Isopeptide Bond Formation Research Articles

Transglutaminase-Catalyzed Bottom-Up Synthesis of Polymer Hydrogel.

Enzyme catalysis has attracted increasing attention for application in the synthesis of polymer hydrogel due to the eco-friendly process and the devisable catalytic reaction. Moreover, bottom-up approaches combining enzyme catalysts and molecular self-assembly have been explored for synthesizing hydrogel with complex architectures. An enzyme widely distributed in nature, transglutaminase (TGase) has been confirmed to catalyze the formation of isopeptide bonds between proteins, which can effectively improve the gelation of proteins. In this mini-review, TGase-catalyzed synthesis of polymer hydrogels, including fibrin hydrogels, polyethylene glycol hydrogels, soy protein hydrogels, collagen hydrogels, gelatin hydrogels and hyaluronan hydrogels, has been reviewed in detail. The catalytic process and gel formation mechanism by TGase have also been considered. Furthermore, future perspectives and challenges in the preparation of polymer hydrogels by TGase are also highlighted.

SpyTag/Catcher chemistry induces the formation of active inclusion bodies in E. coli

SpyTag/Catcher chemistry is usually applied to engineer robust enzymes via head-to-tail cyclization using spontaneous intramolecular isopeptide bond formation. However, the SpyTag/Catcher induced intercellular protein assembly in vivo cannot be ignored. It was found that some active inclusion bodies had generated to different proportions in the expression of six SpyTag/Catcher labeled proteins (CatIBs-STCProtein). Some factors that may affect the formation of CatIBs-STCProtein were discussed, and the subunit quantities were found to be strongly positively related to the formation of protein aggregates. Approximately 85.44% of the activity of the octameric protein leucine dehydrogenase (LDH) was expressed in aggregates, while the activity of the monomeric protein green fluorescence protein (GFP) in aggregates was 12.51%. The results indicated that SpyTag/Catcher can be used to form protein aggregates in E. coli. To facilitate the advantages of CatIBs-STCProtein, we took the CatIBs-STCLDH as an example and further chemically cross-linked with glutaraldehyde to obtain novel cross-linked enzyme aggregates (CLEAs-CatIBs-STCLDH). CLEAs-CatIBs-STCLDH had good thermal stability and organic solvents stability, and its activity remained 51.03% after incubation at 60 °C for 100 mins. Moreover, the crosslinked CatIBs-STCLDH also showed superior stability over traditional CLEAs, and its activity remained 98.70% after 10 cycles of catalysis.

Creation of Multimeric Single-Domain Antibodies Using Bacterial Superglues.

Multimerization of single-domain antibodies (sdAbs) is instrumental for construction of antibody molecules with high avidity, extended in vivo half-life, and tailor-made biological activity. Two-component superglues, based on bacterium-derived peptides (Tags) and small protein domains (Catchers) that form isopeptide bonds when in close proximity, enable the creation of multimers by simply mixing of the individual components. Here, we provide detailed methods for the construction of sdAbs and scaffolds bearing genetically fused superglue components and their assembly into multimeric complexes.

Influence of Transglutaminase Crosslinking on Casein Protein Fractionation during Low Temperature Microfiltration.

Low temperature microfiltration (MF) is applied in dairy processing to achieve higher protein and microbiological quality ingredients and to support ingredient innovation; however, low temperature reduces hydrophobic interactions between casein proteins and increases the solubility of colloidal calcium phosphate, promoting reversible dissociation of micellar β-casein into the serum phase, and thus into permeate, during MF. Crosslinking of casein proteins using transglutaminase was studied as an approach to reduce the permeation of casein monomers, which typically results in reduced yield of protein in the retentate fraction. Two treatments (a) 5 °C/24 h (TA) and (b) 40 °C/90 min (TB), were applied to the feed before filtration at 5 °C, with a 0.1 µm membrane. Flux was high for TA treatment possibly due to the stabilising effect of transglutaminase on casein micelles. It is likely that formation of isopeptide bonds within and on the surface of micelles results in the micelles being less readily available for protein-protein and protein–membrane interactions, resulting in less resistance to membrane pores and flow passage, thereby conferring higher permeate flux. The results also showed that permeation of casein monomers into the permeate was significantly reduced after both enzymatic treatments as compared to control feed due to the reduced molecular mobility of soluble casein, mainly β-casein, caused by transglutaminase crosslinking.

Rapid Production of Bispecific Antibodies from Off-the-Shelf IgGs with High Yield and Purity.

Bispecific antibodies (BsAb) refer to a class of biomacromolecules that are capable of binding two antigens or epitopes simultaneously. This can elicit unique biological effects that cannot be achieved with either individual antibody or two unlinked antibodies. Bispecific antibodies have been used for targeting effector cells to tumor cells, preferential targeting of cells expressing two target biomarkers over cells expressing either target biomarker individually, or to couple two molecular targets on the same cell surface to trigger unique intracellular signaling pathways. Here, we present two related methods that enable direct, rapid assembly of bispecific antibodies from any two "off-the-shelf" Immunoglobulin G (IgG) antibodies, in as little as 1 day. Both workflows can be summarized into two steps: (1) attach a small photoreactive antibody binding domain (pAbBD) fused to SpyCatcher or SpyTag (peptide-protein partners derived from the S. pyogenes fibronectin-binding protein FbaB) to each component IgG, respectively; (2) assemble the BsAb through the spontaneous isopeptide bond formation that occurs between SpyTag and SpyCatcher. These approaches enable production of BsAbs from any two IgG molecules without the need to elucidate their amino acid sequences or genetically alter their structure. Binding assays and T cell-mediated cytolysis assays were performed to validate the binding and functional properties of Trastuzumab × Cetuximab BsAb and Cetuximab × OKT3 BsAb, respectively. This approach enables rapid, low-cost production of highly homogeneous tetravalent BsAbs in a modular fashion, presenting an opportunity to quickly evaluate antibody pairs in a BsAb format for unique or synergistic functionalities.

Detection of Isopeptide Bonds in Monoclonal Antibody Aggregates

PurposeA major difficulty in monoclonal antibody (mAb) therapeutic development is product aggregation. In this study, intermolecular isopeptide bonds in mAb aggregates were characterized for the first time. We aim to propose a mechanism of covalent aggregation in a model antibody using stressed studies at raised temperatures to aid in the understanding of mAb aggregation pathways.MethodsAggregate fractions were generated using raised temperature and were purified using size-exclusion chromatography (SEC). The fractions were tryptically digested and characterized using liquid chromatography hyphenated to tandem mass-spectrometry (LC–MS/MS).ResultsAn increased amount of clipping between aspartic acid and proline in a solvent accessible loop in the constant heavy 2 (CH2) domain of the mAb was observed under these conditions. Detailed peptide mapping revealed 14 isopeptide bonds between aspartic acid at that cleavage site and lysine residues on adjacent antibodies. Two additional isopeptide bonds were identified between the mAb HC N-terminal glutamic acid or a separate aspartic acid to lysine residues on adjacent antibodies.ConclusionsInter-protein isopeptide bonds between the side chains of acidic amino acids (aspartate and glutamate) and lysine were characterized for the first time in mAb aggregates. A chemical mechanism was presented whereby spontaneous isopeptide bond formation could be facilitated via either the aspartic acid side chain or C-terminus.

Cold setting of gelatin–antioxidant peptides composite hydrogels using a new psychrophilic recombinant transglutaminase (rTGase)

This study used a new psychrophilic transglutaminase (TGase) to catalyze the formation of protein and peptide hydrogels by forming isopeptide bonds. The psychrophilic recombinant TGase (rTGase) was overexpressed in E. coli and purified using a Ni-Sepharose column followed by stepwise dialysis to recover the cold-active enzyme (specific activity of 1.63 U/mg). The rTGase catalyzed the formation of cold-set hydrogels at 4 °C, which were comprised of high molecular weight gelatin and antioxidant peptides. The antioxidant bioactivity of the cold-set gelatin–peptides composite hydrogels was significantly improved with rTGase at 4 °C for 10 days. To understand the mechanism of the psychrophilic rTGase on the enhanced bioactivity of cross-linked hydrogels, in silico molecular docking studies were performed to assess the binding of antioxidant peptides into the rTGase active pocket, as well as to analyze the interactions of Gln and Lys residues in the antioxidant peptides with amino acid residues in the rTGase catalytic site. The formation of covalent cross-linkages in the gelatin–gelatin, gelatin–peptide and peptide–peptide matrices, and possible structures of composite hydrogels formed by rTGase catalysis in cold conditions, were deduced. The new psychrophilic rTGase enabled stabilization of the antioxidant bioactivity of protein- and peptide-containing cold-set hydrogels formed at low temperatures.

Vertebrate keratinization evolved into cornification mainly due to transglutaminase and sulfhydryl oxidase activities on epidermal proteins: An immunohistochemical survey.

The epidermis of vertebrates forms an extended organ to protect and exchange gas, water, and organic molecules with aquatic and terrestrial environments. Herein, the processes of keratinization and cornification in aquatic and terrestrial vertebrates were compared using immunohistochemistry. Keratins with low cysteine and glycine contents form the main bulk of proteins in the anamniote epidermis, which undergoes keratinization. In contrast, specialized keratins rich in cysteine-glycine and keratin associated corneous proteins rich in cysteine, glycine, and tyrosine form the bulk of proteins of amniote soft cornification in the epidermis and hard cornification in scales, claws, beak, feathers, hairs, and horns. Transglutaminase (TGase) and sulfhydryl oxidase (SOXase) are the main enzymes involved in cornification. Their evolution was fundamental for the terrestrial adaptation of vertebrates. Immunohistochemistry results revealed that TGase and SOXase were low to absent in fish and amphibian epidermis, while they increased in the epidermis of amniotes with the evolution of the stratum corneum and skin appendages. TGase aids the formation of isopeptide bonds, while SOXase forms disulfide bonds that generate numerous cross-links between keratins and associated corneous proteins, likely increasing the mechanical resistance and durability of the amniote epidermis and its appendages. TGase is low to absent in the beta-corneous layers of sauropsids but is detected in the softer but pliable alpha-layers of sauropsids, mammalian epidermis, medulla, and inner root sheath of hairs. SOXase is present in hard and soft corneous appendages of reptiles, birds, and mammals, and determines cross-linking among corneous proteins of scales, claws, beaks, hairs, and feathers.

Development of a yeast cell surface display method using the SpyTag/SpyCatcher system

Yeast cell surface display (YSD) has been used to engineer various proteins, including antibodies. Directed evolution, which subjects a gene to iterative rounds of mutagenesis, selection and amplification, is useful for protein engineering. In vivo continuous mutagenesis, which continuously diversifies target genes in the host cell, is a promising tool for accelerating directed evolution. However, combining in vivo continuous evolution and YSD is difficult because mutations in the gene encoding the anchor proteins may inhibit the display of target proteins on the cell surface. In this study, we have developed a modified YSD method that utilises SpyTag/SpyCatcher-based in vivo protein ligation. A nanobody fused with a SpyTag of 16 amino acids and an anchor protein fused with a SpyCatcher of 113 amino acids are encoded by separate gene cassettes and then assembled via isopeptide bond formation. This system achieved a high display efficiency of more than 90%, no intercellular protein ligation events, and the enrichment of target cells by cell sorting. These results suggested that our system demonstrates comparable performance with conventional YSD methods; therefore, it can be an appropriate platform to be integrated with in vivo continuous evolution.

One-step purification and immobilization of recombinant proteins using SpyTag/SpyCatcher chemistry.

Based on the specific and spontaneous formation of isopeptide bonds by SpyCatcher/SpyTag, we have developed a one-step method for purification and immobilization of recombinant proteins. The procedure is to immobilize SpyCatcher on glyoxyl agarose gels, and then the SpyCatcher immobilisate can be used to immobilize the SpyTag-fused protein in the crude extract selectively. A mutant of SpyCatcher (mSC), in which a peptide (LysGlyLysGlyLysGly) was added to the C-terminus of SpyCatcher and three lysine residues around the SpyTag/SpyCatcher binding domain were replaced with arginine, was designed to improve the attachment of SpyCatcher to the support. Compared with wild-type SpyCatcher, mSC can be immobilized on the glyoxyl-agarose support more efficiently, which enables the obtained mSC derivative a high binding capacity of the SpyTag-fused protein. The results showed that the target proteins in the crude enzyme extract were purified and immobilized in one step, and the thermal stability of the immobilized target proteins was also remarkably improved.

Structural basis of ubiquitination mediated by protein splicing in early Eukarya

BackgroundInteins are intervening proteins, which are known to perform protein splicing. The reaction results in the production of an intein domain and an inteinless protein, which shows no trace of the insertion. BIL2 is part of the polyubiquitin locus of Tetrahymena thermophila (BUBL), where two bacterial-intein-like (BIL) domains lacking the C + 1 nucleophile, are flanked by two independent ubiquitin-like domains (ubl4/ubl5). MethodsWe solved the X-ray structures of BIL2 in both the inactive and unprecedented, zinc-induced active, forms. Then, we characterized by mass spectrometry the BUBL splicing products in the absence and in the presence of T.thRas-GTPase. Finally, we investigated the effect of ubiquitination on T.thRas-GTPase by molecular dynamics simulations. ResultsThe structural analysis demonstrated that zinc-induced conformational change activates protein splicing. Moreover, mass spectrometry characterization of the splicing products shed light on the possible function of BIL2, which operates as a “single-ubiquitin-dispensing-platform”, allowing the conjugation, via isopeptide bond formation (K(εNH2)-C-ter), of ubl4 to either ubl5 or T.thRas-GTPase. Lastly, we demonstrated that T.thRas-GTPase ubiquitination occurs in proximity of the nucleotide binding pocket and stabilizes the protein active state. ConclusionsWe demonstrated that BIL2 is activated by zinc and that protein splicing induced by this intein does not take place through classical or aminolysis mechanisms but via formation of a covalent isopeptide bond, causing the ubiquitination of endogenous substrates such as T.thRas-GTPase. General significanceIn this “enzyme-free” ubiquitination mechanism the isopeptide formation, which canonically requires E1-E2-E3 enzymatic cascade and constitutes the alphabet of ubiquitin biology, is achieved in a single, concerted step without energy consumption.

Multiplexed Affinity Characterization of Protein Binders Directly from a Crude Cell Lysate by Covalent Capture on Suspension Bead Arrays

The precise determination of affinity and specificity is a crucial step in the development of new protein reagents for therapy and diagnostics. Paradoxically, the selection of protein binders, e.g., antibody fragments, from large combinatorial repertoires is a rapid process compared to the subsequent characterization of selected clones. Here we demonstrate the use of suspension bead arrays (SBA) in combination with flow cytometry to facilitate the post-selection analysis of binder affinities. The array is designed to capture the proteins of interest (POIs) covalently on the surface of superparamagnetic color-coded microbeads directly from expression cell lysate, based on SpyTag-SpyCatcher coupling by isopeptide bond formation. This concept was validated by analyzing the affinities of a typical phage display output, i.e., clones consisting of single-chain variable fragment antibodies (scFvs), as SpyCatcher fusions in 12- and 24-plex SBA formats using a standard three-laser flow cytometer. We demonstrate that the equilibrium dissociation constants (Kd) obtained from multiplexed SBA assays correlate well with experiments performed on a larger scale, while the antigen consumption was reduced >100-fold compared to the conventional 96-well plate format. Protein screening and characterization by SBAs is a rapid and reagent-saving analytical format for combinatorial protein engineering to address specificity maturation and cross-reactivity profiling of antibodies.

Covalent Capture of Collagen Triple Helices Using Lysine-Aspartate and Lysine-Glutamate Pairs.

Collagen mimetic peptides (CMPs) self-assemble into a triple helix reproducing the most fundamental aspect of the collagen structural hierarchy. They are therefore important for both further understanding this complex family of proteins and use in a wide range of biomaterials and biomedical applications. CMP self-assembly is complicated by a number of factors which limit the use of CMPs including their slow rate of folding, relatively poor monomer-trimer equilibrium, and the large number of competing species possible in heterotrimeric helices. All of these problems can be solved through the formation of isopeptide bonds between lysine and either aspartate or glutamate. These amino acids serve two purposes: they first direct self-assemble, allowing for composition and register control within the triple helix, and subsequently can be covalently linked, fixing the composition and register of the assembled structure without perturbing the triple helical conformation. This self-assembly and covalent capture are demonstrated here with four different triple helices. The formation of an isopeptide bond between lysine and glutamate (K-E) is shown to be a faster and higher yielding reaction than lysine with aspartate (K-D). Additionally, K-E amide bonds increase the thermal stability, improve the refolding capabilities, and enhance the triple helical structure as compared to K-E supramolecular interactions, observed by circular dichroism. In contrast, covalent capture of triple helices with K-D amide bonds occurs slower, and the captured triple helices do not have enhanced helical structure. The crystal structure of a triple helix captured through the formation of three K-E isopeptide bonds unequivocally demonstrates the connectivity of the amide bonds formed while also confirming the preservation of the canonical triple helix. The rate of reaction and yield for covalently captured K-E triple helices along with the excellent preservation of triple helical structure demonstrate that this approach can be used to effectively capture and stabilize this important biological motif for biological and biomedical applications.

Expanding the Versatility of Microbial Transglutaminase Using α‐Effect Nucleophiles as Noncanonical Substrates

AbstractThe substrate promiscuity of microbial transglutaminase (mTG) has been exploited in various applications in biotechnology, in particular for the attachment of alkyl amines to glutamine‐containing peptides and proteins. Here, we expand the substrate repertoire to include hydrazines, hydrazides, and alkoxyamines, resulting in the formation of isopeptide bonds with varied susceptibilities to hydrolysis or exchange by mTG. Furthermore, we demonstrate that simple unsubstituted hydrazine and dihydrazides can be used to install reactive hydrazide handles onto the side chain of internal glutamine residues. The distinct hydrazide handles can be further coupled with carbonyls, including ortho‐carbonylphenylboronic acids, to form site‐specific and functional bioconjugates with tunable hydrolytic stability. The extension of the substrate scope of mTG beyond canonical amines thus substantially broadens the versatility of the enzyme, providing a new approach to facilitate novel applications.

Expanding the Versatility of Microbial Transglutaminase Using α-Effect Nucleophiles as Noncanonical Substrates.

The substrate promiscuity of microbial transglutaminase (mTG) has been exploited in various applications in biotechnology, in particular for the attachment of alkyl amines to glutamine-containing peptides and proteins. Here, we expand the substrate repertoire to include hydrazines, hydrazides, and alkoxyamines, resulting in the formation of isopeptide bonds with varied susceptibilities to hydrolysis or exchange by mTG. Furthermore, we demonstrate that simple unsubstituted hydrazine and dihydrazides can be used to install reactive hydrazide handles onto the side chain of internal glutamine residues. The distinct hydrazide handles can be further coupled with carbonyls, including ortho-carbonylphenylboronic acids, to form site-specific and functional bioconjugates with tunable hydrolytic stability. The extension of the substrate scope of mTG beyond canonical amines thus substantially broadens the versatility of the enzyme, providing a new approach to facilitate novel applications.

NMR Spectroscopic Studies Reveal the Critical Role of the Isopeptide Bond in Forming the Otherwise Unstable SpyTag-SpyCatcher Mutant Complexes.

The interplay between protein folding and chemical reaction has been an intriguing subject. In this contribution, we report the study of SpyTag and SpyCatcher reactive mutants using a combination of sodium dodecyl sulfate-polyacrylamide gel electrophoresis, liquid chromatography and mass spectrometry, circular dichroism, and NMR spectroscopy. It was found that the wild-type SpyCatcher is well-folded in solution and docks with SpyTag to form an intermediate that promotes isopeptide bond formation. By contrast, the double mutant SpyCatcherVA is disordered in solution yet remains reactive toward SpyTag, forming a well-folded covalent complex. Control experiments using the catalytically inactive mutants further reveal the critical role of the isopeptide bond in stabilizing the otherwise loose SpyTag-SpyCatcherVA complex, amplifying the effect of the minute sequence disparity. We believe that the synergy between protein folding and isopeptide bonding is an effective way to enhance protein stability and engineer protein-protein interactions.

Chlorpyrifos Oxon-Induced Isopeptide Bond Formation in Human Butyrylcholinesterase.

A newly recognized action of organophosphates (OP) is the ability to crosslink proteins through an isopeptide bond. The first step in the mechanism is covalent addition of the OP to the side chain of lysine. This activates OP-lysine for reaction with a nearby glutamic or aspartic acid to make a gamma glutamyl epsilon lysine bond. Crosslinked proteins are high molecular weight aggregates. Our goal was to identify the residues in the human butyrylcholinesterase (HuBChE) tetramer that were crosslinked following treatment with 1.5 mM chlorpyrifos oxon. High molecular weight bands were visualized on an SDS gel. Proteins in the gel bands were digested with trypsin, separated by liquid chromatography and analyzed in an Orbitrap mass spectrometer. MSMS files were searched for crosslinked peptides using the Batch-Tag program in Protein Prospector. MSMS spectra were manually evaluated for the presence of ions that supported the crosslinks. The crosslink between Lys544 in VLEMTGNIDEAEWEWK544AGFHR and Glu542 in VLEMTGNIDEAEWE542WK satisfied our criteria including that of spatial proximity. Distances between Lys544 and Glu542 were 7.4 and 9.5 Å, calculated from the cryo-EM (electron microscopy) structure of the HuBChE tetramer. Paraoxon ethyl, diazoxon, and dichlorvos had less pronounced effects as visualized on SDS gels. Our proof-of-principle study provides evidence that OP have the ability to crosslink proteins. If OP-induced protein crosslinking occurs in the brain, OP exposure could be responsible for some cases of neurodegenerative disease.

In vitro Crosslinking Reactions and Substrate Incorporation Assays for The Identification of Transglutaminase-2 Protein Substrates.

Transglutaminase (TG2) catalyzes protein crosslinking between glutamyl and lysyl residues. Catalytic activity occurs via a transamidation mechanism resulting in the formation of isopeptide bonds. Since TG2-mediated transamidation is of mechanistic importance for a number of biological processes, assays that enable rapid and efficient identification and characterization of candidate substrates are an important first-step to uncovering the function of crosslinked proteins. Herein we describe an optimized and flexible protocol for in vitro TG2 crosslink reactions and substrate incorporation assays. We have previously employed these techniques in the identification of the protein high mobility group box 1 (HMGB1) as a TG2 substrate. However, the protocol can be adapted for identification of any candidate transamidation substrate.

Suitable fusion of N-terminal heptad repeats to achieve covalently stabilized potent N-peptide inhibitors of HIV-1 infection

N-terminal heptad repeat (NHR)-derived peptide (N-peptide) fusion inhibitors, which are derived from human immunodeficiency virus (HIV) envelope glycoprotein 41 (gp41), are limited by aggregation and unstable trimer conformation. However, they could function as potent inhibitors of viral infection by forming a coiled-coil structure covalently stabilized by interchain disulfide bonds. We previously synthesized N-peptides with potent anti-HIV-1 activity and high stability by coiled-coil fusion and covalent stabilization. Here, we attempted to study the effects of NHRs of chimeric N-peptides by fusing de novo coiled-coil isopeptide bridge-tethered T21 peptides of different NHR lengths. Peptides (T21N23)3 and (T21N36)3 was a more potent HIV-1 fusion inhibitor than (T21N17)3. The site of isopeptide bond formation was precisely controlled and had little influence on N-peptide properties. The N-peptide (T21N36)3, which had a similar conformation as the NHR trimer and interacted well with the C34 peptide, may be useful for screening other C-peptides and small-molecule fusion inhibitors, and for studying the interactions between the NHR trimer and C-terminal heptad repeats.

A comparative analysis of catalytic activity and stability of microbial transglutaminase in controlled denaturing conditions

Microbial transglutaminases (MTGs) catalyzes the formation of Gln-Lys isopeptide bonds and are widely used for the cross-linking of proteins and peptides in food and in biotechnological applications for bioconjugation reactions. In view of its practical utility, a comparative study of the catalytic activity and stability of the enzyme in a wide range of denaturing conditions has been performed through Circular Dichroism (CD), fluorescence and activity assays performed with model substrates. In agreement with previous results, we show that MTG has a significant structural and functional tolerance to pH changes, whereas the enzyme stability and activity decrease in presence of increasing amounts of denaturing agents, such as urea and guanidinium chloride (GdnHCl). Noteworthy, the activity of MTG in denaturing conditions differs markedly from that in pseudo-physiological settings, shifting unexpectedly toward higher substrate specificity. Also, the use of controlled amounts of denaturing agents (1.0–1.5 M urea) largely improves yields and purity of the final products of 10–15% and 25–30%, respectively. These findings widen the range of applicability of the MTG-mediated biocatalysis for industrial and biotechnological purposes.