Isopeptide Bond Formation Research Articles

A SARS-CoV-2 peptide antigen purified from bacteria and displayed in a high-density repetitive manner on a virus-like particle could generate anti-SARS-CoV-2 neutralizing antibodies unlike free peptide

SARS-CoV-2 is an enveloped virus. Proteins of the lipid based envelope are not rigidly arranged as compared to non-enveloped viruses lacking lipid based outer layer. Rigidly arranged multivalent display has been reported to be important for potent immune stimulation. We assembled himeric virus-like-particle (VLP) where the core of the particle is composed of the coat protein of Acinetobacter phage. Peptide-based antigen from receptor binding domain (RBD) of SARS-CoV-2 spike protein has been displayed on the coat based VLP matrix using spy-tag/spy-catcher split inteine conjugation system that allows spontaneous, irreversible isopeptide bond formation. Both the matrix as well as peptide have been purified from bacteria which is an easy platform for protein purification. We used the closed state of spike trimer for epitope mapping as this is the conformation of the spike that the immune system visualizes unlike the open state that appears when the virus tries to attach to receptor. Mice based immunization studies were used to test immunogenicity of the antigens. The work demonstrates that peptide-based antigens displayed in high densities can induce neutralizing antibody production unlike free peptide. Careful choice of peptides can deliver better candidates. Also, small size allows easy improvisation. Production in bacteria offers cheaper and robust purification option.

Atomic Tailoring of Ubiquitin Side Chains Influences E2‐Mediated Ubiquitin Chain Formation

AbstractUbiquitin (Ub) is a small, highly conserved protein essential for eukaryotic biology, and is unique in its formation of polyubiquitin chains by conjugation to one of its seven lysine side chains. Here we report that atomic tailoring of Ub side chains – i. e. the insertion, deletion, or replacement of specific atoms – has significant and unexpected consequences on the enzymatic conjugation of Ub oligomers by isopeptide bond formation mediated by E2 conjugating enzymes. These studies employed chemical synthesis and ligation methods to prepare numerous specifically tailored Ub monomers on multi‐milligram scales. While some modifications including N‐terminal acylation and methionine replacement did not affect protein folding or Ub chain formation with Ube2 K, other modifications had a pronounced effect of oligomerization with Ubc13/Mms2. We observed that Ala46Hse mutation obliterates the ability of this Ub monomer to accept another Ub at Lys63 in Ubc13‐mediated conjugations. Exhaustive replacement of all seven lysines with shorter surrogates Orn, Dab, or Dap essentially blocks Ub chain formation, and in the case of Dap, precludes proper folding of the Ub protein.

Effect of transglutaminase and laccase on pea protein gel properties compared to that of soybean

Enzyme cross-linking is a pivotal method for enhancing the functionality of plant proteins, offering improvements in texture, stability, and nutritional value, which are essential for food applications. Pea protein is increasingly used as it is not identified as allergenic and has fewer other negative health concerns. However, the lower gelling capacity compared with soybean protein has limited its application in plant-based meat or protein-based products. Thus, this study focused on investigating the effects of transglutaminase (TG) and laccase (LAC) and their concentrations on pea protein isolate (PPI) gelation, towards comparison with that of soybean protein isolate (SPI). Results showed that TG and LAC both contribute to the formation of PPI gels while exhibiting different action ways. As the TG content increased, the particle size of PPI significantly increased from 97.27 ± 4.61 μm to 146.67 ± 9.87 μm at 0.8% TG concentration, whereas the particle size remained constant with increasing LAC content. Both TG and LAC significantly enhanced the PPI gel properties, with 0.4% TG addition resulting in gel strength comparable to SPI and 0.8% TG content increasing PPI gel strength by approximately 4.2 times compared to the control, surpassing SPI. Moreover, PPI with TG treated demonstrated superior effects compared to LAC-treated gels at the same concentration. Water retention of gels increased from 89.37 ± 1.97% to 94.90 ± 3.86% with TG concentration increasing, while it decreased with LAC increasing. SDS-PAGE results revealed that TG promoted the formation of larger aggregates in PPI, indicating that legumin and vicilin were the main target sites for TG, while LAC was inapparent on molecular weight. The viscosity of PPI increased from 36.91 ± 2.32 Pa·s to 70.57 ± 1.00 Pa·s with 0.8% TG, while 0.8% LAC increased viscosity to 61.79 ± 1.32 Pa·s. Hydrogen bonds and disulfide bonds play a crucial role in the gel formation process facilitated by TG and LAC. Besides, TG is also expected to contribute through the formation of isopeptide bonds. These findings validated the potential of TG and LAC in improving the quality of PPI gels and provided insights for the application and development of cross-linking enzymes in the protein processing industry.

Techno-Functions and Safety Concerns of Plant-Based Peptides in Food Matrices.

Plant-based peptides (PBPs) benefit functional food development and environmental sustainability. Proteolysis remains the primary method of peptide production because it is a mild and nontoxic technique. However, potential safety concerns still emanate from toxic or allergenic sequences, amino acid racemization, iso-peptide bond formation, Maillard reaction, dose usage, and frequency. The main aim of this review is to investigate the techno-functions of PBPs in food matrices, as well as their safety concerns. The distinctive characteristics of PBPs exhibit their techno-functions for improving food quality and functionality by contributing to several crucial food formulations and processing. The techno-functions of PBPs include solubility, hydrophobicity, bitterness, foaming, oil-binding, and water-holding capacities, which subsequently affect food matrices. The safety and quality of foodstuff containing PBPs depend on the proper source of plant proteins, the selection of processing approaches, and compliance with legal regulations for allergen labeling and safety evaluations. The safety concerns in allergenicity and toxicity were discussed. The conclusion is that food technologists must apply safe limits and consider potential allergenic components generated during the development of food products with PBPs. Therefore, functional food products containing PBPs can be a promising strategy to provide consumers with wholesome health benefits.

Novel soft food gels using beta-lactoglobulin via enzymatic crosslinking as agar gel alternatives

Soft food gels are of high interest for food innovation that can cater to the specific needs of various populations. This study explores the innovative development of soft beta-lactoglobulin (β-LG) gels through a green process that combines heat treatment and transglutaminase (TGase)-catalyzed crosslinking at a moderate pH. The optimal procedure involves heating β-LG solutions, pH 7.5 at 80 °C for 30 min to facilitate protein unfolding and aggregation, followed by TGase catalysis at 50 U TGase/g β-LG. This process produced soft and moist β-LG gels at a low concentration of 5% (w/v). The softness of β-LG gels was characterized through rheological measurements, suggesting a low storage modulus and high frequency dependence. Morphological observations of β-LG gels using TEM showed a loosely arranged network. SDS-PAGE and FTIR analyses indicated the formation of covalent disulfide bonds and isopeptide bonds in β-LG gels through the heat and TGase treatments. The potential of the novel β-LG gels as traditional agar gel substitutes was comparatively analyzed on their textural properties. Both gels showed distinctive jelly-like characteristics, whereas β-LG gels exhibited superior softness and reduced brittleness, positioning them as viable protein-based alternatives to agar gels for various applications in both food and nonfood sectors.

A new preparation method of covalent annular nanodiscs based on MTGase

The preservation of the native conformation and functionality of membrane proteins has posed considerable challenges. While detergents and liposome reconstitution have been traditional approaches, nanodiscs (NDs) offer a promising solution by embedding membrane proteins in phospholipids encircled by an amphipathic helical protein MSP belt. Nevertheless, a drawback of commonly used NDs is their limited homogeneity and stability. In this study, we present a novel approach to construct covalent annular nanodiscs (cNDs) by leveraging microbial transglutaminase (MTGase) to catalyze isopeptide bond formation between the side chains of terminal amino acids, specifically Lysine (K) and Glutamine (Q). This methodology significantly enhances the homogeneity and stability of NDs. Characterization of cNDs and the assembly of membrane proteins within them validate the successful reconstitution of membrane proteins with improved homogeneity and stability. Our findings suggest that cNDs represent a more suitable tool for investigating interactions between membrane proteins and lipids, as well as for analyzing membrane protein structures.

In Situ Transglutaminase Cross-Linking Improves Mechanical Properties of Self-Assembling Peptides for Biomedical Applications.

The development of three-dimensional (3D) biomaterials that mimic natural tissues is required for efficiently restoring physiological functions of injured tissues and organs. In the field of soft hydrogels, self-assembled peptides (SAPs) stand out as distinctive biomimetic scaffolds, offering tunable properties. They have garnered significant attention in nanomedicine due to their innate ability to self-assemble, resulting in the creation of fibrous nanostructures that closely mimic the microenvironment of the extracellular matrix (ECM). This unique feature ensures their biocompatibility and bioactivity, making them a compelling area of study over the past few decades. As they are soft hydrogels, approaches are necessary to enhance the stiffness and resilience of the SAP materials. This work shows an enzymatic strategy to selectively increase the stiffness and resiliency of functionalized SAPs using transglutaminase (TGase) type 2, an enzyme capable of triggering the formation of isopeptide bonds. To this aim, we synthesized a set of SAP sequences and characterized their cross-linking via rheological experiments, atomic force microscopy (AFM), thioflavin-T binding assay, and infrared spectroscopy (ATR-FTIR) tests. The results showed an improvement of the storage modulus of cross-linked SAPs at no cost of the maximum stress-at-failure. Further, in in vitro tests, we examined and validated the TGase capability to cross-link SAPs without hampering seeded neural stem cells (hNSCs) viability and differentiation, potentially leaving the door open for safe in situ cross-linking reactions in vivo.

Isopeptide bond formation mediated by δ-selenolysine for chemical ubiquitination

Isopeptide bond formation mediated by δ-selenolysine for chemical ubiquitination

Catalysis of non-canonical protein ubiquitylation by the ARIH1 ubiquitin ligase.

Protein ubiquitylation typically involves isopeptide bond formation between the C-terminus of ubiquitin to the side-chain amino group on Lys residues. However, several ubiquitin ligases (E3s) have recently been identified that ubiquitylate proteins on non-Lys residues. For instance, HOIL-1 belongs to the RING-in-between RING (RBR) class of E3s and has an established role in Ser ubiquitylation. Given the homology between HOIL-1 and ARIH1, an RBR E3 that functions with the large superfamily of cullin-RING E3 ligases (CRLs), a biochemical investigation was undertaken, showing ARIH1 catalyzes Ser ubiquitylation to CRL-bound substrates. However, the efficiency of ubiquitylation was exquisitely dependent on the location and chemical environment of the Ser residue within the primary structure of the substrate. Comprehensive mutagenesis of the ARIH1 Rcat domain identified residues whose mutation severely impacted both oxyester and isopeptide bond formation at the preferred site for Ser ubiquitylation while only modestly affecting Lys ubiquitylation at the physiological site. The results reveal dual isopeptide and oxyester protein ubiquitylation activities of ARIH1 and set the stage for physiological investigations into this function of emerging importance.

A novel cell-permeable peptide prevents protein SUMOylation and supports the mislocalization and aggregation of TDP-43

SUMOylation is a post-translational modification (PTM) that exerts a regulatory role in different cellular processes, including protein localization, aggregation, and biological activities.It consists of the dynamic formation of covalent isopeptide bonds between a family member of the Small Ubiquitin Like Modifiers (SUMOs) and the target proteins. Interestingly, it is a cellular mechanism implicated in several neurodegenerative pathologies and potentially it could become a new therapeutic target; however, there are very few pharmacological tools to modulate the SUMOylation process.In this study, we have designed and tested the activity of a novel small cell-permeable peptide, COV-1, in a neuroblastoma cell line that specifically prevents protein SUMOylation.COV-1 inhibits UBC9-protein target interaction and efficiently decreases global SUMO-1ylation. Moreover, it can perturb RanGAP-1 perinuclear localization by inducing the downregulation of UBC9. In parallel, we found that COV-1 causes an increase in the ubiquitin degradation system up to its engulfment while enhancing the autophagic flux.Surprisingly, COV-1 modifies protein aggregation, and specifically it mislocalizes TDP-43 within cells, inducing its aggregation and co-localization with SUMO-1.These data suggest that COV-1 could be taken into future consideration as an interesting pharmacological tool to study the cellular cascade effects of SUMOylation prevention.

Visible Light-Induced Specific Protein Reaction Delineates Early Stages of Cell Adhesion.

Light is well-established for control of bond breakage but not for control of specific bond formation in complex environments. We previously engineered the diffusion-limited reactivity of the SpyTag003 peptide with its protein partner SpyCatcher003 through spontaneous isopeptide bond formation. This system enables precise and irreversible assembly of biological building blocks with applications from biomaterials to vaccines. Here we establish a system for the rapid control of this amide bond formation with visible light. We have generated a caged SpyCatcher003, which allows light triggering of covalent bond formation to SpyTag003 in mammalian cells. Photocaging is achieved through site-specific incorporation of an unnatural coumarin-lysine at the reactive site of SpyCatcher003. We showed a uniform specific reaction in cell lysate upon light activation. We then used the spatiotemporal precision of a 405 nm confocal laser for uncaging in seconds, probing the earliest events in mechanotransduction by talin, the key force sensor between the cytoskeleton and the extracellular matrix. Reconstituting talin induced rapid biphasic extension of lamellipodia, revealing the kinetics of talin-regulated cell spreading and polarization. Thereafter we determined the hierarchy of the recruitment of key components for cell adhesion. Precise control over site-specific protein reaction with visible light creates diverse opportunities for cell biology and nanoassembly.

Investigation of the enhancement mechanism of ethanol addition on the gel performance of heat-induced surimi

This study investigated the mechanism of ethanol enhancing the gel properties of heat-induced silver carp (Hypophthalmichthys molitrix) surimi gels. The presence of ethanol reduced the thermal stability of surimi protein, and the denaturation temperature of myosin decreased by approximately 7 °C in the presence of 5% ethanol. The gel strength, textural characteristics, and storage modulus (G′) of the gels improved depending on ethanol levels, while the water holding capacity (WHC) remained steady. Adding ethanol stabilized the α-helix and β-sheet in the protein structures of the gels, reducing the content of the random coil. Appropriate heat denaturation rate induced by ethanol increased protein cross-linking, which may relate to forming more disulfide bonds. In addition, ethanol incorporation promoted the formation of more isopeptide bonds catalyzed by transglutaminase (TGase) and weakened the hydrolysis of surimi proteins by cathepsins, further enhancing the formation of a dense and uniform gel network.

The basal and major pilins in the Corynebacterium diphtheriae SpaA pilus adopt similar structures that competitively react with the pilin polymerase.

Many species of pathogenic gram-positive bacteria display covalently crosslinked protein polymers (called pili or fimbriae) that mediate microbial adhesion to host tissues. These structures are assembled by pilus-specific sortase enzymes that join the pilin components together via lysine-isopeptide bonds. The archetypal SpaA pilus from Corynebacterium diphtheriae is built by the Cd SrtA pilus-specific sortase, which crosslinks lysine residues within the SpaA and SpaB pilins to build the shaft and base of the pilus, respectively. Here, we show that Cd SrtA crosslinks SpaB to SpaA via a K139(SpaB)-T494(SpaA) lysine-isopeptide bond. Despite sharing only limited sequence homology, an NMR structure of SpaB reveals striking similarities with the N-terminal domain of SpaA (N SpaA) that is also crosslinked by Cd SrtA. In particular, both pilins contain similarly positioned reactive lysine residues and adjacent disordered AB loops that are predicted to be involved in the recently proposed "latch" mechanism of isopeptide bond formation. Competition experiments using an inactive SpaB variant and additional NMR studies suggest that SpaB terminates SpaA polymerization by outcompeting N SpaA for access to a shared thioester enzyme-substrate reaction intermediate.

Development of peptide-based biosensors for detecting cross-linking and deamidation activities of transglutaminases.

Transglutaminases (TGs) are a protein family that catalyzes isopeptide bond formation between glutamine and lysine residues of various proteins. There are eight TG isozymes in humans, and each is involved in diverse biological phenomena due to their characteristic distribution. Abnormal activity of TG1 and TG2, which are major TG isozymes, is believed to cause various diseases, such as ichthyosis and celiac disease. To elucidate TGs' mechanisms of action and develop new therapeutic strategies, it is essential to develop bioprobes that can specifically examine the activity of each TG isozyme, which has not been sufficiently studied. We previously have identified several substrate peptide sequences containing Gln residues for each isozyme and developed a method to detect isozyme-specific activities by incorporating a labeled substrate peptide into lysine residues of proteins. We prepared the fluorescein isothiocyanate (FITC)-labeled Gln substrate peptide (FITC-K5 and FITC-T26) and Rhodamine B-labeled Lys substrate peptide (RhoB-Kpep). Each TG reaction specifically cross-linked these probe pairs, and the proximity of FITC and Rhodamine B significantly decreased the fluorescence intensity of FITC depending on the concentration and reaction time of each TG. In this study, we developed a peptide-based biosensor that quickly and easily measures TG isozyme-specific activity. This probe is expected to be helpful in elucidating TG's physiological and pathological functions and in developing compounds that modulate TG activity.

Enzymatic cross-linking of pea and whey proteins to enhance emulsifying and encapsulation properties

Improving the functionality of commercial plant proteins is essential for food application. Here an alternative strategy is provided to modify the structure of pea protein by microbial transglutaminase-induced cross-linking with whey protein for enhancing emulsifying and encapsulation properties. The protein structures and the physicochemical stability of β-carotene-loaded emulsions were investigated subject to protein ratio and cross-linking. Formation of disulphide and isopeptide bonds between heteroproteins according to cross-linking changed protein structures by exposing the hydrophobic sites of proteins with stiffer network. The cross-linked pea/whey protein complexes at protein ratio of 2:1 formed a stable emulsion with a droplet size of 0.10 µm, showing no phase separation for 30 days of storage and high encapsulation efficiency of 92%. These findings provided a novel strategy to design plant/dairy protein networks to protect lipophilic bioactive compounds by replacing half or more dairy protein with plant protein, leading to gradual change towards plant-based diets.

Secondary structure characterization of mixed food protein complexes using microfluidic modulation spectroscopy (MMS)

Microfluidic modulation spectroscopy (MMS) has been recognised as a novel technology for producing high quality and reproducible data at a wide concentration range to characterise protein structure. In this study, the use of MMS for food applications was explored through the structural analysis of pea protein, whey protein, and pea/whey protein complexes prepared by transglutaminase cross-linking. MMS generated reproducible data with >99.5% repeatability in all protein samples. The cross-linking seemed to alter the structure of whey protein with isopeptide and hydrogen bond formation, more significantly than that of pea protein. The larger secondary and tertiary structural changes were observed with higher whey protein content in cross-linked pea/whey protein complex, which eventually increased the surface hydrophobicity. According to the second derivatives analysis by MMS, β-sheet and random coil increased and β-turn decreased in the pea protein, whereas α-helix, β-sheet, and β-turn decreased and random coil increased in the whey protein after cross-linking. In the pea/whey protein complex, β-turn shifted to β-sheet due to cross-linking. There are some discrepancies between the relative secondary structure contents obtained from MMS data compared to the data acquired from circular dichroism. However, MMS has the considerable advantage of being able to directly measure original samples without significant dilution as required for circular dichroism measurements. This study provides evidence that MMS technology could be a robust technique to quantify the secondary structures of typical proteins in food with high reliability.

Extracellular matrix-inspired hydrogel of hyaluronan and gelatin crosslinked via a Link module with a transglutaminase reactive sequence

The extracellular matrix (ECM) is a natural scaffold of cells in the body. It has a complex structure comprising various proteins, such as collagen and hyaladherins, and polysaccharides such as hyaluronan (HA). Here, inspired by the crosslinked ECM structure, we design a genetically engineered Link module—LinkCFQ—by fusing a microbial transglutaminase (MTG)-reactive tag to the Link module, an HA-binding domain of tumor necrosis factor-stimulated gene-6. Although the HA-specific binding property of the Link module is preserved, LinkCFQ demonstrates excellent MTG reactivity with various proteins. Furthermore, an ECM-inspired hydrogel is fabricated from an HA–gelatin mixture crosslinked via HA/Link module interaction and MTG-catalyzed isopeptide bond formation in LinkCFQ. Cell culture and mouse experiments confirm the hydrogel’s biocompatibility and degradability. Our findings provide insights into the design of biomaterials and proteins for tissue engineering, regenerative medicine, drug discovery and delivery, disease models, biofabrication, and medical devices.

Unaided efficient transglutaminase cross-linking of whey proteins strongly impacts the formation and structure of protein alginate particles

There is a dogma within whey protein modification, which dictates the necessity of pretreatment to enzymatic cross-linking of β-lactoglobulin (β-Lg). Here microbial transglutaminase (MTG) cross-linked whey proteins and β-Lg effectively in 50 mM NaHCO3, pH 8.5, without pretreatment. Cross-linked β-Lg spanned 18 to >240 kDa, where 6 of 9 glutamines reacted with 8 of 15 lysines. The initial isopeptide bond formation caused loss of β-Lg native structure with t1/2 = 3 h, while the polymerization occurred with t1/2 = 10 h. Further, cross-linking effects on protein carbohydrate interaction have been overlooked, leaving a gap in understanding of these complex food matrices. Complexation with alginate showed that β-Lg cross-linking decreased onset of particle formation, hydrodynamic diameter, stoichiometry (β-Lg/alginate) and dissociation constant. The complexation was favored at higher temperatures (40 °C), suggesting that hydrophobic interactions were important. Thus, β-Lg was cross-linked without pretreatment and the resulting polymers gave rise to altered complexation with alginate.

Performance and Structure Evaluation of Gln-Lys Isopeptide Bond Crosslinked USYK-SPI Bioplastic Film Derived from Discarded Yak Hair.

To reduce the waste from yak hair and introduce resource recycling into the yak-related industry, an eco-friendly yak keratin-based bioplastic film was developed. We employed yak keratin (USYK) from yak hair, soy protein isolate (SPI) from soybean meal as a film-forming agent, transglutaminase (EC 2.3.2.13, TGase) as a catalytic crosslinker, and glycerol as a plasticizer for USYK-SPI bioplastic film production. The structures of the USYK-SPI bioplastic film were characterized by scanning electron microscopy (SEM), differential scanning calorimetry (DSC), and X-Ray diffraction (XRD). The mechanical properties, the thermal behavior, light transmittance performance, and water vapor permeability (WVP) were measured. The results revealed that the added SPI possibly acted as a reinforcement. The formation of Gln-Lys isopeptide bonds and hydrophobic interactions led to a stable crosslinking structure of USYK-SPI bioplastic film. The thermal and the mechanical behaviors of the USYK-SPI bioplastic film were improved. The enhanced dispersion and formation of co-continuous protein matrices possibly produced denser networks that limited the diffusion of water vapor and volatile compounds in the USYK-SPI bioplastic films. Moreover, the introduction of SPI prompted the relocation of hydrophobic groups on USYK molecules, which gave the USYK-SPI bioplastic film stronger surface hydrophobicity. The SPI and USYK molecules possess aromatic amino residuals (tyrosine, phenylalanine, tryptophan), which can absorb ultraviolet radiation. Thus, the USYK-SPI bioplastic films were shown to have an excellent UV barrier. The synergy effect between USYK and SPI is not only able to improve rigidity and the application performance of keratin-based composite film but can also reduce the cost of the keratin-based composite film through the low-cost of the SPI alternative which partially replaces the high-cost of keratin. The data obtained from this research can provide basic information for further research and practical applications of USYK-SPI bioplastic films. There is an increasing demand for the novel USYK-SPI bioplastic film in exploit packaging material, biomedical materials, eco-friendly wearable electronics, and humidity sensors.

Covalent Capture of a Collagen Mimetic Peptide with an Integrin-Binding Motif.

Collagen mimetic peptides (CMPs) are an excellent model to study the structural and biological properties of the extracellular matrix (ECM) due to ease of synthesis and variability in sequence. To ensure that synthetic materials accurately mimic the structure and function of natural collagen in the ECM, it is necessary to conserve the triple helix. However, CMP folding is subject to equilibrium, and frequently peptides exist in solution as both monomer and triple helix. Additionally, the stability of CMPs is highly dependent on peptide length and amino acid composition, leading to suboptimal performance. Here, we report the utility of covalent capture, a method to (a) direct the folding of a supramolecular triple helix and (b) form isopeptide bonds between the helix strands, in the design of an integrin-binding peptide with a GFOGER motif. Covalent capture effectively locked the triple helix and yielded a peptide with high thermal stability and a rapid folding rate. Compared to supramolecular triple helices bearing the same GFOGER-binding site, cell adhesion was substantially increased. In vitro assays using EDTA/Mg2+ and an anti-α2β1 antibody demonstrated the preservation of the high specificity of the binding event. This covalently captured integrin-binding peptide provides a template for the future design of bioactive ECM mimics, which can overcome limitations of supramolecular approaches for potential drug and biomaterial designs.