Fast-Track Discovery of SARS-CoV-2-Neutralizing Antibodies from Human B Cells by Direct Functional Screening.

As the COVID-19 pandemic revealed, rapid development of vaccines and therapeutic antibodies are crucial to guarantee a quick return to the status quo of society. In early 2020, we deployed our droplet microfluidic single-cell-based platform DROPZYLLA® for the generation of cognate antibody repertoires of convalescent COVID-19 donors. Discovery of SARS-CoV-2-specific antibodies was performed upon display of antibodies on the surface of HEK293T cells by antigen-specific sorting using binding to the SARS-CoV-2 spike and absence of binding to huACE2 as the sort criteria. This efficiently yielded antibodies within 3-6 weeks, of which up to 100% were neutralizing. One of these, MTX-COVAB, displaying low picomolar neutralization IC50 of SARS-CoV-2 and with a neutralization potency on par with the Regeneron antibodies, was selected for GMP manufacturing and clinical development in June 2020. MTX-COVAB showed strong efficacy in vivo and neutralized all identified clinically relevant variants of SARS-CoV-2 at the time of its selection. MTX-COVAB completed GMP manufacturing by the end of 2020, but clinical development was stopped when the Omicron variant emerged, a variant that proved to be detrimental to all monoclonal antibodies already approved. The present study describes the capabilities of the DROPZYLLA® platform to identify antibodies of high virus-neutralizing capacity rapidly and directly.

Glycan variability on a recombinant IgG antibody transiently produced in HEK-293E cells.

In this study, a recombinant monoclonal IgG antibody was produced by transient gene expression (TGE) in suspension-adapted HEK-293E cells. The objective of the study was to determine the variation in recombinant IgG yield and glycosylation in ten independent transfections. In a ten-day batch process, the variation in transient IgG yield in the ten batches was less than 30% with the specific productivity averaging 20.2 ± 2.6 pg/cell/day. We characterized the N-glycosylation profile of each batch of affinity-purified IgG by intact protein and bottom-up mass spectrometry. Four major glycans were identified at Asn(297) in the ten batches with the maximum relative deviation for a single glycoform being 2.5%. In addition, within any single transfection there was little variation in glycoforms over the ten-day culture. Our experimental data indicate that with TGE, the production of recombinant IgG with little batch-to-batch variation in volumetric yield and protein glycosylation is feasible, even in a non-instrumented cultivation system as described here.

Directed evolution of the suicide protein O6-alkylguanine-DNA alkyltransferase for increased reactivity results in an alkylated protein with exceptional stability.

Here we present a biophysical, structural, and computational analysis of the directed evolution of the human DNA repair protein O(6)-alkylguanine-DNA alkyltransferase (hAGT) into SNAP-tag, a self-labeling protein tag. Evolution of hAGT led not only to increased protein activity but also to higher stability, especially of the alkylated protein, suggesting that the reactivity of the suicide enzyme can be influenced by stabilizing the product of the irreversible reaction. Whereas wild-type hAGT is rapidly degraded in cells after alkyl transfer, the high stability of benzylated SNAP-tag prevents proteolytic degradation. Our data indicate that the intrinstic stability of a key α helix is an important factor in triggering the unfolding and degradation of wild-type hAGT upon alkyl transfer, providing new insights into the structure-function relationship of the DNA repair protein.

Alternative splicing gives rise to different isoforms of the Neurospora crassa Tob55 protein that vary in their ability to insert beta-barrel proteins into the outer mitochondrial membrane.

Tob55 is the major component of the TOB complex, which is found in the outer membrane of mitochondria. A sheltered knockout of the tob55 gene was developed in Neurospora crassa. When grown under conditions that reduce the levels of the Tob55 protein, the strain exhibited a reduced growth rate and mitochondria isolated from these cells were deficient in their ability to import beta-barrel proteins. Surprisingly, Western blots of wild-type mitochondrial proteins revealed two bands for Tob55 that differed by approximately 4 kDa in their apparent molecular masses. Sequence analysis of cDNAs revealed that the tob55 mRNA is alternatively spliced and encodes three isoforms of the protein, which are predicted to contain 521, 516, or 483 amino acid residues. Mass spectrometry of proteins isolated from purified outer membrane vesicles confirmed the existence of each isoform in mitochondria. Strains that expressed each isoform of the protein individually were constructed. When cells expressing only the longest form of the protein were grown at elevated temperature, their growth rate was reduced and mitochondria isolated from these cells were deficient in their ability to assembly beta-barrel proteins.

Evolving the substrate specificity of O6-alkylguanine-DNA alkyltransferase through loop insertion for applications in molecular imaging.

We introduce a strategy for evolving protein substrate specificity by the insertion of random amino acid loops into the protein backbone. Application of this strategy to human O6-alkylguanine-DNA alkyltransferase (AGT) led to the isolation of mutants that react with the non-natural substrate O6-propargylguanine. Libraries generated by conventional random or targeted saturation mutagenesis, by contrast, did not yield any mutants with activity towards this new substrate. The strategy of loop insertion to alter enzyme specificity should be general and applicable to other classes of proteins. An important application of the isolated AGT mutant is in molecular imaging, where the mutant and parental AGTs are used to label two different AGT fusion proteins with different fluorophores in the same living cell or in vitro . This allowed the establishment of fluorescence-based assays to detect protein-protein interactions and measure enzymatic activities.

Proteome analysis of mitochondrial outer membrane from Neurospora crassa.

The mitochondrial outer membrane mediates numerous interactions between the metabolic and genetic systems of mitochondria and the rest of the eukaryotic cell. We performed a proteomic study to discover novel functions of components of the mitochondrial outer membrane. Proteins of highly pure outer membrane vesicles (OMV) from Neurospora crassa were identified by a combination of LC-MS/MS of tryptic peptide digests and gel electrophoresis of solubilized OMV proteins, followed by their identification using MALDI-MS PMF. Among the 30 proteins found in at least three of four separate analyses were 23 proteins with known functions in the outer membrane. These included components of the import machinery (the TOM and TOB complexes), a pore-forming component (porin), and proteins that control fusion and fission of the organelle. In addition, proteins playing a role in various biosynthetic pathways, whose intracellular location had not been established previously, could be localized to the mitochondrial outer membrane. Thus, the proteome of the outer membrane can help in identifying new mitochondria-related functions.

Impact of transcription factor Sox8 on oligodendrocyte specification in the mouse embryonic spinal cord.

The myelin-forming oligodendrocytes of the mouse embryonic spinal cord express the three group E Sox proteins Sox8, Sox9, and Sox10. They require Sox9 for their specification from neuroepithelial cells of the ventricular zone and Sox10 for their terminal differentiation and myelination. Here, we show that during oligodendrocyte development, Sox8 is expressed after Sox9, but before Sox10. Loss of Sox8 did not impair oligodendrocyte specification by itself, but enhanced the Sox9-dependent defect. Oligodendrocyte progenitors were still generated in the Sox9-deficient spinal cord, albeit at 20-fold lower rates than in the wildtype. Combined loss of Sox8 and Sox9, in contrast, led to a near complete loss of oligodendrocytes. Other cell types such as ventricular zone cells and radial glia remained unaffected in their numbers as well as their rates of proliferation and apoptosis. Oligodendrocyte development thus relies on the differential contribution of all three group E Sox proteins at various phases.

Role of Tom5 in maintaining the structural stability of the TOM complex of mitochondria.

Transport of nuclear encoded proteins into mitochondria is mediated by multisubunit translocation machineries in the outer and inner membranes of mitochondria. The TOM complex contains receptor and pore components that facilitate the recognition of preproteins and their transfer through the outer membrane. In addition, the complex contains a set of small proteins. Tom7 and Tom6 have been found in Neurospora and yeast, Tom5 has been found so far only in the latter organism. In the present study, we identified Neurospora Tom5 and analyzed its function in comparison to yeast Tom5, which has been proposed to play a role as a receptor-like component. Neurospora Tom5 crosses the outer membrane with its carboxyl terminus facing the intermembrane space like the other small Tom components. The temperature-sensitive growth phenotype of the yeast TOM5 deletion was rescued by overexpression of Neurospora Tom5. On the other hand, Neurospora cells deficient in tom5 did not exhibit any defect in growth. The structural stability of TOM complexes from cells devoid of Tom5 was significantly altered in yeast but not in Neurospora. The efficiency of protein import in Neurospora mitochondria was not affected by deletion of tom5, whereas in yeast it was reduced as compared with wild type. We conclude that the main role of Tom5, rather than being a receptor, is maintaining the structural integrity of the TOM complex.

Mim1, a protein required for the assembly of the TOM complex of mitochondria.

The translocase of the outer mitochondrial membrane (TOM complex) is the general entry site for newly synthesized proteins into mitochondria. This complex is essential for the formation and maintenance of mitochondria. Here, we report on the role of the integral outer membrane protein, Mim1 (mitochondrial import), in the biogenesis of mitochondria. Depletion of Mim1 abrogates assembly of the TOM complex and results in accumulation of Tom40, the principal constituent of the TOM complex, as a low-molecular-mass species. Like all mitochondrial beta-barrel proteins, the precursor of Tom40 is inserted into the outer membrane by the TOB complex. Mim1 is likely to be required for a step after this TOB-complex-mediated insertion. Mim1 is a constituent of neither the TOM complex nor the TOB complex; rather, it seems to be a subunit of another, as yet unidentified, complex. We conclude that Mim1 has a vital and specific function in the assembly of the TOM complex.

Nerve growth factor mediates activation of the Smad pathway in PC12 cells.

Ligand-induced oligomerization of receptors is a key step in initiating growth factor signaling. Nevertheless, complex biological responses often require additional trans-signaling mechanisms involving two or more signaling cascades. For cells of neuronal origin, it was shown that neurotrophic effects evoked by nerve growth factor or other neurotrophins depend highly on the cooperativity with cytokines that belong to the transforming growth factor beta (TGF-beta) superfamily. We found that rat pheochromocytoma cells, which represent a model system for neuronal differentiation, are unresponsive to TGF-beta1 due to limiting levels of its receptor, TbetaRII. However, stimulation with nerve growth factor leads to activation of the Smad pathway independent of TGF-beta. In contrast to TGF-beta signaling, activation of Smad3 by nerve growth factor does not occur via phosphorylation of the C-terminal SSXS-motif, but leads to heteromeric complex formation with Smad4, nuclear translocation of Smad3 and transcriptional activation of Smad-dependent reporter genes. This response is direct and does not require de novo protein synthesis, as shown by cycloheximide treatment. This initiation of transcription is dependent on functional tyrosine kinase receptors and can be blocked by Smad7. These data provide further evidence that the Smad proteins are not exclusively activated by the classical TGF-beta triggered mechanism. The potential of NGF to activate the Smad pathway independent of TGF-beta represents an important regulatory mechanism with special relevance for the development and function of neuronal cells or of other NGF-sensitive cells, in particular those that are TGF-beta-resistant.

Initiation of Smad-dependent and Smad-independent signaling via distinct BMP-receptor complexes.

BACKGROUND: BMP-2 (bone morphogenetic protein-2) signals via two types of transmembrane serine/threonine kinase receptors (BRI and BRII), which form heteromeric complexes prior to and after ligand binding. Within a BMP-bound receptor complex, BRII transphosphorylates and activates BRI-a for further signaling. We investigated which signaling pathway is initiated by BMP-2 via preformed receptor complexes versus BMP-2-induced signaling receptor complexes. METHODS: Immunofluorescence co-patching was used to study the oligomerization of receptors at the surface of live cells. Binding and chemical cross-linking of iodinated BMP-2 followed by immunoprecipitation was used to show association of receptors in the presence of ligand. Western blots with use of anti-phospho-Smad1 antibodies and reporter gene assays with use of SBE-lux were employed to show activation of the Smad pathway. Phosphorylation of p38-MAPK was shown by Western blots. Induction of alkaline phosphatase was determined by staining the cells. The cluster density of receptors was determined with use of image correlation spectroscopy. RESULTS AND CONCLUSION: We showed that the Smad pathway is induced by preformed receptor complexes, whereas BMP-2-induced signaling complexes result in the activation of p38-MAPK. We also found evidence that the clustering of BRI-a at the membrane is altered in the presence of BRII, suggesting that it associates with existing clusters of BRII to initiate efficient Smad signaling. These data clearly demonstrate that it is critical to fully understand receptor oligomerization in order to estimate signaling outcome for distinct receptor and ligand mutants.