SANS reveals lipid-dependent oligomerization of an intramembrane aspartyl protease from H. volcanii.

Reactions that occur within the lipid membrane involve, at minimum, ternary complexes among the enzyme, substrate, and lipid. For many systems, the impact of the lipid in regulating activity or oligomerization state is poorly understood. Here, we used small-angle neutron scattering (SANS) to structurally characterize an intramembrane aspartyl protease (IAP), a class of membrane-bound enzymes that use membrane-embedded aspartate residues to hydrolyze transmembrane segments of biologically relevant substrates. We focused on an IAP ortholog from the halophilic archaeon Haloferax volcanii (HvoIAP). HvoIAP purified in n-dodecyl-ß-D-maltoside (DDM) fractionates on size-exclusion chromatography (SEC) as two fractions. We show that, in DDM, the smaller SEC fraction is consistent with a compact HvoIAP monomer. Molecular dynamics flexible fitting conducted on an AlphaFold2-generated monomer produces a model in which loops are compact alongside the membrane-embedded helices. In contrast, SANS data collected on the second SEC fraction indicate an oligomer consistent with an elongated assembly of discrete HvoIAP monomers. Analysis of in-line SEC-SANS data of the HvoIAP oligomer, the first such experiment to be conducted on a membrane protein at Oak Ridge National Lab (ORNL), shows a diversity of elongated and spherical species, including one consistent with the tetrameric assembly reported for the Methanoculleus marisnigri JR1 IAP crystal structure not observed previously in solution. Reconstitution of monomeric HvoIAP into bicelles increases enzyme activity and results in the assembly of HvoIAP into a species with similar dimensions as the ensemble of oligomers isolated from DDM. Our study reveals lipid-mediated HvoIAP self-assembly and demonstrates the utility of in-line SEC-SANS in elucidating oligomerization states of small membrane proteins.

Lipid environment modulates processivity and kinetics of a presenilin homolog acting on multiple substrates in vitro.

Intramembrane proteases (IPs) hydrolyze peptides in the lipid membrane. IPs participate in a number of cellular pathways including immune response and surveillance, and cholesterol biosynthesis, and they are exploited by viruses for replication. Despite their broad importance across biology, how activity is regulated in the cell to control protein maturation and release of specific bioactive peptides at the right place and right time remains largely unanswered, particularly for the intramembrane aspartyl protease (IAP) subtype. At a molecular biochemical level, different IAP homologs can cleave non-biological substrates, and there is no sequence recognition motif among the nearly 150 substrates identified for just one IAP, presenilin-1, the catalytic component of γ-secretase known for its involvement in the production of amyloid-ß plaques associated with Alzheimer disease. Here we used gel-based assays combined with quantitative mass spectrometry and FRET-based kinetics assays to probe the cleavage profile of the presenilin homolog from the methanogen Methanoculleus marisnigri JR1 as a function of the surrounding lipid-mimicking environment, either detergent micelles or bicelles. We selected four biological IAP substrates that have not undergone extensive cleavage profiling previously, namely, the viral core protein of Hepatitis C virus, the viral core protein of Classical Swine Fever virus, the transmembrane segment of Notch-1, and the tyrosine receptor kinase ErbB4. Our study demonstrates a proclivity toward cleavage of substrates at positions of low average hydrophobicity and a consistent role for the lipid environment in modulating kinetic properties.

Automated Structure- and Sequence-Based Design of Proteins for High Bacterial Expression and Stability.

Upon heterologous overexpression, many proteins misfold or aggregate, thus resulting in low functional yields. Human acetylcholinesterase (hAChE), an enzyme mediating synaptic transmission, is a typical case of a human protein that necessitates mammalian systems to obtain functional expression. We developed a computational strategy and designed an AChE variant bearing 51 mutations that improved core packing, surface polarity, and backbone rigidity. This variant expressed at ∼2,000-fold higher levels in E. coli compared to wild-type hAChE and exhibited 20°C higher thermostability with no change in enzymatic properties or in the active-site configuration as determined by crystallography. To demonstrate broad utility, we similarly designed four other human and bacterial proteins. Testing at most three designs per protein, we obtained enhanced stability and/or higher yields of soluble and active protein in E. coli. Our algorithm requires only a 3D structure and several dozen sequences of naturally occurring homologs, and is available at http://pross.weizmann.ac.il.

Correlation between chemical denaturation and the unfolding energetics of Acanthamoeba actophorin.

The actin filament network is in part remodeled by the action of a family of filament severing proteins that are responsible for modulating the ratio between monomeric and filamentous actin. Recent work on the protein actophorin from the amoeba Acanthamoeba castellani identified a series of site-directed mutations that increase the thermal stability of the protein by 22°C. Here, we expand this observation by showing that the mutant protein is also significantly stable to both equilibrium and kinetic chemical denaturation, and employ computer simulations to account for the increase in thermal or chemical stability through an accounting of atomic-level interactions. Specifically, the potential of mean force (PMF) can be obtained from steered molecular dynamics (SMD) simulations in which a protein is unfolded. However, SMD can be inefficient for large proteins as they require large solvent boxes, and computationally expensive as they require increasingly many SMD trajectories to converge the PMF. Adaptive steered molecular dynamics (ASMD) overcomes the second of these limitations by steering the particle in stages, which allows for convergence of the PMF using fewer trajectories compared with SMD. Use of the telescoping water scheme within ASMD partially overcomes the first of these limitations by reducing the number of waters at each stage to only those needed to solvate the structure within a given stage. In the PMFs obtained from ASMD, the work of unfolding Acto-2 was found to be higher than the Acto-WT by approximately 120 kCal/mol and reflects the increased stability seen in the chemical denaturation experiments. The evolution of the average number of hydrogen bonds and number of salt bridges during the pulling process provides a mechanistic view of the structural changes of the actophorin protein as it is unfolded, and how it is affected by the mutation in concert with the energetics reported through the PMF.

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.

Molecular Insights into Myocilin and Its Glaucoma-Causing Misfolded Olfactomedin Domain Variants.

Numerous human disorders arise due to the inability of a particular protein to adopt its correct three-dimensional structure in the context of the cell, leading to aggregation. A new addition to the list of such protein conformational disorders is the inherited subtype of glaucoma. Different and rare coding mutations in myocilin, found in families throughout the world, are causal for early onset ocular hypertension, a key glaucoma risk factor. Myocilin is expressed at high levels in the trabecular meshwork (TM) extracellular matrix. The TM is the anatomical region of the eye that regulates intraocular pressure, and its dysfunction is associated with most forms of glaucoma. Disease variants, distributed across the 30 kDa olfactomedin domain (mOLF), cause myocilin to be sequestered intracellularly instead of being secreted to the TM extracellular matrix. The working hypothesis is that the intracellular aggregates cause a toxic gain of function: TM cell death is thought to lead to TM matrix dysfunction, hastening elevated intraocular pressure and subsequent vision loss.Our lab has provided molecular underpinnings for myocilin structure and misfolding, placing myocilin-associated glaucoma within the context of amyloid diseases like Alzheimer and diabetes. We have dissected complexities of the modular wild-type (WT) myocilin structure and associated misfolded states. Our data support the model that full-length WT myocilin adopts a Y-shaped dimer-of-dimers conferred by two different coiled-coil regions, generating new hypotheses regarding its mysterious function. The mOLF ß-propellers are paired at each tip of the Y. Disease-associated variants aggregate because mOLFs are less stable, leading to facile aggregation under physiological conditions (37 °C, pH 7.2). Mutant myocilin aggregates exhibit numerous characteristics of amyloid in vitro and in cells, and aggregation proceeds from a partially folded state accessed preferentially by disease variants at physiological conditions. Interestingly, destabilization is not a universal consequence of mutation. We identified counterintuitive, stabilizing point variants that adopt a non-native structure and do not aggregate; however, these variants have not been identified in glaucoma patients. An ongoing effort is predicting the consequence of any given mutation. This effort is relevant to interpreting data from large-scale sequencing projects where clinical and family history data are not available. Finally, our work suggests avenues to develop disease-modifying precision medicines for myocilin-associated glaucoma.

Calcium dysregulation potentiates wild-type myocilin misfolding: implications for glaucoma pathogenesis.

Myocilin is secreted from trabecular meshwork cells to an eponymous extracellular matrix that is critical for maintaining intraocular pressure. Missense mutations found in the myocilin olfactomedin domain (OLF) lead to intracellular myocilin misfolding and are causative for the heritable form of early-onset glaucoma. The OLF domain contains a unique internal, hetero-dinuclear calcium site. Here, we tested the hypothesis that calcium dysregulation causes wild-type (WT) myocilin misfolding reminiscent of that observed for disease variants. Using two cellular models expressing WT myocilin, we show that the Ca2+ ATPase channel blocker thapsigargin inhibits WT myocilin secretion. Intracellular WT myocilin is at least partly insoluble and aggregated in the endoplasmic reticulum (ER), and stains positively with an amyloid dye. By comparing the effect of thapsigargin on WT myocilin to that on a de novo secretion-competent Ca2+-free variant D478S, we discern that non-secretion of WT myocilin is due initially to calcium dysregulation, and is potentiated further by resultant ER stress. In E. coli, depletion of calcium leads to recombinant expression of misfolded isolated WT OLF but the D478S variant is still produced as a folded monomer. Treatment of cells expressing a double mutant composed of D478S and either disease variants P370L or Y437H with thapsigargin promotes its misfolding and aggregation, demonstrating the limits of D478S to correct secretion defects. Taken together, the heterodinuclear calcium site is a liability for proper folding of myocilin. Our study suggests a molecular mechanism by which WT myocilin misfolding may contribute broadly to glaucoma-associated ER stress. This study explores the effect of calcium depletion on myocilin olfactomedin domain folding.

How does a protein's structure spell the difference between health and disease? Our journey to understand glaucoma-associated myocilin.

Over 20 years ago, alterations to the protein myocilin were confirmed to be linked to a heritable form of the prevalent eye disease, glaucoma, and 10 years ago, my lab set out to develop a deeper understanding of myocilin in its normal and diseased state. We have made strides in understanding how genetic mutations in myocilin likely lead to disease, but unlocking myocilin's biological function is still an elusive goalpost. Is normal myocilin unimportant in the human body? Are scientists using the wrong methods to study myocilin biology? Here, I discuss my scientific journey into understanding one small part of the fascinating organ that is the eye.

Common and rare myocilin variants: Predicting glaucoma pathogenicity based on genetics, clinical, and laboratory misfolding data.

Rare variants of the olfactomedin domain of myocilin are considered causative for inherited, early-onset open-angle glaucoma, with a misfolding toxic gain-of-function pathogenic mechanism detailed by 20 years of laboratory research. Myocilin variants are documented in the scientific literature and identified through large-scale genetic sequencing projects such as those curated in the Genome Aggregation Database (gnomAD). In the absence of key clinical and laboratory information, however, the pathogenicity of any given variant is not clear, because glaucoma is a heterogeneous and prevalent age-onset disease, and common variants are likely benign. In this review, we reevaluate the likelihood of pathogenicity for the ~100 nonsynonymous missense, insertion-deletion, and premature termination of myocilin olfactomedin variants documented in the literature. We integrate available clinical, laboratory cellular, biochemical and biophysical data, the olfactomedin domain structure, and population genetics data from gnomAD. Of the variants inspected, ~50% can be binned based on a preponderance of data, leaving many of uncertain pathogenicity that motivate additional studies. Ultimately, the approach of combining metrics from different disciplines will likely resolve outstanding complexities regarding the role of this misfolding-prone protein within the context of a multifactorial and prevalent ocular disease, and pave the way for new precision medicine therapeutics.

A blueprint for academic laboratories to produce SARS-CoV-2 quantitative RT-PCR test kits.

Widespread testing for the presence of the novel coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in individuals remains vital for controlling the COVID-19 pandemic prior to the advent of an effective treatment. Challenges in testing can be traced to an initial shortage of supplies, expertise, and/or instrumentation necessary to detect the virus by quantitative RT-PCR (RT-qPCR), the most robust, sensitive, and specific assay currently available. Here we show that academic biochemistry and molecular biology laboratories equipped with appropriate expertise and infrastructure can replicate commercially available SARS-CoV-2 RT-qPCR test kits and backfill pipeline shortages. The Georgia Tech COVID-19 Test Kit Support Group, composed of faculty, staff, and trainees across the biotechnology quad at Georgia Institute of Technology, synthesized multiplexed primers and probes and formulated a master mix composed of enzymes and proteins produced in-house. Our in-house kit compares favorably with a commercial product used for diagnostic testing. We also developed an environmental testing protocol to readily monitor surfaces for the presence of SARS-CoV-2. Our blueprint should be readily reproducible by research teams at other institutions, and our protocols may be modified and adapted to enable SARS-CoV-2 detection in more resource-limited settings.

Archaeal roots of intramembrane aspartyl protease siblings signal peptide peptidase and presenilin.

Signal peptides help newly synthesized proteins reach the cell membrane or be secreted. As part of a biological process key to immune response and surveillance in humans, and associated with diseases, for example, Alzheimer, remnant signal peptides and other transmembrane segments are proteolyzed by the intramembrane aspartyl protease (IAP) enzyme family. Here, we identified IAP orthologs throughout the tree of life. In addition to eukaryotes, IAPs are encoded in metabolically diverse archaea from a wide range of environments. We found three distinct clades of archaeal IAPs: (a) Euryarchaeota (eg, halophilic Halobacteriales, methanogenic Methanosarcinales and Methanomicrobiales, marine Poseidoniales, acidophilic Thermoplasmatales, hyperthermophilic Archaeoglobus spp.), (b) DPANN, and (c) Bathyarchaeota, Crenarchaeota, and Asgard. IAPs were also present in bacterial genomes from uncultivated members of Candidate Phylum Radiation, perhaps due to horizontal gene transfer from DPANN archaeal lineages. Sequence analysis of the catalytic motif YD…GXGD (where X is any amino acid) in IAPs from archaea and bacteria reveals WD in Lokiarchaeota and many residue types in the X position. Gene neighborhood analysis in halophilic archaea shows IAP genes near corrinoid transporters (btuCDF genes). In marine Euryarchaeota, a putative BtuF-like domain is found in N-terminus of the IAP gene, suggesting a role for these IAPs in metal ion cofactor or other nutrient scavenging. Interestingly, eukaryotic IAP family members appear to have evolved either from Euryarchaeota or from Asgard archaea. Taken together, our phylogenetic and bioinformatics analysis should prompt experiments to probe the biological roles of IAPs in prokaryotic secretomes.

Molecular architecture and modifications of full-length myocilin.

Myocilin, a modular multidomain protein, is expressed broadly in the human body but is best known for its presence in the trabecular meshwork extracellular matrix, and myocilin misfolding is associated with glaucoma. Despite progress in comprehending the structure and misfolding of the myocilin olfactomedin domain, the structure and function of full-length myocilin, and contextual changes in glaucoma, remain unknown. Here we expressed and purified milligram-scale quantities of full-length myocilin from suspension mammalian cell culture (Expi293F), enabling molecular characterization in detail not previously accessible. We systematically characterized disulfide-dependent and -independent oligomerization as well as confirmed glycosylation and susceptibility to proteolysis. We identified oligomeric states with glycosylation sites that are inaccessible to enzymatic removal. Low-resolution single particle 2D class averaging from conventional transmission electron microscopy imaging confirms an extended arrangement of tetramers, truncated products consistent with dimers, and a higher-ordered state consistent with octamer. Taken together, our study reveals new myocilin misfolded states and layers of intrinsic heterogeneity, expands our knowledge of olfactomedin-family proteins and lays the foundation for a better molecular understanding of myocilin structure and its still enigmatic biological function.

Stable calcium-free myocilin olfactomedin domain variants reveal challenges in differentiating between benign and glaucoma-causing mutations.

Nonsynonymous gene mutations can be beneficial, neutral, or detrimental to the stability, structure, and biological function of the encoded protein, but the effects of these mutations are often not readily predictable. For example, the ß-propeller olfactomedin domain of myocilin (mOLF) exhibits a complex interrelationship among structure(s), stability, and aggregation. Numerous mutations within mOLF are linked to glaucoma; the resulting variants are less stable, aggregation-prone, and sequestered intracellularly, causing cytotoxicity. Here, we report the first stable mOLF variants carrying substitutions in the calcium-binding site that exhibit solution characteristics indistinguishable from those of glaucoma variants. Crystal structures of these stable variants at 1.8-2.0-Å resolution revealed features that we could not predict by molecular dynamics simulations, including loss of loop structure, helix unwinding, and a blade shift. Double mutants that combined a stabilizing substitution and a selected glaucoma-causing single-point mutant rescued in vitro folding and stability defects. In the context of full-length myocilin, secretion of stable single variants was indistinguishable from that of the WT protein, and the double mutants were secreted to varying extents. In summary, our finding that mOLF can tolerate particular substitutions that render the protein stable despite a conformational switch emphasizes the complexities in differentiating between benign and glaucoma-causing variants and provides new insight into the possible biological function of myocilin.

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.

Both positional and chemical variables control in vitro proteolytic cleavage of a presenilin ortholog.

Mechanistic details of intramembrane aspartyl protease (IAP) chemistry, which is central to many biological and pathogenic processes, remain largely obscure. Here, we investigated the in vitro kinetics of a microbial intramembrane aspartyl protease (mIAP) fortuitously acting on the renin substrate angiotensinogen and the C-terminal transmembrane segment of amyloid precursor protein (C100), which is cleaved by the presenilin subunit of γ-secretase, an Alzheimer disease (AD)-associated IAP. mIAP variants with substitutions in active-site and putative substrate-gating residues generally exhibit impaired, but not abolished, activity toward angiotensinogen and retain the predominant cleavage site (His-Thr). The aromatic ring, but not the hydroxyl substituent, within Tyr of the catalytic Tyr-Asp (YD) motif plays a catalytic role, and the hydrolysis reaction incorporates bulk water as in soluble aspartyl proteases. mIAP hydrolyzes the transmembrane region of C100 at two major presenilin cleavage sites, one corresponding to the AD-associated Aß42 peptide (Ala-Thr) and the other to the non-pathogenic Aß48 (Thr-Leu). For the former site, we observed more favorable kinetics in lipid bilayer-mimicking bicelles than in detergent solution, indicating that substrate-lipid and substrate-enzyme interactions both contribute to catalytic rates. High-resolution MS analyses across four substrates support a preference for threonine at the scissile bond. However, results from threonine-scanning mutagenesis of angiotensinogen demonstrate a competing positional preference for cleavage. Our results indicate that IAP cleavage is controlled by both positional and chemical factors, opening up new avenues for selective IAP inhibition for therapeutic interventions.

Atomic Structure of a Fluorescent Ag8 Cluster Templated by a Multistranded DNA Scaffold.

Multinuclear silver clusters encapsulated by DNA exhibit size-tunable emission spectra and rich photophysics, but their atomic organization is poorly understood. Herein, we describe the structure of one such hybrid chromophore, a green-emitting Ag8 cluster arranged in a Big Dipper-shape bound to the oligonucleotide A2C4. Three 3' cytosine metallo-base pairs stabilize a parallel A-form-like duplex with a 5' adenine-rich pocket, which binds a metallic, trapezoidal-shaped Ag5 moiety via Ag-N bonds to endo- and exocyclic nitrogens of cytosine and adenine. The unique DNA configuration, constrained coordination environment, and templated Ag8 cluster arrangement highlight the reciprocity between the silvers and DNA in adopting this structure. These first atomic details of a DNA-encapsulated Ag cluster fluorophore illuminate many aspects of biological assembly, nanoscience, and metal cluster photophysics.

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.

Solution Structure of an Intramembrane Aspartyl Protease via Small Angle Neutron Scattering.

Intramembrane aspartyl proteases (IAPs) comprise one of four families of integral membrane proteases that hydrolyze substrates within the hydrophobic lipid bilayer. IAPs include signal peptide peptidase, which processes remnant signal peptides from nascent polypeptides in the endoplasmic reticulum, and presenilin, the catalytic component of the γ-secretase complex that processes Notch and amyloid precursor protein. Despite their broad biomedical reach, basic structure-function relationships of IAPs remain active areas of research. Characterization of membrane-bound proteins is notoriously challenging due to their inherently hydrophobic character. For IAPs, oligomerization state in solution is one outstanding question, with previous proposals for monomer, dimer, tetramer, and octamer. Here we used small angle neutron scattering (SANS) to characterize n-dodecyl-ß-D-maltopyranoside (DDM) detergent solutions containing and absent a microbial IAP ortholog. A unique feature of SANS is the ability to modulate the solvent composition to mask all but the enzyme of interest. The signal from the IAP was enhanced by deuteration and, uniquely, scattering from DDM and buffers were matched by the use of both tail-deuterated DDM and D2O. The radius of gyration calculated for IAP and the corresponding ab initio consensus model are consistent with a monomer. The model is slightly smaller than the crystallographic IAP monomer, suggesting a more compact protein in solution compared with the crystal lattice. Our study provides direct insight into the oligomeric state of purified IAP in surfactant solution, and demonstrates the utility of fully contrast-matching the detergent in SANS to characterize other intramembrane proteases and their membrane-bound substrates.

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.