Acceptors stability modulates the efficiency of post-translational protein N-glycosylation.

N-glycosylation is the most common protein modification in the eukaryotic secretory pathway. It involves the attachment a high mannose glycan to Asn residues in the context of Asn-X-Ser/Thr/Cys, a motif known as N-glycosylation sequon. This process is mediated by STT3A and STT3B, the catalytic subunits of the oligosaccharyltransferase complexes. STT3A forms part of complexes associated with the SEC61 translocon and functions co-translationally. Vacant sequons have another opportunity for glycosylation by complexes carrying STT3B. Local sequence information plays an important role in determining N-glycosylation efficiency, but non-local factors can also have a significant impact. For instance, certain proteins associated with human genetic diseases exhibit abnormal N-glycosylation levels despite having wild-type acceptor sites. Here, we investigated the effect of protein stability on this process. To this end, we generated a family of 40 N-glycan acceptors based on superfolder GFP, and we measured their efficiency in HEK293 cells and in two derived cell lines lacking STT3B or STT3A. Sequon occupancy was highly dependent on protein stability, improving as the thermodynamic stability of the acceptor proteins decreases. This effect is mainly due to the activity of the STT3B-based OST complex. These findings can be integrated into a simple kinetic model that distinguishes local information within sequons from global information of the acceptor proteins.

Development of high-affinity nanobodies specific for NaV1.4 and NaV1.5 voltage-gated sodium channel isoforms.

Voltage-gated sodium channels, NaVs, are responsible for the rapid rise of action potentials in excitable tissues. NaV channel mutations have been implicated in several human genetic diseases, such as hypokalemic periodic paralysis, myotonia, and long-QT and Brugada syndromes. Here, we generated high-affinity anti-NaV nanobodies (Nbs), Nb17 and Nb82, that recognize the NaV1.4 (skeletal muscle) and NaV1.5 (cardiac muscle) channel isoforms. These Nbs were raised in llama (Lama glama) and selected from a phage display library for high affinity to the C-terminal (CT) region of NaV1.4. The Nbs were expressed in Escherichia coli, purified, and biophysically characterized. Development of high-affinity Nbs specifically targeting a given human NaV isoform has been challenging because they usually show undesired crossreactivity for different NaV isoforms. Our results show, however, that Nb17 and Nb82 recognize the CTNaV1.4 or CTNaV1.5 over other CTNav isoforms. Kinetic experiments by biolayer interferometry determined that Nb17 and Nb82 bind to the CTNaV1.4 and CTNaV1.5 with high affinity (KD ∼ 40-60 nM). In addition, as proof of concept, we show that Nb82 could detect NaV1.4 and NaV1.5 channels in mammalian cells and tissues by Western blot. Furthermore, human embryonic kidney cells expressing holo NaV1.5 channels demonstrated a robust FRET-binding efficiency for Nb17 and Nb82. Our work lays the foundation for developing Nbs as anti-NaV reagents to capture NaVs from cell lysates and as molecular visualization agents for NaVs.

Protein fibrillation lag times during kinetic inhibition.

Protein aggregation is linked to more than 30 human pathologies, including Alzheimer's and Parkinson's diseases. Since small oligomers that form at the beginning of the fibrillation process probably are the most toxic elements, therapeutic strategies involving fibril fragmentation could be detrimental. An alternative approach, named kinetic inhibition, aims to prevent fibril formation by using small ligands that stabilize the parent protein. The factors that govern fibrillation lag times during kinetic inhibition are largely unknown, notwithstanding their importance for designing effective long-term therapies. Inhibitor-bound species are not likely to be incorporated into the core of mature fibrils, although their presence could alter the kinetics of the fibrillation process. For instance, inhibitor-bound species may act as capping elements that impair the nucleation process and/or fibril growth. Here, we address this issue by studying the effect of two natural inhibitors on the fibrillation behavior of lysozyme at neutral pH. We analyzed a set of 79 fibrillation curves obtained in lysozyme alone and a set of 37 obtained in the presence of inhibitors. We calculated the concentrations of the relevant species at the beginning of the curves using the inhibitor-binding constants measured under the same experimental conditions. We found that inhibitor-bound protein species do not affect fibrillation onset times, which are mainly determined by the concentration of unbound protein species present in equilibrium. In this system, knowledge of the fibrillation kinetics and inhibitor affinities suffices to predict the effect of kinetic inhibitors on fibrillation lag times. In addition, we developed a new methodology to better estimate fibrillation lag times from experimental curves.

The allosteric modulation of thyroxine-binding globulin affinity is entropy driven.

BACKGROUND: Thyroxine-binding globulin (TBG) is a non-inhibitory member of the serpin family of proteins whose main structural element is the reactive center loop (RCL), that, upon cleavage by proteases, is inserted into the protein core adopting a ß-strand conformation (stressed to relaxed transition, S-to-R). After S-to-R transition thyroxine (T4) affinity decreases. However, crystallographic studies in the presence or absence of the hormone in different states are unable to show significant differences in the structure and interactions of the binding site. Experimental results also suggest the existence of several S states (differing in the number of inserted RCL residues), associated with a differential affinity. METHODS: To shed light into the molecular basis that regulates T4 affinity according to the degree of RCL insertion in TBG, we performed extended molecular dynamics simulations combined with several thermodynamic analysis of the T4 binding to TBG in three different S states, and in the R state. RESULTS: Our results show that, despite T4 binding in the protein by similar interactions in all states, a good correlation between the degree of RCL insertion and the binding affinity, driven by a change in TBG conformational entropy, was observed. CONCLUSION: TBG allosteric regulation is entropy driven. The presence of multiple S states may allow more efficient T4 release due to protease activity. GENERAL SIGNIFICANCE: The presented results are clear examples of how computer simulation methods can reveal the thermodynamic basis of allosteric effects, and provide a general framework for understanding serpin allosteric affinity regulation.

Development of a hyperimmune equine serum therapy for COVID-19 in Argentina.

The disease named COVID-19, caused by the SARS-CoV-2 coronavirus, is currently generating a global pandemic. Vaccine development is no doubt the best long-term immunological approach, but in the current epidemiologic and health emergency there is a need for rapid and effective solutions. Convalescent plasma is the only antibody-based therapy available for COVID-19 patients to date. Equine polyclonal antibodies (EpAbs) put forward a sound alternative. The new generation of processed and purified EpAbs containing highly purified F(ab')2 fragments demonstrated to be safe and well tolerated. EpAbs are easy to manufacture allowing a fast development and scaling up for a treatment. Based on these ideas, we present a new therapeutic product obtained after immunization of horses with the receptor-binding domain of the viral Spike glycoprotein. Our product shows around 50 times more potency in in vitro seroneutralization assays than the average of convalescent plasma. This result may allow us to test the safety and efficacy of this product in a phase 2/3 clinical trial to be conducted in July 2020 in the metropolitan area of Buenos Aires, Argentina.

La enfermedad denominada COVID-19 es causada por el coronavirus SARS-CoV-2 y es actualmente considerada una pandemia a nivel global. El desarrollo de vacunas es sin duda la mejor estrategia a largo plazo, pero debido a la emergencia sanitaria, existe una necesidad urgente de encontrar soluciones rápidas y efectivas para el tratamiento de la enfermedad. Hasta la fecha, el uso de plasma de convalecientes es la única inmunoterapia disponible para pacientes hospitalizados con COVID-19. El uso de anticuerpos policlonales equinos (EpAbs) es otra alternativa terapéutica interesante. La nueva generación de EpAbs incluyen el procesamiento y purificación de los mismos y la obtención de fragmentos F(ab')2 con alta pureza y un excelente perfil de seguridad en humanos. Los EpAbs son fáciles de producir, lo cual permite el desarrollo rápido y la elaboración a gran escala de un producto terapéutico. En este trabajo mostramos el desarrollo de un suero terapéutico obtenido luego de la inmunización de caballos utilizando el receptor-binding domain de la glicoproteína Spike del virus. Nuestro producto mostró ser alrededor de 50 veces más potente en ensayos de seroneutralización in vitro que el promedio de los plasmas de convalecientes. Estos resultados nos permitirían testear la seguridad y eficacia de nuestro producto en ensayos clínicos de fase 2/3 a realizarse a partir de julio de 2020 en la zona metropolitana de Buenos Aires, Argentina.

N-glycosylation Triggers a Dual Selection Pressure in Eukaryotic Secretory Proteins.

Nearly one third of the eukaryotic proteome traverses the secretory pathway and most of these proteins are N-glycosylated in the lumen of the endoplasmic reticulum. N-glycans fulfill multiple structural and biological functions, and are crucial for productive folding of many glycoproteins. N-glycosylation involves the attachment of an oligosaccharide to selected asparagine residues in the sequence N-X-S/T (X ≠ P), a motif known as an N-glycosylation'sequon'. Mutations that create novel sequons can cause disease due to the destabilizing effect of a bulky N-glycan. Thus, an analogous process must have occurred during evolution, whenever ancestrally cytosolic proteins were recruited to the secretory pathway. Here, we show that during evolution N-glycosylation triggered a dual selection pressure on secretory pathway proteins: while sequons were positively selected in solvent exposed regions, they were almost completely eliminated from buried sites. This process is one of the sharpest evolutionary signatures of secretory pathway proteins, and was therefore critical for the evolution of an efficient secretory pathway.

Development of a hyperimmune equine serum therapy for COVID-19 in Argentina

The disease named COVID-19, caused by the SARS-CoV-2 coronavirus, is currently generating a global pandemic. Vaccine development is no doubt the best long-term immunological approach, but in the current epidemiologic and health emergency there is a need for rapid and effective solutions. Convalescent plasma is the only antibody-based therapy available for COVID-19 patients to date. Equine polyclonal antibodies (EpAbs) put forward a sound alternative. The new generation of processed and purified EpAbs containing highly purified F(ab’)2 fragments demonstrated to be safe and well tolerated. EpAbs are easy to manufacture allowing a fast development and scaling up for a treatment. Based on these ideas, we present a new therapeutic product obtained after immunization of horses with the receptor-binding domain of the viral Spike glycoprotein. Our product shows around 50 times more potency in in vitro seroneutralization assays than the average of convalescent plasma. This result may allow us to test the safety and efficacy of this product in a phase 2/3 clinical trial to be conducted in July 2020 in the metropolitan area of Buenos Aires, Argentina.

La enfermedad denominada COVID-19 es causada por el coronavirus SARS-CoV-2 y es actualmente considerada una pandemia a nivel global. El desarrollo de vacunas es sin duda la mejor estrategia a largo plazo, pero debido a la emergencia sanitaria, existe una necesidad urgente de encontrar soluciones rápidas y efectivas para el tratamiento de la enfermedad. Hasta la fecha, el uso de plasma de convalecientes es la única inmunoterapia disponible para pacientes hospitalizados con COVID-19. El uso de anticuerpos policlonales equinos (EpAbs) es otra alternativa terapéutica interesante. La nueva generación de EpAbs incluyen el procesamiento y purificación de los mismos y la obtención de fragmentos F(ab’)2 con alta pureza y un excelente perfil de seguridad en humanos. Los EpAbs son fáciles de producir, lo cual permite el desarrollo rápido y la elaboración a gran escala de un producto terapéutico. En este trabajo mostramos el desarrollo de un suero terapéutico obtenido luego de la inmunización de caballos utilizando el receptor-binding domain de la glicoproteína Spike del virus. Nuestro producto mostró ser alrededor de 50 veces más potente en ensayos de seroneutralización in vitro que el promedio de los plasmas de convalecientes. Estos resultados nos permitirían testear la seguridad y eficacia de nuestro producto en ensayos clínicos de fase 2/3 a realizarse a partir de julio de 2020 en la zona metropolitana de Buenos Aires, Argentina.