Tremendous progress in the last few years has been made to explain how seemingly harmless environmental proteins from different origins can induce potent Th2‐biased inflammatory responses. Convergent findings have shown the key roles of allergens displaying proteolytic activity in the initiation and progression of the allergic response. Through their propensity to activate IgE‐independent inflammatory pathways, certain allergenic proteases are now considered as initiators for sensitization to themselves and to non‐protease allergens. The protease allergens degrade junctional proteins of keratinocytes or airway epithelium to facilitate allergen delivery across the epithelial barrier and their subsequent uptake by antigen‐presenting cells. Epithelial injuries mediated by these proteases together with their sensing by protease‐activated receptors (PARs) elicit potent inflammatory responses resulting in the release of pro‐Th2 cytokines (IL‐6, IL‐25, IL‐1β, TSLP) and danger‐associated molecular patterns (DAMPs; IL‐33, ATP, uric acid). Recently, protease allergens were shown to cleave the protease sensor domain of IL‐33 to produce a super‐active form of the alarmin. At the same time, proteolytic cleavage of fibrinogen can trigger TLR4 signaling, and cleavage of various cell surface receptors further shape the Th2 polarization. Remarkably, the sensing of protease allergens by nociceptive neurons can represent a primary step in the development of the allergic response. The goal of this review is to highlight the multiple innate immune mechanisms triggered by protease allergens that converge to initiate the allergic response.

The cysteine protease enzyme legumain hydrolyzes peptide bonds with high specificity after asparagine and under more acidic conditions after aspartic acid [ Baker , E. N. J. Mol. Biol. 1980 , 141 , 441−484; Baker , E. N. ; J. Mol. Biol. 1977 , 111 , 207 – 210; Drenth , J. ; Biochemistry 1976 , 15 , 3731 – 3738; Menard , R. ; J. Cell. Biochem. 1994 , 137; Polgar , L. Eur. J. Biochem. 1978 , 88 , 513 – 521; Storer , A. C. ; Methods Enzymol. 1994 , 244 , 486 – 500. Remarkably, legumain additionally exhibits ligase activity that prevails at pH > 5.5. The atomic reaction mechanisms including their pH dependence are only partly understood. Here we present a density functional theory (DFT)-based quantum mechanics/molecular mechanics (QM/MM) study of the detailed reaction mechanism of both activities for human legumain in solution. Contrasting the situation in other papain-like proteases, our calculations reveal that the active site Cys189 must be present in the protonated state for a productive nucleophilic attack and simultaneous rupture of the scissile peptide bond, consistent with the experimental pH profile of legumain-catalyzed cleavages. The resulting thioester intermediate (INT1) is converted by water attack on the thioester into a second intermediate, a diol (INT2), which is released by proton abstraction by Cys189. Surprisingly, we found that ligation is not the exact reverse of the proteolysis but can proceed via two distinct routes. Whereas the transpeptidation route involves aminolysis of the thioester (INT1), at pH 6 a cysteine-independent, histidine-assisted ligation route was found. Given legumain’s important roles in immunity, cancer, and neurodegenerative diseases, our findings open up possibilities for targeted drug design in these fields.

Background: Over 100 million people worldwide suffer from birch pollen allergy. Bet v 1 has been identified as the major birch pollen allergen. However, the molecular mechanisms of birch allergic sensitization, including the roles of Bet v 1 and other components of the birch pollen extract, remain incompletely understood. Here, we examined how known birch pollen–derived molecules influence the endolysosomal processing of Bet v 1, thereby shaping its allergenicity.Methods: We analyzed the biochemical and immunological interaction of ligands with Bet v 1. We then investigated the proteolytic processing of Bet v 1 by endosomal extracts in the presence and absence of ligands, followed by a detailed kinetic analysis of Bet v 1 processing by individual endolysosomal proteases as well as the T‐cell epitope presentation in BMDCs.Results: We identified E₁ phytoprostanes as novel Bet v 1 ligands. Pollen‐derived ligands enhanced the proteolytic resistance of Bet v 1, affecting degradation kinetics and preferential cleavage sites of the endolysosomal proteases cathepsin S and legumain. E₁ phytoprostanes exhibited a dual role by stabilizing Bet v 1 and inhibiting cathepsin protease activity.Conclusion: Bet v 1 can serve as a transporter of pollen‐derived, bioactive compounds. When carried to the endolysosome, such compounds can modulate the proteolytic activity, including its processing by cysteine cathepsins. We unveil a paradigm shift from an allergen‐centered view to a more systemic view that includes the host endolysosomal enzymes.

The widespread occurrence of SARS-CoV-2 has had a profound effect on society and a vaccine is currently being developed. Angiotensin-converting enzyme 2 (ACE2) is the primary host cell receptor that interacts with the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein. Although pneumonia is the main symptom in severe cases of SARS-CoV-2 infection, the expression levels of ACE2 in the lung is low, suggesting the presence of another receptor for the spike protein. In order to identify the additional receptors for the spike protein, we screened a receptor for the SARS-CoV-2 spike protein from the lung cDNA library. We cloned L-SIGN as a specific receptor for the N-terminal domain (NTD) of the SARS-CoV-2 spike protein. The RBD of the spike protein did not bind to L-SIGN. In addition, not only L-SIGN but also DC-SIGN, a closely related C-type lectin receptor to L-SIGN, bound to the NTD of the SARS-CoV-2 spike protein. Importantly, cells expressing L-SIGN and DC-SIGN were both infected by SARS-CoV-2. Furthermore, L-SIGN and DC-SIGN induced membrane fusion by associating with the SARS-CoV-2 spike protein. Serum antibodies from infected patients and a patient-derived monoclonal antibody against NTD inhibited SARS-CoV-2 infection of L-SIGN or DC-SIGN expressing cells. Our results highlight the important role of NTD in SARS-CoV-2 dissemination through L-SIGN and DC-SIGN and the significance of having anti-NTD neutralizing antibodies in antibody-based therapeutics.