Critical Calibration of Mass Photometry for Higher-Mass Samples Such as Adeno-Associated Virus Vectors.

Mass photometry (MP) is a label-free, single-molecule technique that can determine molecular mass distribution with very low sample consumption in a short time. Because of the established experimental instrument and analytical software, MP measurements may be readily obtained; thus, the application of MP is expanding, especially in the fields of bioscience and biotechnology. However, because the MP data quality is strongly focus-dependent, optical settings must be intrinsically strict. In this study, we report the importance of the critical calibration of the mass photometer, which is required for the accurate estimation of high-molecular mass samples, such as adeno-associated virus vectors. Additionally, a method for optimizing the instrument settings, including the calibration of the stage, is presented.

A Collaborative Study on the Classification of Silicone Oil Droplets and Protein Particles Using Flow Imaging Method.

In this study, we conducted a collaborative study on the classification between silicone oil droplets and protein particles detected using the flow imaging (FI) method toward proposing a standardized classifier/model. We compared four approaches, including a classification filter composed of particle characteristic parameters, principal component analysis, decision tree, and convolutional neural network in the performance of the developed classifier/model. Finally, the points to be considered were summarized for measurement using the FI method, and for establishing the classifier/model using machine learning to differentiate silicone oil droplets and protein particles.

Pro108Ser mutation of SARS-CoV-2 3CLpro reduces the enzyme activity and ameliorates the clinical severity of COVID-19.

Recently, an international randomized controlled clinical trial showed that patients with SARS-CoV-2 infection treated orally with the 3-chymotrypsin-like protease (3CLpro) inhibitor PF-07321332 within three days of symptom onset showed an 89% lower risk of COVID-19-related hospital admission/ death from any cause as compared with the patients who received placebo. Lending support to this critically important result of the aforementioned trial, we demonstrated in our study that patients infected with a SARS-Cov-2 sub-lineage (B.1.1.284) carrying the Pro108Ser mutation in 3CLpro tended to have a comparatively milder clinical course (i.e., a smaller proportion of patients required oxygen supplementation during the clinical course) than patients infected with the same sub-lineage of virus not carrying the mutation. Characterization of the mutant 3CLpro revealed that the Kcat/Km of the 3CLpro enzyme containing Ser108 was 58% lower than that of Pro108 3CLpro. Hydrogen/deuterium-exchange mass spectrometry (HDX-MS) revealed that the reduced activity was associated with structural perturbation surrounding the substrate-binding region of the enzyme, which is positioned behind and distant from the 108th amino acid residue. Our findings of the attenuated clinical course of COVID-19 in patients infected with SARS-CoV-2 strains with reduced 3CLpro enzymatic activity greatly endorses the promising result of the aforementioned clinical trial of the 3CLpro inhibitor.

Glycyrrhizin Derivatives Suppress Cancer Chemoresistance by Inhibiting Progesterone Receptor Membrane Component 1.

Progesterone receptor membrane component 1 (PGRMC1) is highly expressed in various cancer cells and contributes to tumor progression. We have previously shown that PGRMC1 forms a unique heme-stacking functional dimer to enhance EGF receptor (EGFR) activity required for cancer proliferation and chemoresistance, and the dimer dissociates by carbon monoxide to attenuate its biological actions. Here, we determined that glycyrrhizin (GL), which is conventionally used to ameliorate inflammation, specifically binds to heme-dimerized PGRMC1. Binding analyses using isothermal titration calorimetry revealed that some GL derivatives, including its glucoside-derivative (GlucoGL), bind to PGRMC1 potently, whereas its aglycone, glycyrrhetinic acid (GA), does not bind. GL and GlucoGL inhibit the interaction between PGRMC1 and EGFR, thereby suppressing EGFR-mediated signaling required for cancer progression. GL and GlucoGL significantly enhanced EGFR inhibitor erlotinib- or cisplatin (CDDP)-induced cell death in human colon cancer HCT116 cells. In addition, GL derivatives suppressed the intracellular uptake of low-density lipoprotein (LDL) by inhibiting the interaction between PGRMC1 and the LDL receptor (LDLR). Effects on other pathways cannot be excluded. Treatment with GlucoGL and CDDP significantly suppressed tumor growth following xenograft transplantation in mice. Collectively, this study indicates that GL derivatives are novel inhibitors of PGRMC1 that suppress cancer progression, and our findings provide new insights for cancer treatment.

Relation of Colloidal and Conformational Stabilities to Aggregate Formation in a Monoclonal Antibody.

Aggregation of therapeutic monoclonal antibodies has a potential risk of immunogenicity, requiring minimization of aggregate formation. We have developed a fitting formula for antibody aggregation at 40°C based on physicochemical parameters, including colloidal and conformational stabilities. An IgG1 monoclonal antibody, MAb-T, was formulated in 24 combinations of different buffer types and pH with or without sodium chloride. The fitting formula for monomer loss was successfully established by nonlinear regression analysis of the results from accelerated stability testing. Calculated monomer fraction values by the fitting formula were strongly correlated with experimental values (R2 = 0.92). The model includes secondary virial coefficient, B22, as the representative parameter of colloidal stability, and aggregation temperature, Tagg, representing conformational stability. Then, we examined charge state, conformational flexibility, and thermal unfolding profile of MAb-T to clarify the molecular basis for the different aggregation propensities in sodium acetate buffer and in sodium citrate buffer at the same pH and buffer concentration. We concluded that the accumulation of citrate anions on the surface of MAb-T is the primary source of the less colloidal and conformational stabilities, resulting in the higher aggregation propensity in sodium citrate buffer.

Identification of IgG1 Aggregation Initiation Region by Hydrogen Deuterium Mass Spectrometry.

Antibody aggregates are a potential risk for immunogenicity; therefore, rational approaches to improve associated aggregation properties need to be developed. Here, we report the amino acid region responsible for aggregation initiation. Two types of therapeutic IgG1 antibody monomer samples were prepared: IgG1 mAb40-3M stored at 40°C for 3 months, which existed in monodisperse state, and the monomer mAb65-5m, which was dissociated from small soluble aggregates by heating at 65°C for 5 min. Hydrogen deuterium exchange mass spectrometry of mAb40-3M identified 2 sites in the Fc region (site 1, F239-M256; site 2, S428-G450) with increased exchange rates. Site 1 includes a region reported as being susceptible to structural change induced by stress. Exposure of site 1 was undetected after 2 months of storage at 40°C but was subsequently detectable after 3 months. As site 2 is spatially close to site 1, the structural change of site 1 could propagate site 2. Besides these 2 regions, hydrogen deuterium exchange mass spectrometry of mAb65-5m identified an exposure of I257-W281 in Fc (site 3), within which a peptide sequence with high aggregation tendency was discovered. We thus concluded that exposure of site 3 is a trigger for the association of a partially denatured antibody.

An Assessment of the Ability of Submicron- and Micron-Size Silicone Oil Droplets in Dropped Prefillable Syringes to Invoke Early- and Late-Stage Immune Responses.

A number of biopharmaceuticals are available as lyophilized formulations along with a prefilled syringe (PFS) containing water for injection (WFI). Submicron- and micron-size droplets of lubricating silicone oil (SO) applied to the inner surface of the PFS barrel might migrate into the WFI, to which protein pharmaceuticals can adsorb, potentially inducing an immune response. In the present study, we subjected siliconized cyclo-olefin polymer PFSs filled with WFI to dropping stress to simulate actual shipping conditions as well as evaluated the risk associated with the released SO droplets. The results confirmed the undesirable effects of SO on therapeutic proteins, including adsorption to SO droplets and increased secretion of several innate cytokines from human peripheral blood mononuclear cells of a small donor panel. Assessment of immunogenicity in vivo using BALB/c mice revealed a slight increase in the plasma concentrations of antidrug antibodies over 21 days in response to SO-containing antibody samples compared to the absence of SO. These results indicate that SO droplets form complexes with pharmaceutical proteins that can potentially invoke early- and late-stage immune responses. Therefore, the use of SO-free cyclo-olefin polymer PFSs as primary containers for WFI could contribute to the enhanced safety of reconstituted biopharmaceuticals.

Crystal structure of the dog allergen Can f 6 and structure-based implications of its cross-reactivity with the cat allergen Fel d 4.

Several dog allergens cause allergic reactions in humans worldwide. Seven distinct dog allergens, designated Canis familiaris allergen 1 to 7 (Can f 1-Can f 7), have been identified thus far. Can f 6 shows high sequence similarity and cross-reactivity with Fel d 4 and Equ c 1, major cat and horse allergens, respectively. This study was conducted on the allergenic epitopes of Can f 6 based on its structural characterization. We demonstrated that sera from 18 out of 38 (47%) dog-sensitized patients reacted to recombinant Can f 6 protein (rCan f 6). We then determined the crystal structure of rCan f 6 by X-ray crystallography, which exhibited a conserved tertiary structural architecture found in lipocalin family proteins. Based on the tertiary structure and sequence similarities with Fel d 4 and Equ c 1, we predicted three IgE-recognizing sites that are possibly involved in cross-reactivity. Substituting three successive amino acids in these sites to triple alanine decreased IgE reactivity to the allergen. However, the degree of reduction in IgE reactivity largely depended on the site mutated and the serum used, suggesting that Can f 6 is a polyvalent allergen containing multiple epitopes and Can f 6-reactive sera contain varied amounts of IgE recognising individual Can f 6 epitopes including those predicted in this study. We also demonstrated that the predicted epitopes are partly involved in IgE cross-reactivity to Fel d 4. Interestingly, the effect of the mutation depended on whether the protein was structured or denatured, indicating that the bona fide tertiary structure of Can f 6 is essential in determining its IgE epitopes.

Physicochemical improvement of rabbit derived single-domain antibodies by substitutions with amino acids conserved in camelid antibodies.

Recently, we showed that immunized rabbit heavy chain variable regions (rVHs) can have strong antigen binding activity comparable to that of the camelid variable domain of the heavy chain of heavy chain antibody (VHH). These rVHs lack the light chain variable regions (rVLs), which exist in the authentic Fab format; thus, molecular surfaces at the interface region of rVHs are exposed to solvent. This physical feature may change physicochemical properties, such as causing reduced stability. By overcoming potential physicochemical issues through engineering the interface region, rVHs could become more useful as single-domain antibodies. In this study, we substituted amino acid residues conserved at the interface region of rVHs with those of VHHs. These substitutions included V37F, involving substitution of a residue in the hydrophobic core with a bulkier hydrophobic amino acid, and G44E/L45R, involving double substitutions of highly exposed residues with more hydrophilic ones. As expected, biophysical and structural characterizations showed that the V37F substitution markedly enhanced the thermal stability through increased hydrophobic packing, while G44E/L45R substitutions greatly reduced hydrophobicity of the interface. The quadruple substitutions of V37F/G44E/L45R/F91Y resulted in not only enhancements of thermal stability and reduction in hydrophobicity, both in an additive manner, but also synergistic improvement of purification yield. This quadruple mutant exhibited greatly reduced non-specific binding with improved colloidal stability owing to the reduced hydrophobicity. The approach used in this study should further enhance the utility of rVHs and promote research and development of single-domain antibodies.

Sedimentation velocity analytical ultracentrifugation for characterization of therapeutic antibodies.

Sedimentation velocity analytical ultracentrifugation (SV-AUC) coupled with direct computational fitting of the observed concentration profiles (sedimentating boundary) have been developed and widely used for the characterization of macromolecules and nanoparticles in solution. In particular, size distribution analysis by SV-AUC has become a reliable and essential approach for the characterization of biopharmaceuticals including therapeutic antibodies. In this review, we describe the importance and advantages of SV-AUC for studying biopharmaceuticals, with an emphasis on strategies for sample preparation, data acquisition, and data analysis. Recent discoveries enabled by AUC with a fluorescence detection system and potential future applications are also discussed.

NMR Detection of Semi-Specific Antibody Interactions in Serum Environments.

Although antibody functions are executed in heterogeneous blood streams characterized by molecular crowding and promiscuous intermolecular interaction, detailed structural characterizations of antibody interactions have thus far been performed under homogeneous in vitro conditions. NMR spectroscopy potentially has the ability to study protein structures in heterogeneous environments, assuming that the target protein can be labeled with NMR-active isotopes. Based on our successful development of isotope labeling of antibody glycoproteins, here we apply NMR spectroscopy to characterize antibody interactions in heterogeneous extracellular environments using mouse IgG-Fc as a test molecule. In human serum, many of the HSQC peaks originating from the Fc backbone exhibited attenuation in intensity of various magnitudes. Similar spectral changes were induced by the Fab fragment of polyclonal IgG isolated from the serum, but not by serum albumin, indicating that a subset of antibodies reactive with mouse IgG-Fc exists in human serum without preimmunization. The metaepitopes recognized by serum polyclonal IgG cover the entire molecular surface of Fc, including the binding sites to Fc receptors and C1q. In-serum NMR observation will offer useful tools for the detailed characterization of biopharamaceuticals, including therapeutic antibodies in physiologically relevant heterogeneous environments, also giving deeper insight into molecular recognition by polyclonal antibodies in the immune system.

Analytical ultracentrifugation with fluorescence detection system reveals differences in complex formation between recombinant human TNF and different biological TNF antagonists in various environments.

A number of studies have attempted to elucidate the binding mechanism between tumor necrosis factor (TNF) and clinically relevant antagonists. None of these studies, however, have been conducted as close as possible to physiologic conditions, and so the relationship between the size distribution of TNF-antagonist complexes and the antagonists' biological activity or adverse effects remains elusive. Here, we characterized the binding stoichiometry and sizes of soluble TNF-antagonist complexes for adalimumab, infliximab, and etanercept that were formed in human serum and in phosphate-buffered saline (PBS). Fluorescence-detected sedimentation velocity analytical ultracentrifugation analyses revealed that adalimumab and infliximab formed a range of complexes with TNF, with the major complexes consisting of 3 molcules of the respective antagonist and one or 2 molcules of TNF. Considerably greater amounts of high-molecular-weight complexes were detected for infliximab in human serum. The emergence of peaks with higher sedimentation coefficients than the adalimumab monomer as a function of added human serum albumin (HSA) concentration in PBS suggested weak reversible interactions between HSA and immunoglobulins. Etanerept exclusively formed 1:1 complexes with TNF in PBS, and a small amount of complexes with higher stoichiometry was detected in human serum. Consistent with these biophysical characterizations, a reporter assay showed that adalimumab and infliximab, but not etanercept, exerted FcγRIIa- and FcγRIIIa-mediated cell signaling in the presence of TNF and that infliximab exhibited higher potency than adalimumab. This study shows that assessing distribution profiles in serum will contribute to a more comprehensive understanding of the in vivo behavior of therapeutic proteins.

Mass spectrometric analysis of protein-ligand interactions.

The interactions of small molecules with proteins (protein-ligand interactions) mediate various biological phenomena including signal transduction and protein transcription and translation. Synthetic compounds such as drugs can also bind to target proteins, leading to the inhibition of protein-ligand interactions. These interactions typically accompany association-dissociation equilibrium according to the free energy difference between free and bound states; therefore, the quantitative biophysical analysis of the interactions, which uncovers the stoichiometry and dissociation constant, is important for understanding biological reactions as well as for rational drug development. Mass spectrometry (MS) has been used to determine the precise molecular masses of molecules. Recent advancements in MS enable us to determine the molecular masses of protein-ligand complexes without disrupting the non-covalent interactions through the gentle desolvation of the complexes by increasing the vacuum pressure of a chamber in a mass spectrometer. This method is called MS under non-denaturing conditions or native MS and allows the unambiguous determination of protein-ligand interactions. Under a few assumptions, MS has also been applied to determine the dissociation constants for protein-ligand interactions. The structural information of a protein-ligand interaction, such as the location of the interaction and conformational change in a protein, can also be analyzed using hydrogen/deuterium exchange MS. In this paper, we briefly describe the history, principle, and recent applications of MS for the study of protein-ligand interactions.

Structural characterization of the circadian clock protein complex composed of KaiB and KaiC by inverse contrast-matching small-angle neutron scattering.

The molecular machinery of the cyanobacterial circadian clock consists of three proteins: KaiA, KaiB, and KaiC. Through interactions among the three Kai proteins, the phosphorylation states of KaiC generate circadian oscillations in vitro in the presence of ATP. Here, we characterized the complex formation between KaiB and KaiC using a phospho-mimicking mutant of KaiC, which had an aspartate substitution at the Ser431 phosphorylation site and exhibited optimal binding to KaiB. Mass-spectrometric titration data showed that the proteins formed a complex exclusively in a 6:6 stoichiometry, indicating that KaiB bound to the KaiC hexamer with strong positive cooperativity. The inverse contrast-matching technique of small-angle neutron scattering enabled selective observation of KaiB in complex with the KaiC mutant with partial deuteration. It revealed a disk-shaped arrangement of the KaiB subunits on the outer surface of the KaiC C1 ring, which also serves as the interaction site for SasA, a histidine kinase that operates as a clock-output protein in the regulation of circadian transcription. These data suggest that cooperatively binding KaiB competes with SasA with respect to interaction with KaiC, thereby promoting the synergistic release of this clock-output protein from the circadian oscillator complex.

Interaction mode between catalytic and regulatory subunits in glucosidase II involved in ER glycoprotein quality control.

The glycoside hydrolase family 31 (GH31) α-glucosidases play vital roles in catabolic and regulated degradation, including the α-subunit of glucosidase II (GIIα), which catalyzes trimming of the terminal glucose residues of N-glycan in glycoprotein processing coupled with quality control in the endoplasmic reticulum (ER). Among the known GH31 enzymes, only GIIα functions with its binding partner, regulatory ß-subunit (GIIß), which harbors a lectin domain for substrate recognition. Although the structural data have been reported for GIIα and the GIIß lectin domain, the interaction mode between GIIα and GIIß remains unknown. Here, we determined the structure of a complex formed between GIIα and the GIIα-binding domain of GIIß, thereby providing a structural basis underlying the functional extension of this unique GH31 enzyme.

Structural and binding properties of laminarin revealed by analytical ultracentrifugation and calorimetric analyses.

One of the ß-1,3-glucans, laminarin, has been widely used as a substrate for enzymes including endo-1,3-ß-glucanase. To obtain quantitative information about the molecular interaction between laminarin and endo-1,3-ß-glucanase, the structural properties of laminarin should be determined. The results from pioneering work using analytical ultracentrifugation for carbohydrate analysis showed that laminarin from Laminaria digitata predominantly exists as a single-chain species with approximately 5% of triple-helical species. Differential scanning calorimetry experiments did not show a peak assignable to the transition from triple-helix to single-chain, supporting the notion that a large proportion of laminarin is the single-chain species. The interaction of laminarin with an inactive variant of endo-1,3-ß-glucanase from Cellulosimicrobium cellulans, E119A, was quantitatively analyzed using isothermal titration calorimetry. The binding was enthalpically driven and the binding affinity was approximately 10(6) M(-1). The results from binding stoichiometric analysis indicated that on average, E119A binds to laminarin in a 2:1 ratio. This seems to be reasonable, because laminarin mainly exists as a monomer, the apparent molecular mass of laminarin is 3.6 kDa, and E119A would have substrate-binding subsites corresponding to 6 glucose units. The analytical ultracentrifugation experiments could detect different complex species of laminarin and endo-1,3-ß-glucanase.

Haem-dependent dimerization of PGRMC1/Sigma-2 receptor facilitates cancer proliferation and chemoresistance.

Progesterone-receptor membrane component 1 (PGRMC1/Sigma-2 receptor) is a haem-containing protein that interacts with epidermal growth factor receptor (EGFR) and cytochromes P450 to regulate cancer proliferation and chemoresistance; its structural basis remains unknown. Here crystallographic analyses of the PGRMC1 cytosolic domain at 1.95 Å resolution reveal that it forms a stable dimer through stacking interactions of two protruding haem molecules. The haem iron is five-coordinated by Tyr113, and the open surface of the haem mediates dimerization. Carbon monoxide (CO) interferes with PGRMC1 dimerization by binding to the sixth coordination site of the haem. Haem-mediated PGRMC1 dimerization is required for interactions with EGFR and cytochromes P450, cancer proliferation and chemoresistance against anti-cancer drugs; these events are attenuated by either CO or haem deprivation in cancer cells. This study demonstrates protein dimerization via haem-haem stacking, which has not been seen in eukaryotes, and provides insights into its functional significance in cancer.

Small-molecule inhibition of PTPRZ reduces tumor growth in a rat model of glioblastoma.

Protein tyrosine phosphatase receptor-type Z (PTPRZ) is aberrantly over-expressed in glioblastoma and a causative factor for its malignancy. However, small molecules that selectively inhibit the catalytic activity of PTPRZ have not been discovered. We herein performed an in vitro screening of a chemical library, and identified SCB4380 as the first potent inhibitor for PTPRZ. The stoichiometric binding of SCB4380 to the catalytic pocket was demonstrated by biochemical and mass spectrometric analyses. We determined the crystal structure of the catalytic domain of PTPRZ, and the structural basis of the binding of SCB4380 elucidated by a molecular docking method was validated by site-directed mutagenesis studies. The intracellular delivery of SCB4380 by liposome carriers inhibited PTPRZ activity in C6 glioblastoma cells, and thereby suppressed their migration and proliferation in vitro and tumor growth in a rat allograft model. Therefore, selective inhibition of PTPRZ represents a promising approach for glioma therapy.

Disassembly of the self-assembled, double-ring structure of proteasome α7 homo-tetradecamer by α6.

The 20S core particle of the eukaryotic proteasome is composed of two α- and two ß-rings, each of which is a hetero-heptamer composed of seven homologous but distinct subunits. Although formation of the eukaryotic proteasome is a highly ordered process assisted by assembly chaperones, α7, an α-ring component, has the unique property of self-assembling into a homo-tetradecamer. We used biophysical methods to characterize the oligomeric states of this proteasome subunit and its interaction with α6, which makes direct contacts with α7 in the proteasome α-ring. We determined a crystal structure of the α7 tetradecamer, which has a double-ring structure. Sedimentation velocity analytical ultracentrifugation and mass spectrometric analysis under non-denaturing conditions revealed that α7 exclusively exists as homo-tetradecamer in solution and that its double-ring structure is disassembled upon the addition of α6, resulting in a 1:7 hetero-octameric α6-α7 complex. Our findings suggest that proteasome formation involves the disassembly of non-native oligomers, which are assembly intermediates.

Structural Basis for Dimer Formation of Human Condensin Structural Maintenance of Chromosome Proteins and Its Implications for Single-stranded DNA Recognition.

Eukaryotic structural maintenance of chromosome proteins (SMC) are major components of cohesin and condensins that regulate chromosome structure and dynamics during cell cycle. We here determine the crystal structure of human condensin SMC hinge heterodimer with ~30 residues of coiled coils. The structure, in conjunction with the hydrogen exchange mass spectrometry analyses, revealed the structural basis for the specific heterodimer formation of eukaryotic SMC and that the coiled coils from two different hinges protrude in the same direction, providing a unique binding surface conducive for binding to single-stranded DNA. The characteristic hydrogen exchange profiles of peptides constituted regions especially across the hinge-hinge dimerization interface, further suggesting the structural alterations upon single-stranded DNA binding and the presence of a half-opened state of hinge heterodimer. This structural change potentially relates to the DNA loading mechanism of SMC, in which the hinge domain functions as an entrance gate as previously proposed for cohesin. Our results, however, indicated that this is not the case for condensins based on the fact that the coiled coils are still interacting with each other, even when DNA binding induces structural changes in the hinge region, suggesting the functional differences of SMC hinge domain between condensins and cohesin in DNA recognition.