Synthesis of Broad-Specificity Activity-Based Probes for Exo-β-Mannosidases

Exo--mannosidases are a broad class of stereochemically retaining hydrolases that are essential for the breakdown of complex carbohydrate substrates found in all kingdoms of life. Yet the detection of exo--mannosidases...

Deep Mutational Engineering of broadly-neutralizing and picomolar affinity nanobodies to accommodate SARS-CoV-1 & 2 antigenic polymorphism

We report in this study the molecular engineering of nanobodies that bind with picomolar affinity to both SARS-CoV-1 and SARS-CoV-2 Receptor Binding Domains (RBD) and are highly neutralizing. We applied Deep Mutational Engineering to VHH72, a nanobody initially specific for SARS-CoV-1 RBD with little cross-reactivity to SARS-CoV-2 antigen. We first identified all the individual VHH substitutions that increase binding to SARS-CoV-2 RBD and then screened highly focused combinatorial libraries to isolate engineered nanobodies with improved properties. The corresponding VHH-Fc molecules show high affinities for SARS-CoV-2 antigens from various emerging variants and SARS-CoV-1, block the interaction between ACE2 and RBD and neutralize the virus with high efficiency. Its rare specificity across sarbecovirus relies on its peculiar epitope outside the immunodominant regions. The engineered nanobodies share a common motif of three amino acids, which contribute to the broad specificity of recognition. These nanobodies appears as promising therapeutic candidates to fight SARS-CoV-2 infection.

Afadin couples RAS GTPases to the polarity rheostat Scribble

AFDN/Afadin is required for establishment and maintenance of cell-cell contacts and is a unique effector of RAS GTPases. The biological consequences of RAS signalling to AFDN are unknown. Here, we use proximity-based proteomics to generate an interaction map for the long and short isoforms of AFDN, identifying the polarity protein SCRIB/Scribble as the top hit. We reveal that the first PDZ domain of SCRIB and the AFDN FHA domain mediate a direct but non-canonical interaction between these important adhesion and polarity proteins. Further, the dual RA domains of AFDN have broad specificity for RAS and RAP GTPases, and KRAS co-localizes with and promotes AFDN-SCRIB complex formation. Knockout of AFDN or SCRIB in MCF7 epithelial cells disrupts MAPK and PI3K activation and inhibits cell motility in a growth factor-dependent manner. These data have important implications for understanding why cells with activated RAS have reduced cell contacts and polarity defects, and finally begin to characterize AFDN as a RAS effector.

Synthesis of Zearalenone Immunogen and Comparative Analysis of Antibody Characteristics

Background. This study aimed to explore the zearalenone (ZEN) immunogen synthesis method, immunogenicity, and antibody characteristics and to lay a foundation for the establishment of immunoassay methods for ZEN single residue and ZEN and its analogs total residue. Methods. Based on the molecular structure and active sites of ZEN, oxime active ester (OAE), condensation mixed anhydride (CMA), formaldehyde (FA), and 1,4-butanediol diglycidyl ether method (BDE) were designed and used for immunogen (ZEN-BSA) synthesis. The immunogens were identified by infrared (IR) and ultraviolet (UV) spectra and gel electrophoresis (SDS-PAGE) and were then used to immunize Balb/c mice to prepare ZEN polyclonal antibody (ZEN pAb). The titers and sensitivity of the ZEN pAb were determined by indirect noncompetitive ELISA (inELISA) and indirect competitive ELISA (icELISA), respectively, and its specificity was assessed by the cross-reaction test (CR). Results. ZEN-BSA was successfully synthesized, and the molecular binding ratios of ZEN to BSA were 17.2 : 1 (OAE), 14.6 : 1 (CMA), 9.7 : 1 (FA), and 8.3 : 1 (BDE), respectively. The highest inELISA titers of ZEN pAb of each group were 1 : (6.4 × 103) (OAE), 1 : (3.2 × 103) (CMA), 1 : (1.6 × 103) (FA), and 1 : (1.6 × 103) (BDE), respectively. The 50% inhibition concentrations (IC50) for ZEN by icELISA of each group were 11.67 μg/L (OAE), 16.29 μg/L (CMA), 20.92 μg/L (FA) and 24.36 μg/L (BDE), respectively. ZEN pAb from the mice immunized with ZEN-BSA (OAE) and ZEN-BSA (CMA) had class broad specificity to ZEN and its analogs. The CRs of ZEN pAb with α-ZAL, β-ZAL, α-ZOL, β-ZOL, and ZON were 36.53%, 16.98%, 64.33%, 20.16%, and 10.66%, respectively. ZEN pAb from the mice immunized with ZEN-BSA (FA) and ZEN-BSA (BDE) had high specificity for ZEN. The CRs of ZEN pAb with its analogs were all less than 1.0%. Conclusion. This study demonstrated that the preparation of the class broad-specificity antibodies of ZEN and its analogs can be achieved by immunizing animals with the immunogen ZEN-BSA prepared by the OAE method, while the preparation of highly specific antibodies can be achieved by immunizing animals with the immunogen ZEN-BSA prepared by the FA method. These findings lay the material and technical foundation for immunoassay of ZEN single residue and ZEN and its analogs total residue.

A Broad-Specificity Chitinase from Penicillium oxalicum k10 Exhibits Antifungal Activity and Biodegradation Properties of Chitin

Penicillium oxalicum k10 isolated from soil revealed the hydrolyzing ability of shrimp chitin and antifungal activity against Sclerotinia sclerotiorum. The k10 chitinase was produced from a powder chitin-containing medium and purified by ammonium sulfate precipitation and column chromatography. The purified chitinase showed maximal activity toward colloidal chitin at pH 5 and 40 °C. The enzymatic activity was enhanced by potassium and zinc, and it was inhibited by silver, iron, and copper. The chitinase could convert colloidal chitin to N-acetylglucosamine (GlcNAc), (GlcNAc)2, and (GlcNAc)3, showing that this enzyme had endocleavage and exocleavage activities. In addition, the chitinase prevented the mycelial growth of the phytopathogenic fungi S. sclerotiorum and Mucor circinelloides. These results indicate that k10 is a potential candidate for producing chitinase that could be useful for generating chitooligosaccharides from chitinous waste and functions as a fungicide.

SARS-CoV-2 501Y.V2 (B.1.351) elicits cross-reactive neutralizing antibodies

Neutralization escape by SARS-CoV-2 variants, as has been observed in the 501Y.V2 (B.1.351) variant, has impacted the efficacy of first generation COVID-19 vaccines. Here, the antibody response to the 501Y.V2 variant was examined in a cohort of patients hospitalized with COVID-19 in early 2021 - when over 90% of infections in South Africa were attributed to 501Y.V2. Robust binding and neutralizing antibody titers to the 501Y.V2 variant were detected and these binding antibodies showed high levels of cross-reactivity for the original variant, from the first wave. In contrast to an earlier study where sera from individuals infected with the original variant showed dramatically reduced potency against 501Y.V2, sera from 501Y.V2-infected patients maintained good cross-reactivity against viruses from the first wave. Furthermore, sera from 501Y.V2-infected patients also neutralized the 501Y.V3 (P.1) variant first described in Brazil, and now circulating globally. Collectively these data suggest that the antibody response in patients infected with 501Y.V2 has a broad specificity and that vaccines designed with the 501Y.V2 sequence may elicit more cross-reactive responses.