Gastrobodies are engineered antibody mimetics resilient to pepsin and hydrochloric acid

Protein-based targeting reagents, such as antibodies and non-antibody scaffold proteins, are rapidly inactivated in the upper gastrointestinal (GI) tract. Hydrochloric acid in gastric juice denatures proteins and activates pepsin, concentrations of which reach 1 mg/mL in the mammalian stomach. Two stable scaffold proteins (nanobody and nanofitin), previously developed to be protease-resistant, were completely digested in less than 10 min at 100-fold lower concentration of pepsin than found in the stomach. Here we present gastrobodies, a protein scaffold derived from Kunitz soybean trypsin inhibitor (SBTI). SBTI is highly resistant to the challenges of the upper GI tract, including digestive proteases, pH 2 and bile acids. Computational prediction of SBTI’s evolvability identified two nearby loops for randomization, to create a potential recognition surface which was experimentally validated by alanine scanning. We established display of SBTI on full-length pIII of M13 phage. Phage selection of gastrobody libraries against the glucosyltransferase domain of Clostridium difficile toxin B (GTD) identified hits with nanomolar affinity and enzyme inhibitory activity. Anti-GTD binders retained high stability to acid, digestive proteases and heat. Gastrobodies show resilience to exceptionally harsh conditions, which should provide a foundation for targeting and modulating function within the GI tract.

Harnessing the functional diversity of plant cystatins to design inhibitor variants highly active against herbivorous arthropod digestive proteases

Protein engineering approaches have been proposed to improve the inhibitory properties of plant cystatins against herbivorous arthropod digestive proteases. These approaches typically involve the site-directed mutagenesis of functionally relevant amino acids, the production and selection of improved inhibitory variants by molecular phage display procedures, or the design of bi/multifunctional translational fusions integrating one or several cystatin inhibitory domains. Here, we propose a new approach where the function-related structural elements of a cystatin are substituted by the corresponding elements of an alternative cystatin. Cys protease inhibitory assays were first performed with 20 representative plant cystatins and model Cys proteases, including herbivorous arthropod digestive proteases, to appreciate the extent of functional variability among plant cystatin protein family members. The most, and less, potent of these cystatins were then used as donors of structural elements to create hybrids of tomato cystatin SlCYS8 used as a model recipient inhibitor. Our data confirm the wide variety of cystatin protease inhibitory profiles among plant taxa. They also demonstrate the usefulness of these proteins as a pool of discrete structural elements for the design of cystatin variants with improved potency against herbivorous pest digestive Cys proteases.

Partial characterization of digestive proteases in Pacific red snapper Lutjanus peru Nichols & Murphy, 1922 (Perciformes: Lutjanidae)

Pacific red snapper (Lutjanus peru) is an important commercial species in Mexico with great aquaculture potential; however, digestive physiology is still unknown. Therefore, the objective of the present work was to characterize the digestive proteases of L. peru juvenile using biochemical and electrophoretic techniques. Results showed a higher acid protease activity than the alkaline proteases, trypsin, chymotrypsin, and leucine aminopeptidase (LAP). The optimum temperature for acid proteases was between 30 to 40°C. Trypsin activity showed two maximum peaks of temperature (30 and 50°C), while alkaline proteases, chymotrypsin, and LAP had optimum temperatures of 50, 50 to 60, and 40°C, respectively. Moreover, the optimum pH of acid proteases was between 2 and 3. Also, alkaline proteases, trypsin, chymotrypsin showed pH optimums at pH 6, 9, and 5, respectively, although LAP showed two optimum pH values at 6 and 9. Acid protease zymogram showed three isoforms, totally inhibited by pepstatin A. Alkaline protease zymogram revealed six bands (125.4, 67.2, 57.9, 48.6, 29.8, and 26.9 kDa), which were inhibited by specific serine-proteases and metalloproteases inhibitors. In conclusion, the main digestion in L. peru depends on stomach proteases, which are characteristic of carnivorous fish, followed by intestinal digestion supported mainly by chymotrypsin.

Transcriptomic and proteomic analysis of putative digestive proteases in the salivary gland and gut of Empoasca (Matsumurasca) onukii Matsuda

Background Infestation by tea green leafhoppers (Empoasca (Matsumurasca) onukii) can cause a series of biochemical changes in tea leaves. As a typical cell-rupture feeder, E. onukii secretes proteases while using its stylet to probe the tender shoots of tea plants (Camellia sinensis). This study identified and analyzed proteases expressed specifically in the salivary gland (SG) and gut of E. onukii through enzymatic activity assays complemented with an integrated analysis of transcriptomic and proteomic data. Results In total, 129 contigs representing seven types of putative proteases were identified. Transcript abundance of digestive proteases and enzymatic activity assays showed that cathepsin B-like protease, cathepsin L-like protease, and serine proteases (trypsin- and chymotrypsin-like protease) were highly abundant in the gut but moderately abundant in the SG. The abundance pattern of digestive proteases in the SG and gut of E. onukii differed from that of other hemipterans, including Nilaparvata lugens, Laodelphax striatellus, Acyrthosiphum pisum, Halyomorpha halys and Nephotettix cincticeps. Phylogenetic analysis showed that aminopeptidase N-like proteins and serine proteases abundant in the SG or gut of hemipterans formed two distinct clusters. Conclusions Altogether, this study provides insightful information on the digestive system of E. onukii. Compared to five other hemipteran species, we observed different patterns of proteases abundant in the SG and gut of E. onukii. These results will be beneficial in understanding the interaction between tea plants and E. onukii.

Comparative characterization of digestive proteases in redhead cichlid (Vieja melanurus) and twoband cichlid (Vieja bifasciata) (Percoidei: Cichlidae)

ABSTRACT In the Southeast of Mexico, there are many native cichlids with commercial interest such as redhead cichlid (Vieja melanurus) and twoband cichlid (V. bifasciata), which have a great local demand and excellent meat quality. However, it is necessary to implement their culture based on nutrition studies and digestive biochemistry. This study’s objective was to characterize these two cichlids’ digestive proteases (pH, temperature, and inhibitors) through biochemistry techniques. Results showed that V. melanurus and V. bifasciata have a digestive capacity analogous to other omnivore fishes, where the optimal pH values of stomach proteases (4 and 2, respectively) and intestinal proteases (6 and 12, respectively), the optimal temperature of acid (35°C and 55°C, respectively) and alkaline proteases (45°C and 55°C, respectively) are quite similar. Both species presented high thermal and pH stabilities. Inhibition showed that V. melanurus is more sensitive to specific inhibitors for alkaline proteases than V. bifasciata. In conclusion, V. bisfasciata and V. melanurus have different digestive protease patterns. Both species can hydrolyze different protein ingredients to formulate a specific diet. Nevertheless, V. bifasciata is more resistant to the presence of inhibitors, which allow it to include vegetable proteins in its diet.

Transcriptomic and proteomic analysis of putative digestive proteases in the salivary gland and gut of Empoasca (Matsumurasca) onukii Matsuda

Background: Infestation by tea green leafhoppers, Empoasca (Matsumurasca) onukii , could cause a series of biochemical changes in tea leaves. As a typical cell-rupture feeder, E. onukii secretes proteases while probing with its stylet into the tender shoots of tea plants ( Camellia sinensis ). This study identified and analyzed proteases specifically expressed in the salivary gland (SG) and gut of E. onukii through enzymatic activity assays, complemented with an integrated analysis of transcriptome and proteome data.Results: In total, 129 contigs representing seven types of putative proteases were identified. Transcript abundance of digestive proteases and enzymatic activity assays showed that cathepsin B-like protease, cathepsin L-like protease, and serine proteases (trypsin- and chymotrypsin-like protease) were highly abundant in the gut while moderately abundant in the SG. The abundance pattern of digestive proteases in the SG and gut of E. onukii differed from that of other hemipterans including Nilaparvata lugens , Laodelphax striatellus , Acyrthosiphum pisum , Halyomorpha halys and Nephotettix cincticeps . Phylogenetic analysis showed that aminopeptidase N-like proteins and serine proteases abundant in the SG or gut of hemipterans were distributed to two distinct clusters.Conclusions: Altogether, this study provide insightful information on the digestive system of E. onukii and observed different patterns of proteases abundant in the SG and gut of E. onukii , in comparison with other five hemipteran species. These results will be beneficial in understanding the interaction between tea plants and E. onukii .

Partial Characterization of Protease Inhibitors of Ulva ohnoi and Their Effect on Digestive Proteases of Marine Fish

This piece of research evaluates the presence of protease inhibitors in the macroalga Ulva ohnoi and provides an initial overview of their mode of action. The ability of Ulva protease inhibitors to inhibit digestive proteases of three marine fish species, as well as their capacity to hamper the hydrolysis of a reference protein by those fish proteases, were assessed. In addition, thermal stability and the mode of inhibition on trypsin and chymotrypsin were also studied. Dose-response inhibition curves and in vitro protein hydrolysis assays revealed a noticeable inhibition of fish enzymes when Ulva concentration increased in the assay. The thermal treatment of Ulva reduced markedly the inhibitory effect on fish digestive protease. Finally, Lineweaver–Burk plots indicated that trypsin and chymotrypsin inhibition consisted of a mixed-type inhibition mechanism in which the inhibitory effect depends on Ulva concentration. Overall, the results confirmed the presence of protease inhibitors in Ulva, though heat treatment was enough for inactivating these compounds.