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.

Recombinant Destabilase from Hirudo medicinalis Is Able to Dissolve Human Blood Clots In Vitro

Leeches are amazing animals that can be classified as conditionally poisonous animals since the salivary cocktail they produce is injected directly into the victim, and its components have strictly defined biological purposes, such as preventing blood clot formation. Thrombolytic drugs are mainly aimed at treating newly formed blood clots. Aged clots are stabilized by a large number of isopeptide bonds that prevent the action of thrombolytics. These bonds are destroyed by destabilase, an enzyme of the leech’s salivary glands. Here, we conducted a pilot study to evaluate the feasibility and effectiveness of the use of destabilase in relation to blood clots formed during real pathological processes. We evaluated the isopeptidase activity of destabilase during the formation of a stabilized fibrin clot. We showed that destabilase does not affect the internal and external coagulation cascades. We calculated the dose–response curve and tested the ability of destabilase to destroy isopeptide bonds in natural blood clots. The effect of aged and fresh clots dissolving ability after treatment with destabilase coincided with the morphological characteristics of clots during surgery. Thus, recombinant destabilase can be considered as a potential drug for the treatment of aged clots, which are difficult to treat with known thrombolytics.

Immunotargeting of Nanocrystals by SpyCatcher Conjugation of Engineered Antibodies

Inorganic nanocrystals such as quantum dots (QDs) and upconverting nanoparticles (UCNPs) are uniquely suited for quanti-tative live-cell imaging and are typically functionalized with ligands to study specific receptors or cellular targets. Antibod-ies (Ab) are among the most useful targeting reagents owing to their high affinities and specificities, but common nanocrys-tal labeling methods may orient Ab incorrectly, be reversible or denaturing, or lead to Ab-NP complexes too large for some applications. Here, we show that SpyCatcher proteins, which bind and spontaneously form covalent isopeptide bonds with cognate SpyTag peptides, can conjugate engineered Ab to nanoparticle surfaces with control over stability, orientation, and stoichiometry. Compact SpyCatcher-functionalized QDs and UCNPs may be labeled with short-chain variable fragment Ab (scFv) engineered to bind urokinase-type plasminogen activator receptors (uPAR) that are overexpressed in many human can-cers. Confocal imaging of anti-uPAR scFv-QD conjugates shows the Ab mediates specific binding and internalization by breast cancer cells expressing uPAR. Time-lapse imaging of photostable scFv-UCNP conjugates show that Ab binding caus-es uPAR internalization with a ∼20-minute half-life on the cell surface, and uPAR is internalized to endolysosomal com-partments distinct from general membrane stains and without significant recycling to the cell surface. The controlled and stable conjugation of engineered Ab to NPs enables targeting of diverse receptors for live-cell study of their distribution, trafficking, and physiology.

Detection of Isopeptide Bonds in Monoclonal Antibody Aggregates

Purpose A 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. Methods Aggregate 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). Results An 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. Conclusions Inter-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.

An ester bond underlies the mechanical strength of a pathogen surface protein

Gram-positive bacteria can resist large mechanical perturbations during their invasion and colonization by secreting various surface proteins with intramolecular isopeptide or ester bonds. Compared to isopeptide bonds, ester bonds are prone to hydrolysis. It remains elusive whether ester bonds can completely block mechanical extension similarly to isopeptide bonds, or whether ester bonds dissipate mechanical energy by bond rupture. Here, we show that an ester-bond containing stalk domain of Cpe0147 is inextensible even at forces > 2 nN. The ester bond locks the structure to a partially unfolded conformation, in which the ester bond remains largely water inaccessible. This allows the ester bond to withstand considerable mechanical forces and in turn prevent complete protein unfolding. However, the protecting effect might be reduced at non-physiological basic pHs or low calcium concentrations due to destabilizing the protein structures. Inspired by this design principle, we engineer a disulfide mutant resistant to mechanical unfolding under reducing conditions.

Factor XIII-A: An Indispensable “Factor” in Haemostasis and Wound Healing

Factor XIII (FXIII) is a transglutaminase enzyme that catalyses the formation of ε-(γ-glutamyl)lysyl isopeptide bonds into protein substrates. The plasma form, FXIIIA2B2, has an established function in haemostasis, with fibrin being its principal substrate. A deficiency in FXIII manifests as a severe bleeding diathesis emphasising its crucial role in this pathway. The FXIII-A gene (F13A1) is expressed in cells of bone marrow and mesenchymal lineage. The cellular form, a homodimer of the A subunits denoted FXIII-A, was perceived to remain intracellular, due to the lack of a classical signal peptide for its release. It is now apparent that FXIII-A can be externalised from cells, by an as yet unknown mechanism. Thus, three pools of FXIII-A exist within the circulation: plasma where it circulates in complex with the inhibitory FXIII-B subunits, and the cellular form encased within platelets and monocytes/macrophages. The abundance of this transglutaminase in different forms and locations in the vasculature reflect the complex and crucial roles of this enzyme in physiological processes. Herein, we examine the significance of these pools of FXIII-A in different settings and the evidence to date to support their function in haemostasis and wound healing.

Genetics, Structure, and Function of Group A Streptococcal Pili

Streptococcus pyogenes (Group A Streptococcus; GAS) is an exclusively human pathogen. This bacterial species is responsible for a large variety of infections, ranging from purulent but mostly self-limiting oropharynx/skin diseases to streptococcal sequelae, including glomerulonephritis and rheumatic fever, as well as life-threatening streptococcal toxic-shock syndrome. GAS displays a wide array of surface proteins, with antigenicity of the M protein and pili utilized for M- and T-serotyping, respectively. Since the discovery of GAS pili in 2005, their genetic features, including regulation of expression, and structural features, including assembly mechanisms and protein conformation, as well as their functional role in GAS pathogenesis have been intensively examined. Moreover, their potential as vaccine antigens has been studied in detail. Pilus biogenesis-related genes are located in a discrete section of the GAS genome encoding fibronectin and collagen binding proteins and trypsin-resistant antigens (FCT region). Based on the heterogeneity of genetic composition and DNA sequences, this region is currently classified into nine distinguishable forms. Pili and fibronectin-binding proteins encoded in the FCT region are known to be correlated with infection sites, such as the skin and throat, possibly contributing to tissue tropism. As also found for pili of other Gram-positive bacterial pathogens, GAS pilin proteins polymerize via isopeptide bonds, while intramolecular isopeptide bonds present in the pilin provide increased resistance to degradation by proteases. As supported by findings showing that the main subunit is primarily responsible for T-serotyping antigenicity, pilus functions and gene expression modes are divergent. GAS pili serve as adhesins for tonsillar tissues and keratinocyte cell lines. Of note, a minor subunit is considered to have a harpoon function by which covalent thioester bonds with host ligands are formed. Additionally, GAS pili participate in biofilm formation and evasion of the immune system in a serotype/strain-specific manner. These multiple functions highlight crucial roles of pili during the onset of GAS infection. This review summarizes the current state of the art regarding GAS pili, including a new mode of host-GAS interaction mediated by pili, along with insights into pilus expression in terms of tissue tropism.

Streptococcus pneumoniae Type 1 Pilus – A Multifunctional Tool for Optimized Host Interaction

Streptococcus pneumoniae represents a major Gram-positive human pathogen causing bacterial pneumonia, otitis media, meningitis, and other invasive diseases. Several pneumococcal isolates show increasing resistance rates against antibacterial agents. A variety of virulence factors promote pneumococcal pathogenicity with varying importance in different stages of host infection. Virulence related hair-like structures (“pili”) are complex, surface located protein arrays supporting proper host interaction. In the last two decades different types of pneumococcal pili have been identified: pilus-1 (P1) and pilus-2 (P2) are formed by the catalytic activity of sortases that covalently assemble secreted polypeptide pilin subunits in a defined order and finally anchor the resulting pilus in the peptidoglycan. Within the long pilus fiber the presence of intramolecular isopeptide bonds confer high stability to the sequentially arranged individual pilins. This mini review will focus on S. pneumoniae TIGR4 P1 molecular architecture, the subunits it builds and provides insights into P1 sortase-mediated assembly. The complex P1 architecture (anchor-/backbone-/tip-subunits) allows the specific interaction with various target structures facilitating different steps of colonization, invasion and spreading within the host. Optimized pilin subunit confirmation supports P1 function under physiological conditions. Finally, aspects of P1- host interplay are summarized, including recent insights into P1 mechanobiology, which have important implications for P1 mediated pathogenesis.

Prediction of Neddylation Sites Using the Composition of k-spaced Amino Acid Pairs and Fuzzy SVM

Introduction: Neddylation is the process of ubiquitin-like protein NEDD8 attaching substrate lysine via isopeptide bonds. As a highly dynamic and reversible post-translational modification, lysine neddylation has been found to be involved in various biological processes and closely associated with many diseases. Objective: The accurate identification of neddylation sites is necessary to elucidate the underlying molecular mechanisms of neddylation. As traditional experimental methods are often expensive and time-consuming, it is imperative to design computational methods to identify neddylation sites. Methods: In this study, a novel predictor named CKSAAP_NeddSite is developed to detect neddylation sites. An effective feature encoding technology, the composition of k-spaced amino acid pairs, is used to encode neddylation sites. And the F-score feature selection method is adopted to remove the redundant features. Moreover, a fuzzy support vector machine algorithm is employed to overcome the class imbalance and noise problem. Results: As illustrated by 10-fold cross-validation, CKSAAP_NeddSite achieves an AUC of 0.9848. Independent tests also show that CKSAAP_NeddSite significantly outperforms existing neddylation sites predictor. Therefore, CKSAAP_NeddSite can be a useful bioinformatics tool for the prediction of neddylation sites. Feature analysis shows that some residues around neddylation sites may play an important role in the prediction. Conclusion: The results of analysis and prediction could offer useful information for elucidating the molecular mechanisms of neddylation. A user-friendly web-server for CKSAAP_NeddSite is established at 123.206.31.171/CKSAAP_NeddSite.

Microbial Transglutaminase as a Tool to Improve the Features of Hydrocolloid-Based Bioplastics

Several proteins from animal and plant origin act as microbial transglutaminase substrate, a crosslinking enzyme capable of introducing isopeptide bonds into proteins between the aminoacids glutamines and lysines. This feature has been widely exploited to modify the biological properties of many proteins, such as emulsifying, gelling, viscosity, and foaming. Besides, microbial transglutaminase has been used to prepare bioplastics that, because made of renewable molecules, are able to replace the high polluting plastics of petrochemical origin. In fact, most of the time, it has been shown that the microbial enzyme strengthens the matrix of protein-based bioplastics, thus, influencing the technological characteristics of the derived materials. In this review, an overview of the ability of many proteins to behave as good substrates of the enzyme and their ability to give rise to bioplastics with improved properties is presented. Different applications of this enzyme confirm its important role as an additive to recover high value-added protein containing by-products with a double aim (i) to produce environmentally friendly materials and (ii) to find alternative uses of wastes as renewable, cheap, and non-polluting sources. Both principles are in line with the bio-economy paradigm.