Interspecific Analysis of Sea Urchin Adhesive Composition Emphasizes Variability of Glycans Conjugated With Putative Adhesive Proteins

Sea urchins possess specialized adhesive organs, tube feet. Although initially believed to function as suckers, it is currently accepted that they rely on adhesive and de-adhesive secretions to attach and detach repeatedly from the substrate. Given the biotechnological potential of their strong reversible adhesive, sea urchins are under investigation to identify the protein and glycan molecules responsible for its surface coupling, cohesion and polymerization properties. However, this characterization has only focused on a single species, Paracentrotus lividus. To provide a broader insight into sea urchins adhesion, a comparative study was performed using four species belonging to different taxa and habitats: Diadema africanum, Arbacia lixula, Paracentrotus lividus and Sphaerechinus granularis. Their tube feet external morphology and histology was studied, together with the ultrastructure of their adhesive secretory granules. In addition, one antibody and five lectins were used on tube foot histological sections and extracts, and on adhesive footprints to detect the presence of adhesion-related (glyco)proteins like those present in P. lividus in other species. Results confirmed that the antibody raised against P. lividus Nectin labels the adhesive organs and footprints in all species. This result was further confirmed by a bioinformatic analysis of Nectin-like sequences in ten additional species, increasing the comparison to seven families and three orders. The five tested lectins (GSL II, WGA, STL, LEL, and SBA) demonstrated that there is high interspecific variability of the glycans involved in sea urchin adhesion. However, there seems to be more conservation among taxonomically closer species, like P. lividus and S. granularis. In these species, lectin histochemistry and lectin blots indicated the presence of high molecular weight putative adhesive glycoproteins bearing N-acetylglucosamine residues in the form of chitobiose in the adhesive epidermis and footprints. Our results emphasize a high selective pressure for conservation of functional domains in large putative cohesive proteins and highlight the importance of glycosylation in sea urchin adhesion with indications of taxonomy-related conservation of the conjugated glycans.

(Un)expected Similarity of the Temporary Adhesive Systems of Marine, Brackish, and Freshwater Flatworms

Many free-living flatworms have evolved a temporary adhesion system, which allows them to quickly attach to and release from diverse substrates. In the marine Macrostomum lignano, the morphology of the adhesive system and the adhesion-related proteins have been characterised. However, little is known about how temporary adhesion is performed in other aquatic environments. Here, we performed a 3D reconstruction of the M. lignano adhesive organ and compared it to the morphology of five selected Macrostomum, representing two marine, one brackish, and two freshwater species. We compared the protein domains of the two adhesive proteins, as well as an anchor cell-specific intermediate filament. We analysed the gene expression of these proteins by in situ hybridisation and performed functional knockdowns with RNA interference. Remarkably, there are almost no differences in terms of morphology, protein regions, and gene expression based on marine, brackish, and freshwater habitats. This implies that glue components produced by macrostomids are conserved among species, and this set of two-component glue functions from low to high salinity. These findings could contribute to the development of novel reversible biomimetic glues that work in all wet environments and could have applications in drug delivery systems, tissue adhesives, or wound dressings.

Effect of glyprolines on serum caspase levels under “social” stress

Objective. To investigate the effect of glyprolines on the levels of initiating and effector caspases in the serum of white rats under "social" stress. Materials and methods. The study was conducted on 90 white male rats of 6 months of age. All manipulations with animals were carried out in accordance with international and domestic requirements for working with laboratory animals. When modeling "social" stress, groups of animals with aggressive and submissive behavior were formed. Laboratory animals, taking into account the types of behavior, were divided into groups (n=10): a group of intact males (control); a group of animals exposed to" social " stress for 20 days (stress); groups of individuals who received intraperitoneal Selank (Thr-Lys-Pro-Arg-Pro-Gly-Pro), Pro-Gly-Pro, Pro-Gly-Pro-Leu at doses of 100 mcg/kg / day from the 1st day of stress exposure within a 20- day course. The effect of neuropeptides on the activity of apoptosis processes was evaluated by determining the level of initiating and effector caspases (caspase-8 and caspase-3) (ELISA Kit for Caspase-8 and ELISA Kit for Caspase-3; USA) in the blood serum of white rats by enzyme immunoassay. Results. According to the results of the study, it was found that under conditions of "social" stress, an increase in apoptotic processes was observed, accompanied by an increase in the level of caspase-3 and caspase-8 in the blood serum of white rats. The introduction of the studied compounds against the background of stress contributed to a decrease in the level of the studied indicators, which is most likely due to the presence of antiapoptotic action in glyprolins due to inhibition of the caspase-dependent cascade of apoptosis reactions, as a result of which the destruction of cellular structures occurs by hydrolysis of nuclear lamina, cleavage of adhesive proteins, destruction of the cytoskeleton. Conclusion. Thus, the conducted study established the presence of Thr-Lys-Pro-Arg-Pro-Gly-Pro (Selank), Pro-Gly-Pro and Pro-Gly-Pro-Leu under conditions of stress-induced antiapoptotic activity due to inhibition of the caspase-dependent cascade of apoptosis reactions.

Facile fabrication and nanoscale assembly of polydopamine-functionalized, flexible chitosan films

Substrates that are simultaneously thin, strong, optically transparent, and biocompatible have diverse applications in a range of fundamental and applied fields. While nature-derived materials offer advantages of sustainability and inherent biocompatibility compared to synthetic polymers, their brittleness and swelling, as well as surface charge and chemical functionalization non-conducive to cell growth, can hinder widespread application. In this work, we discuss the fabrication and systematic characterization of polydopamine-coated chitosan thin films. Chitosan is a widely used, partially deacetylated form of chitin, derived from crustaceans and arthropods. Polydopamine (PDA) is derived from chemistries mimicking mussel foot adhesive proteins. A facile dip-coating process of thin and flexible, uncrosslinked chitosan films in aqueous dopamine solutions leads to dramatic changes in physical and chemical properties. We show how the PDA forms time-dependent assemblies on the film surfaces, affecting surface roughness, hydrophilicity, and mechanical strength. Coating the surface for even a few seconds provides functional changes to the films. Our results shows that the optimal coating time is on the order of few hours, whereby the films are optically transparent with excellent extensibility and Young’s modulus, while further coating reduces the benefits of this surface coating. These materials are biocompatible, serving as substrates for cell adhesion and growth while maintaining good viability. Overall, these findings give insight to the effects of PDA assembly on surfaces, and illustrate how a simple, quick, and robust bioinspired coating process can prime substrates for biomedical applications such as tissue engineering, biosensing, and wound healing.

Characterization of nit sheath protein functions and transglutaminase-mediated cross-linking in the human head louse, Pediculus humanus capitis

Background Head louse females secrete liquid glue during oviposition, which is solidified to form the nit sheath over the egg. Recently, two homologous proteins, named louse nit sheath protein (LNSP) 1 and LNSP 2, were identified as adhesive proteins but the precise mechanism of nit sheath solidification is unknown. Methods We determined the temporal transcriptome profiles of the head louse accessory glands plus oviduct, from which putative major structural proteins and those with functional importance were deduced. A series of RNA interference (RNAi) experiments and treatment of an inhibitor were conducted to elucidate the function and action mechanism of each component. Results By transcriptome profiling of genes expressed in the louse accessory glands plus uterus, the LNSP1 and LNSP2 along with two hypothetical proteins were confirmed to be the major structural proteins. In addition, several proteins with functional importance, including transglutaminase (TG), defensin 1 and defensin 2, were identified. When LNSP1 was knocked down via RNA interference, most eggs became nonviable via desiccation, suggesting its role in desiccation resistance. Knockdown of LNSP2, however, resulted in oviposition failure, which suggests that LNSP2 may serve as the basic platform to form the nit sheath and may have an additional function of lubrication. Knockdown of TG also impaired egg hatching, demonstrating its role in the cross-linking of nit sheath proteins. The role of TG in cross-linking was further confirmed by injecting or hair coating of GGsTop, a TG inhibitor. Conclusions Both LNSP1 and LNSP2 are essential for maintaining egg viability besides their function as glue. The TG-mediated cross-linking plays critical roles in water preservation that are essential for ensuring normal embryogenesis. TG-mediated cross-linking mechanism can be employed as a therapeutic target to control human louse eggs, and any topically applied TG inhibitors can be exploited as potential ovicidal agents. Graphical abstract

A Novel Recombinant Chimeric Proteinous Bio-adhesive Consisting of Mussel Foot Protein 3, 5 and Curli Proteins GvpA and CsgA Expressed in Pichia Pastoris

Despite various efforts to produce strong recombinant bio-adhesive proteins for medical purposes, efficient production of a safe and feasible bio-glue is not yet a commercial reality due to the weak properties or low expression level. Here, a feasible expression system has been developed to produce strong recombinant fusion protein using Mfp3 and Mfp5 along with two curli proteins, GvpA and CsgA expressed under the control of alcohol oxidase (AOX1) promoter for high-level production in P. pastoris using pPICZα vector. Purified chimeric proteins were first evaluated using western blotting and its remaining dopa were measured in the modified proteins by NBT assay. We further elucidated mechanistic properties of obtained adhesive protein assembly in various pH based on its different subunits using atomic force microscopy (AFM) when adsorbed onto the mica surface. We found that combinational structural features of subunits and post-translational changes during expression in yeast host have led to potent adherence due to higher dopa residues majorly in acidic condition and tetrad complex that is higher than that of earlier reports in prokaryotic systems. We believe that our obtained chimeric protein resulted through fusion of GvpA and CsgA proteins with dopa-containing Mfp proteins expressed in the methylotrophic yeast, P. pastoris, not only presents a candidate for future biomedical applications, but also provides novel biological clues using for high-performance bioinspired biomaterials designation.

On the Nanomechanical and Viscoelastic Properties of Coatings Made of Recombinant Sea Star Adhesive Proteins

To attach to surfaces in the sea, sea stars produce proteinaceous adhesive secretions. Sfp1 is a major constituent of this adhesive, where it is present in the form of four subunits (named Sfp1α to δ) displaying specific protein-, carbohydrate- and metal-binding domains. Recently, two recombinant proteins inspired from Sfp1 have been produced: one corresponding to the C-terminal part of Sfp1β and the other to the full-length Sfp1δ. Adsorption ability tests showed that both recombinant proteins were able to adsorb and to form coatings on different surfaces in artificial seawater as well as in Tris buffer supplemented with NaCl or CaCl2. In this study, we used Atomic Force Microscopy (AFM) to characterize the nanomechanical properties of these coatings with an emphasis on functional characteristics such as adhesive properties and modulus of elasticity. We used AFM techniques which are the most appropriate to characterize the coating microstructure combined with the mapping of its nanomechanical properties.