Recombinant antibodies recognize conformation-dependent epitopes of the leucine zipper of misfolding-prone myocilin.

Recombinant antibodies with well-characterized epitopes and known conformational specificities are critical reagents to support robust interpretation and reproducibility of immunoassays across biomedical research. For myocilin, a protein prone to misfolding that is associated with glaucoma and an emerging player in other human diseases, currently available antibodies are unable to differentiate among the numerous disease-associated protein states. This fundamentally constrains efforts to understand the connection between myocilin structure, function, and disease. To address this concern, we used protein engineering methods to develop new recombinant antibodies that detect the N-terminal leucine zipper structural domain of myocilin and that are cross-reactive for human and mouse myocilin. After harvesting spleens from immunized mice and in vitro library panning, we identified two antibodies, 2A4 and 1G12. 2A4 specifically recognizes a folded epitope while 1G12 recognizes a range of conformations. We matured antibody 2A4 for improved biophysical properties, resulting in variant 2H2. In a human IgG1 format, 2A4, 1G12, and 2H2 immunoprecipitate full-length folded myocilin present in the spent media of human trabecular meshwork (TM) cells, and 2H2 can visualize myocilin in fixed human TM cells using fluorescence microscopy. These new antibodies should find broad application in glaucoma and other research across multiple species platforms.

Optically Modulated and Optically Activated Delayed Fluorescent Proteins through Dark State Engineering.

Modulating fluorescent protein emission holds great potential for increasing readout sensitivity for applications in biological imaging and detection. Here, we identify and engineer optically modulated yellow fluorescent proteins (EYFP, originally 10C, but renamed EYFP later, and mVenus) to yield new emitters with distinct modulation profiles and unique, optically gated, delayed fluorescence. The parent YFPs are individually modulatable through secondary illumination, depopulating a long-lived dark state to dynamically increase fluorescence. A single point mutation introduced near the chromophore in each of these YFPs provides access to a second, even longer-lived modulatable dark state, while a different double mutant renders EYFP unmodulatable. The naturally occurring dark state in the parent YFPs yields strong fluorescence modulation upon long-wavelength-induced dark state depopulation, allowing selective detection at the frequency at which the long wavelength secondary laser is intensity modulated. Distinct from photoswitches, however, this near IR secondary coexcitation repumps the emissive S1 level from the long-lived triplet state, resulting in optically activated delayed fluorescence (OADF). This OADF results from secondary laser-induced, reverse intersystem crossing (RISC), producing additional nanosecond-lived, visible fluorescence that is delayed by many microseconds after the primary excitation has turned off. Mutation of the parent chromophore environment opens an additional modulation pathway that avoids the OADF-producing triplet state, resulting in a second, much longer-lived, modulatable dark state. These Optically Modulated and Optically Activated Delayed Fluorescent Proteins (OMFPs and OADFPs) are thus excellent for background- and reference-free, high sensitivity cellular imaging, but time-gated OADF offers a second modality for true background-free detection. Our combined structural and spectroscopic data not only gives additional mechanistic details for designing optically modulated fluorescent proteins but also provides the opportunity to distinguish similarly emitting OMFPs through OADF and through their unique modulation spectra.

Antibodies Used to Detect Glaucoma-Associated Myocilin: More or Less Than Meets the Eye?

Antibodies are key reagents used in vision research, indeed across biomedical research, but they often do not reveal the whole story about a sample. It is important for researchers to be aware of aspects of antibodies that may affect or limit data interpretation. Federal agencies now require funded grants to demonstrate how they will authenticate reagents used. There is also a push for recombinant antibodies, enabled by phage display technology awarded the 2018 Nobel Prize in Chemistry, which allow for thorough validation and a fixed DNA sequence. Here, we discuss how issues surrounding antibodies are pertinent to detecting myocilin, a protein found in trabecular meshwork and associated with a portion of hereditary glaucoma. Confirmation of myocilin expression in tissues and cell culture has been adopted as validation standard in trabecular meshwork research; thus, a discussion of antibody characteristics and fidelity is critical. Further, based on our basic structural understanding of myocilin architecture and its biophysical aggregation properties, we provide a wish list for the characteristics of next-generation antibody reagents for vision researchers. In the long term, well-characterized antibodies targeting myocilin will enable new insights into its function and involvement in glaucoma pathogenesis.

Exploring substituent diversity on pyrrolidine-aryltriazole iminosugars: Structural basis of ß-glucocerebrosidase inhibition.

The synthesis of a library of pyrrolidine-aryltriazole hybrids through CuAAC between two epimeric dihydroxylated azidomethylpyrrolidines and differently substituted phenylacetylenes is reported. The evaluation of the new compounds as inhibitors of lysosomal ß-glucocerebrosidase showed the importance of the substitution pattern of the phenyl moiety in the inhibition. Crystallization and docking studies revealed key interactions of the pyrrolidine motif with aminoacid residues of the catalytic site while the aryltriazole moiety extended along a hydrophobic surface groove. Some of these compounds were able to increase the enzyme activity in Gaucher patient fibroblasts, acting as a new type of chemical chaperone for Gaucher disease.

Differential Misfolding Properties of Glaucoma-Associated Olfactomedin Domains from Humans and Mice.

Mutations in myocilin, predominantly within its olfactomedin (OLF) domain, are causative for the heritable form of open angle glaucoma in humans. Surprisingly, mice expressing Tyr423His mutant myocilin, corresponding to a severe glaucoma-causing mutation (Tyr437His) in human subjects, exhibit a weak, if any, glaucoma phenotype. To address possible protein-level discrepancies between mouse and human OLFs, which might lead to this outcome, biophysical properties of mouse OLF were characterized for comparison with those of human OLF. The 1.55 Å resolution crystal structure of mouse OLF reveals an asymmetric 5-bladed ß-propeller that is nearly indistinguishable from previous structures of human OLF. Wild-type and selected mutant mouse OLFs mirror thermal stabilities of their human OLF counterparts, including characteristic stabilization in the presence of calcium. Mouse OLF forms thioflavin T-positive aggregates with a similar end-point morphology as human OLF, but amyloid aggregation kinetic rates of mouse OLF are faster than human OLF. Simulations and experiments support the interpretation that kinetics of mouse OLF are faster because of a decreased charge repulsion arising from more neutral surface electrostatics. Taken together, phenotypic differences observed in mouse and human studies of mutant myocilin could be a function of aggregation kinetics rates, which would alter the lifetime of putatively toxic protofibrillar intermediates.

Epitope mapping of commercial antibodies that detect myocilin.

The presence of myocilin is often used in the process of validating trabecular meshwork (TM) cells and eye tissues, but the antibody reagents used for detection are poorly characterized. Indeed, for over a century, researchers have been using antibodies to track proteins of interest in a variety of biological contexts, but many antibodies remain ill-defined at the molecular level and in their target epitope. Such issues have prompted efforts from major funding agencies to validate reagents and combat reproducibility issues across biomedical sciences. Here we characterize the epitopes recognized by four commercial myocilin antibodies, aided by structurally and biochemically characterized myocilin fragments. All four antibodies recognize enriched myocilin secreted from human TM cell media. The detection of myocilin fragments by ELISA and Western blot reveal a variety of epitopes across the myocilin polypeptide chain. A more precise understanding of myocilin antibody targets, including conformational specificity, should aid the community in standardizing protocols across laboratories and in turn, lead to a better understanding of eye physiology and disease.

Structure and Misfolding of the Flexible Tripartite Coiled-Coil Domain of Glaucoma-Associated Myocilin.

Glaucoma-associated myocilin is a member of the olfactomedins, a protein family involved in neuronal development and human diseases. Molecular studies of the myocilin N-terminal coiled coil demonstrate a unique tripartite architecture: a Y-shaped parallel dimer-of-dimers with distinct tetramer and dimer regions. The structure of the dimeric C-terminal 7-heptad repeats elucidates an unexpected repeat pattern involving inter-strand stabilization by oppositely charged residues. Molecular dynamics simulations reveal an alternate accessible conformation in which the terminal inter-strand disulfide limits the extent of unfolding and results in a kinked configuration. By inference, full-length myocilin is also branched, with two pairs of C-terminal olfactomedin domains. Selected variants within the N-terminal region alter the apparent quaternary structure of myocilin but do so without compromising stability or causing aggregation. In addition to increasing our structural knowledge of naturally occurring extracellular coiled coils and biomedically important olfactomedins, this work broadens the scope of protein misfolding in the pathogenesis of myocilin-associated glaucoma.

De novo design of antibody complementarity determining regions binding a FLAG tetra-peptide.

Computational antibody engineering efforts to date have focused on improving binding affinities or biophysical characteristics. De novo design of antibodies binding specific epitopes could greatly accelerate discovery of therapeutics as compared to conventional immunization or synthetic library selection strategies. Here, we employed de novo complementarity determining region (CDR) design to engineer targeted antibody-antigen interactions using previously described in silico methods. CDRs predicted to bind the minimal FLAG peptide (Asp-Tyr-Lys-Asp) were grafted onto a single-chain variable fragment (scFv) acceptor framework. Fifty scFvs comprised of designed heavy and light or just heavy chain CDRs were synthesized and screened for peptide binding by phage ELISA. Roughly half of the designs resulted in detectable scFv expression. Four antibodies, designed entirely in silico, bound the minimal FLAG sequence with high specificity and sensitivity. When reformatted as soluble antigen-binding fragments (Fab), these clones expressed well, were predominantly monomeric and retained peptide specificity. In both formats, the antibodies bind the peptide only when present at the amino-terminus of a carrier protein and even conservative peptide amino acid substitutions resulted in a complete loss of binding. These results support in silico CDR design of antibody specificity as an emerging antibody engineering strategy.

Residues Controlling Facial Selectivity in an Alkene Reductase and Semirational Alterations to Create Stereocomplementary Variants.

A systematic saturation mutagenesis campaign was carried out on an alkene reductase from Pichia stipitis (OYE 2.6) to develop variants with reversed stereoselectivities. Wild-type OYE 2.6 reduces three representative Baylis-Hillman adducts to the corresponding S products with almost complete stereoselectivities and good catalytic efficiencies. We created and screened 13 first-generation, site-saturation mutagenesis libraries, targeting residues found near the bound substrate. One variant (Tyr78Trp) showed high R selectivity toward one of the three substrates, but no change (cyclohexenone derivative) and no catalytic activity (acrylate derivative) for the other two. Subsequent rounds of mutagenesis retained the Tyr78Trp mutation and explored other residues that impacted stereoselectivity when altered in a wild-type background. These efforts yielded double and triple mutants that possessed inverted stereoselectivities for two of the three substrates (conversions >99% and at least 91% ee (R)). To understand the reasons underlying the stereochemical changes, we solved crystal structures of two key mutants: Tyr78Trp and Tyr78Trp/Ile113Cys, the latter with substrate partially occupying the active site. By combining these experimental data with modeling studies, we have proposed a rationale that explains the impacts of the most useful mutations.