Perbandingan Regresi B-Splines dan P-Splines pada Hubungan Indeks Pembangunan Manusia dan Persentase Penduduk Miskin Kabupaten/Kota di Indonesia: Comparison of B-Splines and P-Splines Regression on the Relationship between Human Development Index and The Percentage of Poor Districts/Cities in Indonesia

Poverty is basically a manifestation of the opportunity imbalance that every human being has, which occurs due to the imbalance of capabilities possessed. Measuring poverty by using dimensions of capability such as education, health and quality of life standards, can be a reference for identifying the characteristics of actual poverty. One of the measuring that takes into account the capability dimensions is the Human Development Index (HDI). Knowing the close relationship between HDI and the percentage of poor people, in this study a comparison of B-Splines and P-Splines nonparametric quantile regression methods was used to modeling the relationship between HDI and the percentage of poor population according to districts or city in Indonesia in 2017. The results of the analysis showed that the distribution data with curve fittings using non-parametric regression B-Splines and P-Splines produce smooth curves reaching all existing data distributions. The comparison of MSE B-Splines and P-splines models showed that the B-splines regression model for the HDI variable and the percentage of poor people gave the smallest MSE value of 0.1928378, so that it was the best model to analyze the relationship between HDI data and the percentage of poor people in district or city ​​in Indonesia in 2017

Negative interference with antibody-dependent cellular cytotoxicity mediated by rituximab from its interactions with human serum proteins

Although interactions of small molecular drugs with serum proteins have been widely studied from pharmacokinetic and pharmacodynamic perspectives, there have been few reports on the effects of serum components on therapeutic antibody functions. This study reports the effect of abundant serum proteins on antibody-dependent cellular cytotoxicity (ADCC) mediated by rituximab and Fcγ receptor III (FcγRIII). Human serum albumin (HSA) and the Fab fragment from the pooled serum polyclonal IgG were found to compromise ADCC as non-competitive inhibitors. Our nuclear magnetic resonance data provided direct evidence for the interactions of HSA with both the Fab and Fc regions of rituximab and also with the extracellular region of FcγRIII (sFcγRIII). The degree of involvement in the interaction decreased in the order of rituximab-Fab > rituximab-Fc > sFcγRIII, suggesting preferential binding of HSA to net positively charged proteins. Although much less pronounced than the effect of HSA, polyclonal IgG-Fab specifically interacted with rituximab-Fc. The NMR data also showed that the serum protein interactions cover the Fc surface extensively, suggesting that they can act as pan-inhibitors against various Fc receptor-mediated functions and pharmacokinetics. Our findings highlight the importance of considering serum–protein interactions in the design and application of antibody-based drugs with increased efficacy and safety

Silkworm Pupae Function as Efficient Producers of Recombinant Glycoproteins with Stable-Isotope Labeling

Baculovirus-infected silkworms are promising bioreactors for producing recombinant glycoproteins, including antibodies. Previously, we developed a method for isotope labeling of glycoproteins for nuclear magnetic resonance (NMR) studies using silkworm larvae reared on an artificial diet containing 15N-labeled yeast crude protein extract. Here, we further develop this method by introducing a technique for the expression of isotope-labeled glycoproteins by silkworm pupae, which has several potential advantages relative to larvae-based techniques in terms of production yield, ease of handling, and storage. Here, we fed fifth instar larvae an artificial diet with an optimized composition containing [methyl-13C]methionine, leading to pupation. Nine-day-old pupae were then injected with recombinant Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid for expression of recombinant human immunoglobulin G (IgG). From the whole-body homogenates of pupae, 0.35 mg/pupa of IgG was harvested, which is a yield that is five times higher than can be obtained from larvae. Recombinant IgG, thus prepared, exhibited mainly three kinds of pauci-mannose-type oligosaccharides and had a 13C-enrichment ratio of approximately 80%. This enabled selective observation of NMR signals originating from the methionyl methyl group of IgG, confirming its conformational integrity. These data demonstrate the utility of silkworm pupae as factories for producing recombinant glycoproteins with amino-acid-selective isotope labeling

The Fab portion of immunoglobulin G contributes to its binding to Fcγ receptor III

Most cells active in the immune system express receptors for antibodies which mediate a variety of defensive mechanisms. These receptors interact with the Fc portion of the antibody and are therefore collectively called Fc receptors. Here, using high-speed atomic force microscopy, we observe interactions of human, humanized, and mouse/human-chimeric immunoglobulin G1 (IgG1) antibodies and their cognate Fc receptor, FcγRIIIa. Our results demonstrate that not only Fc but also Fab positively contributes to the interaction with the receptor. Furthermore, hydrogen/deuterium exchange mass spectrometric analysis reveals that the Fab portion of IgG1 is directly involved in its interaction with FcγRIIIa, in addition to the canonical Fc-mediated interaction. By targeting the previously unidentified receptor-interaction sites in IgG-Fab, our findings could inspire therapeutic antibody engineering

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

Quantitative Visualization of the Interaction between Complement Component C1 and Immunoglobulin G: The Effect of C_H1 Domain Deletion

Immunoglobulin G (IgG) adopts a modular multidomain structure that mediates antigen recognition and effector functions, such as complement-dependent cytotoxicity. IgG molecules are self-assembled into a hexameric ring on antigen-containing membranes, recruiting the complement component C1q. In order to provide deeper insights into the initial step of the complement pathway, we report a high-speed atomic force microscopy study for the quantitative visualization of the interaction between mouse IgG and the C1 complex composed of C1q, C1r, and C1s. The results showed that the C1q in the C1 complex is restricted regarding internal motion, and that it has a stronger binding affinity for on-membrane IgG2b assemblages than C1q alone, presumably because of the lower conformational entropy loss upon binding. Furthermore, we visualized a 1:1 stoichiometric interaction between C1/C1q and an IgG2a variant that lacks the entire CH1 domain in the absence of an antigen. In addition to the canonical C1q-binding site on Fc, their interactions are mediated through a secondary site on the CL domain that is cryptic in the presence of the CH1 domain. Our findings offer clues for novel-modality therapeutic antibodies

Characterization of conformational deformation-coupled interaction between immunoglobulin G1 Fc glycoprotein and a low-affinity Fcγ receptor by deuteration-assisted small-angle neutron scattering

A recently developed integrative approach combining varied types of experimental data has been successfully applied to three-dimensional modelling of larger biomacromolecular complexes. Deuteration-assisted small-angle neutron scattering (SANS) plays a unique role in this approach by making it possible to observe selected components in the complex. It enables integrative modelling of biomolecular complexes based on building-block structures typically provided by X-ray crystallography. In this integrative approach, it is important to be aware of the flexible properties of the individual building blocks. Here we examine the ability of SANS to detect a subtle conformational change of a multidomain protein using the Fc portion of human immunoglobulin G (IgG) interacting with a soluble form of the low-affinity Fcγ receptor IIIb (sFcγRIIIb) as a model system. The IgG-Fc glycoprotein was subjected to SANS in the absence and presence of 75%-deuterated sFcγRIIIb, which was matched out in D2O solution. This inverse contrast-matching technique enabled selective observation of SANS from IgG-Fc, thereby detecting its subtle structural deformation induced by the receptor binding. The SANS data were successfully interpreted by considering previously reported crystallographic data and an equilibrium between free and sFcγRIIIb-bound forms. Our SANS data thus demonstrate the applicability of SANS in the integrative approach dealing with biomacromolecular complexes composed of weakly associated building blocks with conformational plasticity

On-Membrane Dynamic Interplay between Anti-GM1 IgG Antibodies and Complement Component C1q

Guillain–Barré syndrome, an autoimmune neuropathy characterized by acute limb weakness, is often preceded by Campylobacter jejuni infection. Molecular mimicry exists between the bacterial lipo-oligosaccharide and human ganglioside. Such C. jejuni infection induces production of immunoglobulin G1 (IgG1) autoantibodies against GM1 and causes complement-mediated motor nerve injury. For elucidating the molecular mechanisms linking autoantigen recognition and complement activation, we characterized the dynamic interactions of anti-GM1 IgG autoantibodies on ganglioside-incorporated membranes. Using high-speed atomic force microscopy, we found that the IgG molecules assemble into a hexameric ring structure on the membranes depending on their specific interactions with GM1. Complement component C1q was specifically recruited onto these IgG rings. The ring formation was inhibited by an IgG-binding domain of staphylococcal protein A bound at the cleft between the CH2 and CH3 domains. These data indicate that the IgG assembly is mediated through Fc–Fc interactions, which are promoted under on-membrane conditions due to restricted translational diffusion of IgG molecules. Reduction and alkylation of the hinge disulfide impaired IgG ring formation, presumably because of an increase in conformational entropic penalty. Our findings provide mechanistic insights into the molecular processes involved in Guillain–Barré syndrome and, more generally, into antigen-dependent interplay between antibodies and complement components on membranes

Overall structure of fully assembled cyanobacterial KaiABC circadian clock complex by an integrated experimental-computational approach

巨大タンパク質複合体による概日リズム制御 --小角散乱と計算科学の統合アプローチ--. 京都大学プレスリリース. 2022-03-11.A-B-C is not as easy as 1-2-3 but assembles like clockwork. 京都大学プレスリリース. 2022-03-11.In the cyanobacterial circadian clock system, KaiA, KaiB and KaiC periodically assemble into a large complex. Here we determined the overall structure of their fully assembled complex by integrating experimental and computational approaches. Small-angle X-ray and inverse contrast matching small-angle neutron scatterings coupled with size-exclusion chromatography provided constraints to highlight the spatial arrangements of the N-terminal domains of KaiA, which were not resolved in the previous structural analyses. Computationally built 20 million structural models of the complex were screened out utilizing the constrains and then subjected to molecular dynamics simulations to examine their stabilities. The final model suggests that, despite large fluctuation of the KaiA N-terminal domains, their preferential positionings mask the hydrophobic surface of the KaiA C-terminal domains, hindering additional KaiA-KaiC interactions. Thus, our integrative approach provides a useful tool to resolve large complex structures harboring dynamically fluctuating domains