here we show that selection using a cDNA display system can be performed in a high percentage of serum and that this might be an option to select molecules with high potency and stability in a biological context. Specifically, we chose interleukin-17A as a target protein and performed in vitro selection of cyclic peptide aptamers from a library of approximately 1012 members in the presence of serum. The selected molecules had nanomolar affinity to the target and were stable in serum. Interestingly, we found that a component of the DNA linker that connected the peptide and cDNA may play a pivotal role in target binding.

cDNA Display-Mediated Immuno-PCR (cD-IPCR): An Ultrasensitive Immunoassay for Biomolecular Detection.

Immuno-PCR (IPCR) is a sensitive antigen detection by means of specific antibody-DNA conjugates. To ensure the successful conjugation of a protein (an antibody) with a reporter DNA, immuno-PCR method based on cDNA display (cD-IPCR) has been introduced. The cDNA display molecule is a 1:1 covalent complex of a polypeptide and its encoding cDNA at the single molecule level, which is directly used for antigen detection and subsequent qPCR. This method can be applied to detect various antigens in biological samples, if sequences of their single-domain antibodies (VHHs) or peptide aptamers are known.

Photocrosslinking of cDNA Display Molecules with Their Target Proteins as a New Strategy for Peptide Selection.

Binding peptides for given target molecules are often selected in vitro during drug discovery and chemical biology research. Among several display technologies for this purpose, complementary DNA (cDNA) display (a covalent complex of a peptide and its encoding cDNA linked via a specially designed puromycin-conjugated DNA) is unique in terms of library size, chemical stability, and flexibility of modification. However, selection of cDNA display libraries often suffers from false positives derived from non-specific binding. Although rigorous washing is a straightforward solution, this also leads to the loss of specific binders with moderate affinity because the interaction is non-covalent. To address this issue, herein, we propose a method to covalently link cDNA display molecules with their target proteins using light irradiation. We designed a new puromycin DNA linker that contains a photocrosslinking nucleic acid and prepared cDNA display molecules using the linker. Target proteins were also labeled with a short single-stranded DNA that should transiently hybridize with the linker. Upon ultraviolet (UV) light irradiation, cDNA display molecules encoding correct peptide aptamers made stable crosslinked products with the target proteins in solution, while display molecules encoding control peptides did not. Although further optimization and improvement is necessary, the results pave the way for efficient selection of peptide aptamers in multimolecular crowding biosystems.

In Vitro Construction of Large-scale DNA Libraries from Fragments Containing Random Regions using Deoxyinosine-containing Oligonucleotides and Endonuclease V.

Efficient and precise construction of DNA libraries is a fundamental starting point for directed evolution of polypeptides. Recently, several in vitro selection methods have been reported that do not rely on cells for protein expression, where peptide libraries in the order of 1013 species are used for in vitro affinity selection. To maximize their potential, simple yet versatile construction of DNA libraries from several fragments containing random regions without bacterial transformation is essential. To address this issue, we herein propose a novel DNA construction methodology based on the use of polymerase chain reaction (PCR) primers containing a single deoxyinosine (I) residue near their 5' end. Treatment of the PCR products with endonuclease V generates 3' overhangs with customized lengths and sequences, which can be ligated accurately and efficiently with other fragments having exactly complementary overhangs. As a proof of concept, we constructed an artificial gene library of single-domain antibodies from four DNA fragments.

cDNA Display: A Stable and Simple Genotype-Phenotype Coupling Using a Cell-Free Translation System.

A cDNA display method was developed based on the mRNA display method to increase its stability and efficiency for the directed evolution of various kinds of peptides and proteins. In this method, the puromycin-linker is a key molecule to realize smart genotype-phenotype coupling. A recently improved puromycin-linker and its use were explained in detail for the in vitro selection of peptides and proteins using the cDNA display method.

In vitro selection of anti-gliadin single-domain antibodies from a naïve library for cDNA-display mediated immuno-PCR.

Gluten intolerance, or adverse intestinal reactions to gluten, is a fairly common problem among certain groups of people. Celiac disease is the most severe form of gluten intolerance, which can lead to permanent damage in the digestive system. Since lifelong avoidance of gluten is the only available treatment, development of reliable techniques to identify gluten contamination in food is important. Gliadin, a component of gluten, is known to play a major role in gluten toxicity. In this study, cDNA display method was used to select specific single-domain antibodies against toxic gliadin from an alpaca-derived naïve VHH library. The cDNA display method is a promising in vitro display technique, which uniquely converts an unstable mRNA-protein fusion molecule to a stable mRNA/cDNA-protein fusion molecule using a well-designed puromycin linker. Three candidate VHHs were selected and the affinities of the VHHs were observed by pulldown assay and indirect ELISA method. In addition, a novel cDNA display mediated immuno-PCR method (cD-IPCR) was successfully applied to detect gliadin in food. We believe this work demonstrates the potential application of the cDNA display method in selecting binders against toxic and heterogeneous targets such as gliadin with an immunization-free preparation manner.

Selection of Peptides that Associate with Dye-Conjugated Solid Surfaces in a pH-Dependent Manner Using cDNA Display.

Peptides that recognize artificial materials including synthetic polymers and small molecules are drawing attention in the fields of biotechnology and chemical biology. In particular, reversible peptide aptamers that associate with the target molecules only under specific conditions are interesting. In this work, peptide aptamers that recognize a phenolphthalein derivative (PhP: a pH-sensitive organic dye) immobilized on a solid surface in a pH-dependent manner were selected using an in vitro display method (cDNA display). Considering the hydrophobic and aromatic nature of PhP, we prepared a biased DNA library (3A library) that encodes more aromatic amino acids than the standard random codon and performed seven rounds of selection from >1010 peptide species. The selected peptides including LVFLIWWM (LV59) associated with PhP-modified solid support (sepharose resin and magnetic beads) in neutral buffer but readily dissociated under basic conditions where PhP undergoes large structural change from lactone to quinoid, which is accompanied by increase of hydrophilicity and anionic charge. Control experiments suggested that LV59 recognized both phenol and lactone moieties, and the association under neutral pH is mainly driven by π-stacking and hydrophobic interaction between the peptide and PhP. Notably, however, total hydrophobicity and number of aromatic rings did not completely explain the affinity, and sequence specificity was observed to some extent. After further optimization, this interaction pair would be practically useful for protein purification.

A novel immuno-PCR method using cDNA display.

Immuno-PCR (IPCR) provides sensitive and versatile detection of a variety of antigens by conjugating a PCR-amplifiable DNA reporter to a specific antibody or an aptamer. Several methodologies have been developed to prepare appropriate DNA-antibody conjugates, but in most cases, it remains difficult to label polypeptides with high site-specificity and fixed stoichiometry. To address this issue, we first demonstrated the feasibility of IPCR based on cDNA display, a 1:1 covalent complex of a polypeptide and its encoding cDNA via puromycin at the single molecule level. Several other in vitro display technologies (e.g., ribosome display, mRNA display) have similar simple nucleic acid-peptide linkage. However, they should be unsuitable for diagnostic applications because of their lability against heat and RNase. The newly developed system here, termed cDNA display mediated immuno-PCR (cD-IPCR), proved to work in direct- and sandwich-type detection of target proteins. Detection of a target in serum was also possible, using a VHH (variable domain of the heavy chain of a heavy chain antibody) antibody as a binding molecule. Although further improvement on sensitivity and quantitativity is necessary before the method becomes useful, we believe this work demonstrated a potential of cD-IPCR as an alternative novel format of IPCR.

cDNA Display Mediated Immuno-PCR (cD-IPCR): A Novel PCR-based Antigen Detection Method.

Immuno-PCR (IPCR) is a powerful method in antigen detection where a PCR-amplifiable DNA reporter is conjugated to a specific antibody or an aptamer for the target molecule. In the development and application of IPCR, successful conjugation of a protein (an antibody) with a reporter DNA becomes challenging. To address this issue, we recently demonstrated the feasibility of IPCR based on cDNA display, a 1:1 covalent complex of a polypeptide and its encoding cDNA at the single molecule level. The cDNA display molecule for IPCR is generated first by transcribing the DNA that encodes the detection antibody into an mRNA by in vitro transcription. A puromycin DNA linker is then ligated to the mRNA and then in vitro translation and reverse-transcription are performed to generate the cDNA display molecule. The molecule is then directly used in antigen detection and subsequent qPCR. This method can be applied to detect various antigens in biological samples, if sequences of their single-domain antibodies (VHHs) or peptide aptamers are known.

In Vitro Selection of Single-Domain Antibody (VHH) Using cDNA Display.

Single-domain antibody (e.g., Nanobody, VHH antibody) is a promising scaffold for therapeutic and diagnostic reagents. To expand the range of target molecules, in vitro selection using cell-free display technologies such as cDNA display is useful and powerful because of their huge libraries and robust stability. We provide technical details for in vitro selection of single-domain antibodies using cDNA display.

Anti-survivin single-domain antibodies derived from an artificial library including three synthetic random regions by in vitro selection using cDNA display.

Single-domain antibodies (variable domain of the heavy chain of a heavy chain antibody; VHH) are promising reagents for therapeutics and diagnostics because of their stability, cost-effective production and material workability as a small antibody. Currently, general acquisition of a VHH using immunization of camelids is inconvenient from the standpoint of animal protection, cost and the process is time-consuming. Thus, a straightforward and efficient method for screening VHHs against a target molecule is required. In this study, we examined whether in vitro selection of a VHH against a target protein could be performed by a cDNA display method with an artificial VHH library that had the three complementarity-determining regions (CDRs) randomized by chemical synthesis. The results revealed that a particular VHH against survivin, which is a member of the inhibitor of apoptosis family, was selected with affinity in the range of 10-7 to 10-8 M. The in vitro selection of a VHH using cDNA display with an artificial synthesized library without animal immunization was shown to be effective for rapid and inexpensive screening of VHHs against a target protein.

Iodoacetyl-functionalized pullulan: A supplemental enhancer for single-domain antibody-polyclonal antibody sandwich enzyme-linked immunosorbent assay for detection of survivin.

Survivin, an inhibitor of the apoptosis protein family, is a potent tumor marker for diagnosis and prognosis. The enzyme-linked immunosorbent assay (ELISA) is one of the methods that has been used for detection of survivin. However, ELISA has several disadvantages caused by the use of conventional antibodies, and we have therefore been trying to develop a novel ELISA system using camelid single-domain antibodies (VHHs) as advantageous replacements. Here we report a supplemental approach to improve the VHH-polyclonal antibody sandwich ELISA for survivin detection. Iodoacetyl-functionalized pullulan was synthesized, and its thiol reactivity was characterized by a model reaction with l-cysteine. The thiophilic pullulan was applied to an immunoassay asan additive upon coating of standard assay plates with an anti-survivin VHH fusion protein with C-terminal cysteine. The results showed that the mole ratio of the additive to VHH had a significant effect on the consequent response. Mole ratios of 0.07, 0.7, and 7 led to 90% lower, 15% higher, and 69% lower responses, respectively, than the response of a positive control in which no additive was used. The background levels observed in any additive conditions were as low as that of a negative control lacking both VHH and the additive. These results indicate the applicability of the thiol-reactive pullulan as a response enhancer to VHH-based ELISA.

In vitro selection of random peptides against artificial lipid bilayers: a potential tool to immobilize molecules on membranes.

The first in vitro selection of binding peptides against artificial lipid membranes from a random peptide library using an in vitro display method (cDNA display) is reported. The selected peptide, LB-1, has both amphiphilic and cationic regions, and proteins fused to LB-1 can be immobilized on the liposome surface.

Enhanced mRNA-protein fusion efficiency of a single-domain antibody by selection of mRNA display with additional random sequences in the terminal translated regions.

In vitro display technologies such as mRNA and cDNA display are powerful tools to create and select functional peptides. However, in some cases, efficiency of mRNA-protein fusion is very low, which results in decreased library size and lower chance of successful selection. In this study, to improve mRNA-protein fusion efficiency, we prepared an mRNA display library of a protein with random N- and C-terminal coding regions consisting of 12 nucleotides (i.e. four amino acids), and performed an electrophoresis mobility shift assay (EMSA)-based selection of successfully formed mRNA display molecules. A single-domain antibody (Nanobody, or VHH) was used as a model protein, and as a result, a pair of sequences was identified that increased mRNA-protein fusion efficiency of this protein by approximately 20%. Interestingly, enhancement of the fusion efficiency induced by the identified sequences was protein-specific, and different results were obtained for other proteins including VHHs with different CDRs. The results suggested that conformation of mRNA as a whole, rather than the amino acid sequence of the translated peptide, is an important factor to determine mRNA-protein fusion efficiency.