Novel high-yield potato protease inhibitor panels block a wide array of proteases involved in viral infection and crucial tissue damage.

Viruses critically rely on various proteases to ensure host cell entry and replication. In response to viral infection, the host will induce acute tissue inflammation pulled by granulocytes. Upon hyperactivation, neutrophil granulocytes may cause undue tissue damage through proteolytic degradation of the extracellular matrix. Here, we assess the potential of protease inhibitors (PI) derived from potatoes in inhibiting viral infection and reducing tissue damage. The original full spectrum of potato PI was developed into five fractions by means of chromatography and hydrolysis. Individual fractions showed varying inhibitory efficacy towards a panel of proteases including trypsin, chymotrypsin, ACE2, elastase, and cathepsins B and L. The fractions did not interfere with SARS-CoV-2 infection of Vero E6 cells in vitro. Importantly, two of the fractions fully inhibited elastin-degrading activity of complete primary human neutrophil degranulate. These data warrant further development of potato PI fractions for biomedical purposes, including tissue damage crucial to SARS-CoV-2 pathogenesis. KEY MESSAGES: Protease inhibitor fractions from potato differentially inhibit a series of human proteases involved in viral replication and in tissue damage by overshoot inflammation. Protease inhibition of cell surface receptors such as ACE2 does not prevent virus infection of Vero cells in vitro. Protease inhibitors derived from potato can fully inhibit elastin-degrading primary human neutrophil proteases. Protease inhibitor fractions can be produced at high scale (hundreds of thousands of kilograms, i.e., tons) allowing economically feasible application in lower and higher income countries.

Rheological properties of patatin gels compared with ß-lactoglobulin, ovalbumin, and glycinin.

BACKGROUND: The thermal unfolding and rheological properties of patatin gels were compared with those of commonly used proteins (ß-lactoglobulin, ovalbumin, glycinin). RESULTS: A significant difference between these proteins was observed in both the denaturation temperature (59 °C for patatin; about 20 °C lower than the other proteins) and the onset temperature of gel formation (50-60 °C, compared to 70-85 °C for the other proteins). At low ionic strength the minimal concentration was only 6% (w/v) for patatin, compared to 8-11% for the other proteins. This effect was attributed to the relatively high exposed hydrophobicity of patatin as determined by hydrophobic interaction chromatography. For gels compared at 'iso-strength', the frequency dependence was found to be close to identical, while small differences were observed in the strain at fracture. CONCLUSIONS: Patatin was found to form gels with comparable small-deformational rheological properties as typical food proteins. In addition, at concentrations where the elastic modulus was similar for all proteins, the frequency and strain dependence were also comparable. From this it is concluded that patatin is a promising protein to be used in food applications as a gelling agent.

Comprehensive overview of the quality of plant- And animal-sourced proteins based on the digestible indispensable amino acid score.

Indispensable amino acid (IAA) composition and standardized ileal digestibility (SID) of five animal- and 12 plant-based proteins were used to calculate their respective Digestible Indispensable Amino Score (DIAAS) according to the three age categories defined by the Food and Agriculture Organization (FAO). Mean IAA content and mean SID obtained from each protein dataset were subsequently used to simulate optimal nutritional quality of protein mixtures. Datasets revealed considerable variation in DIAAS within the same protein source and among different protein sources. Among the selected protein sources, and based on the 0.5- to 3-year-old reference pattern, pork meat, casein, egg, and potato proteins are classified as excellent quality proteins with an average DIAAS above 100. Whey and soy proteins are classified as high-quality protein with an average DIAAS ≥75. Gelatin, rapeseed, lupin, canola, corn, hemp, fava bean, oat, pea, and rice proteins are classified in the no quality claim category (DIAAS <75). Potato, soy, and pea proteins can complement a broad range of plant proteins, leading to higher DIAAS when supplied in the form of protein mixtures and at specific ratios. Such complementarity highlights the potential to achieve an optimal nutritional efficiency with plant proteins alone.

Exopolysaccharide structure is not a determinant of host-plant specificity in nodulation of Vicia sativa roots.

Exopolysaccharide (EPS)-deficient strains of the root nodule symbiote Rhizobium leguminosarum induce formation of abortive infection threads in Vicia sativa subsp. nigra roots. As a result, the nodule tissue remains uninfected. Formation of an infection thread can be restored by coinoculation of the EPS-deficient mutant with a Nod factor-deficient strain, which produces a similar EPS structure. This suggests that EPS contributes to host-plant specificity of nodulation. Here, a comparison was made of i) coinoculation with heterologous strains with different EPS structures, and ii) introduction of the pRL1JI Sym plasmid or a nod gene-encoding fragment in the same heterologous strains. Most strains not complementing in coinoculation experiments were able to nodulate V. sativa roots as transconjugants. Apparently, coinoculation is a delicate approach in which differences in root colonization ability or bacterial growth rate easily affect successful infection-thread formation. Obviously, lack of infection-thread formation in coinoculation studies is not solely determined by EPS structure. Transconjugation data show that different EPS structures can allow infection-thread formation and subsequent nodulation of V. sativa roots.

A novel polar surface polysaccharide from Rhizobium leguminosarum binds host plant lectin.

Rhizobium bacteria produce different surface polysaccharides which are either secreted in the growth medium or contribute to a capsule surrounding the cell. Here, we describe isolation and partial characterization of a novel high molecular weight surface polysaccharide from a strain of Rhizobium leguminosarum that nodulates Pisum sativum (pea) and Vicia sativa (vetch) roots. Carbohydrate analysis showed that the polysaccharide consists for 95% of mannose and glucose, with minor amounts of galactose and rhamnose. Lectin precipitation analysis revealed high binding affinity of pea and vetch lectin for this polysaccharide, in contrast to the other known capsular and extracellular polysaccharides of this strain. Expression of the polysaccharide was independent of the presence of a Sym plasmid or the nod gene inducer naringenin. Incubation of R. leguminosarum with labelled pea lectin showed that this polysaccharide is exclusively localized on one of the poles of the bacterial cell. Vetch roots incubated with rhizobia and labelled pea lectin revealed that this bacterial pole is involved in attachment to the root surface. A mutant strain deficient in the production of this polysaccharide was impaired in attachment and root hair infection under slightly acidic conditions, in contrast to the situation at slightly alkaline conditions. Our data are consistent with the hypothesis that rhizobia can use (at least) two mechanisms for docking at the root surface, with use of a lectin-glycan mechanism under slightly acidic conditions.

Involvement of exo5 in production of surface polysaccharides in Rhizobium leguminosarum and its role in nodulation of Vicia sativa subsp. nigra.

Analysis of two exopolysaccharide-deficient mutants of Rhizobium leguminosarum, RBL5808 and RBL5812, revealed independent Tn5 transposon integrations in a single gene, designated exo5. As judged from structural and functional homology, this gene encodes a UDP-glucose dehydrogenase responsible for the oxidation of UDP-glucose to UDP-glucuronic acid. A mutation in exo5 affects all glucuronic acid-containing polysaccharides and, consequently, all galacturonic acid-containing polysaccharides. Exo5-deficient rhizobia do not produce extracellular polysaccharide (EPS) or capsular polysaccharide (CPS), both of which contain glucuronic acid. Carbohydrate composition analysis and nuclear magnetic resonance studies demonstrated that EPS and CPS from the parent strain have very similar structures. Lipopolysaccharide (LPS) molecules produced by the mutant strains are deficient in galacturonic acid, which is normally present in the core and lipid A portions of the LPS. The sensitivity of exo5 mutant rhizobia to hydrophobic compounds shows the involvement of the galacturonic acid residues in the outer membrane structure. Nodulation studies with Vicia sativa subsp. nigra showed that exo5 mutant rhizobia are impaired in successful infection thread colonization. This is caused by strong agglutination of EPS-deficient bacteria in the root hair curl. Root infection could be restored by simultaneous inoculation with a Nod factor-defective strain which retained the ability to produce EPS and CPS. However, in this case colonization of the nodule tissue was impaired.