Biology subjects

Kametani, Y.

Publications and source records attributed to Kametani, Y..

5 recordsLinked to original sources

bioRxiv2026

Hydration Energetics Shape Antibody Discrimination between Sulfotyrosine and Phosphotyrosine

Chemically similar post-translational modifications can mediate distinct biological functions, but how proteins distinguish between them remains unclear. Sulfotyrosine (sTyr) and phosphotyrosine (pTyr) exemplify this problem because they have similar sizes, local geometries, and electrostatic properties but function in different biological contexts. Here, we used the monoclonal antibody PSG2, which recognizes sTyr independently of the surrounding peptide sequence, to examine how a protein distinguishes these modifications. The crystal structure of PSG2 bound to an sTyr-containing peptide revealed a deep electropositive pocket with no modeled water molecules in direct contact with the sulfate group. Gas-phase density functional theory calculations favored pTyr over sTyr, showing that direct protein-ligand interactions alone are insufficient to explain PSG2 selectivity. Explicit first-shell hydration calculations showed that pTyr has a larger desolvation penalty than sTyr, and accounting for this difference reversed the calculated energetic order. Isothermal titration calorimetry showed favorable enthalpic and entropic contributions to sTyr binding, whereas no detectable heat signal was observed for pTyr. These results show that PSG2 distinguishes sTyr from pTyr through the balance between direct protein-ligand interactions and ligand desolvation.

biophysics↗

bioRxiv2025

Human Interleukin-4-Dependent Facilitation of Human IgG Production in PBL-NOG-hIL-4-Tg mice

Immune-humanized mice provide valuable experimental models for evaluating immune-based therapies, yet the induction of human antigen-specific IgG production remains limited due to species-specific incompatibilities. Our previous work demonstrated that NOG-hIL-4-Tg mice, which express human interleukin-4 (IL-4), support human T and B cell maintenance and enable antigen-specific IgG production following transplantation of human peripheral blood mononuclear cells (PBMCs). In this study, we investigated how IL-4 enhances antibody responses in this model. Flow cytometry and histological analysis revealed that human B cell maintenance was associated with specific plasma hIL-4 concentration ranges at supraphysiological levels. T and B cell receptor repertoire analysis using next-generation sequencing showed that clonal diversity remained largely conserved for one month and decreased to three months post-engraftment. Immunoglobulin repertoire profiling confirmed IgG class switching in an IL-4-concentration-dependent manner. Among the IgG subclasses, IgG3 increased during the first and second months and then decreased thereafter. In contrast, IgG1 tended to increase over time; however, the proportion of IgG subclass varied among individual donors. Following immunization with two distinct peptide antigens, the mice produced enhanced levels of antigen-reactive IgG. However, many B cell clones also exhibited weak responses to unrelated third-party antigens, likely reflecting insufficient affinity maturation. Histological evaluation showed tertiary lymphoid structure (TLS)-like accumulations of B and T cells in the spleen, although fully developed germinal centers were absent. Taken together, these findings demonstrate that NOG-hIL-4-Tg mice maintain human B cells within a regulated IL-4 environment, promote early formation of splenic tertiary lymphoid structures, and support IgG production characterized by clonal expansion, class switching, and somatic hypermutation. These results confirm that human IL-4 expression supports human antibody responses in the PBL-NOG-hIL-4-Tg mouse system. (278)

immunology↗

bioRxiv2025

Formation of a μ-oxo nucleophile enables efficient hydrolysis by a trinuclear metal center in Family II inorganic pyrophosphatase

Efficient catalysis by metalloproteins relies on precise spatial arrangement of metal ions and active-site residues. Family II inorganic pyrophosphatase (PPase) from Shewanella species features a trinuclear metal center and displays higher catalytic activity than binuclear counterparts. Here we elucidate its hydrolytic mechanism using X-ray crystal structure-based extended X-ray absorption fine structure (XCS-EXAFS), site-directed mutagenesis, and density functional theory (DFT) calculations. We identify a catalytic -oxo nucleophile, formed via proton transfer from a bridging -hydroxide to Asp14 and subsequent hydrogen-bond rearrangement to Asp72, as the key species in SN2-type hydrolysis. This conversion defines the rate-limiting step with an activation barrier of 15.5 kcal/mol. Molecular orbital analysis reveals that the trinuclear cluster promotes -oxo formation, aligns the nucleophile for attack, and stabilizes the transition state. The side-chain rotation of the conserved Asp14 is crucial for catalysis. Our results highlight how metalloenzymes exploit geometric and electronic tuning to achieve high reactivity through evolutionarily optimized architectures.

biophysics↗

bioRxiv2023

Complex relationship among vessel diameter, shear stress and blood pressure controlling vessel pruning during angiogenesis

Blood vessel pruning during angiogenesis is the optimization process of the branching pattern to improve the transport properties of a vascular network. Recent studies show that part of endothelial cells (ECs) subjected to lower shear stress migrate toward vessels with higher shear stress in opposition to the blood flow for vessel regression. While dynamic changes of blood flow and local mechano-stress could coordinately modulate EC migration for vessel regression within the closed circulatory system, the effect of complexity of haemodynamic forces and vessel properties on vessel pruning remains elusive. Here, we reconstructed a 3-dimentsional (3D) vessel structure from 2D confocal images of the growing vessels in the mouse retina, and numerically obtained the local information of blood flow, shear stress and blood pressure in the vasculature. Moreover, we developed a predictive model for vessel pruning based on machine learning. We found that the combination of shear stress and blood pressure with vessel radius was tightly corelated to vessel pruning sites. Our results highlighted that orchestrated contribution of local haemodynamic parameters was important for the vessel pruning. Authors SummaryBlood vessel networks formed by angiogenic vessel growth subsequently undergo extensive vascular remodeling process by regression of selected vascular branches. Optimization of the branching pattern in the vasculature is critical to ensure sufficient blood supply throughout the entire tissue. Recent studies have highlighted a strong relationship between the vessel remodeling and the shear stress acting on the vessel wall. However, its detailed mechanisms remain elusive due to the difficulties of estimating local haemodynamic parameters and relating them to vessel remodeling. Here, we have numerically simulated local haemodynamic parameters within the vascular network of the postnatal day 6 (P6) mouse retinal vasculature. Then, the relationship among the local shear stress, blood pressure, and vessel radius with the vessel pruning was examined. Moreover, we developed a predictive model for the vessel pruning based on the local haemodynamic parameters by a machine learning technique. Importantly, our results indicate that the combination of shear stress and blood pressure with vessel radius is tightly correlated to vessel pruning sites. Given the ongoing clinical approach to suppress tumor growth via blood vessel normalization, our results provide important knowledge for developing future medicine such as nanomedicine based on drug delivery systems.

developmental biology↗

bioRxiv2020

Numerical evaluation reveals the effect of branching morphology on vessel transport properties during angiogenesis

Blood flow governs transport of oxygen and nutrients into tissues. Hypoxic tissues secrete VEGFs to promote angiogenesis during development and in tissue homeostasis. In contrast, tumors enhance pathologic angiogenesis during growth and metastasis, suggesting suppression of tumor angiogenesis could limit tumor growth. In line with these observations, various factors have been identified to control vessel formation in the last decades. However, their impact on the vascular transport properties of oxygen remain elusive. Here, we take a computational approach to examine the effects of vascular branching on blood flow in the growing vasculature. First of all, we reconstruct the 3D vascular model from the 2D confocal images of the growing vasculature at P6 mouse retina, then simulate blood flow in the vasculature, which is applied for the gene targeting mouse models causing hypo- or hyper-branching vascular formation. Interestingly, hyper-branching morphology attenuates effective blood flow at the angiogenic front and promotes tissue hypoxia. In contrast, vascular hypo-branching enhances blood supply at the angiogenic front of the growing vasculature. Oxygen supply by newly formed blood vessels improves local hypoxia and decreases VEGF expression at the angiogenic front during angiogenesis. Consistent with the simulation results indicating improved blood flow in the hypo-branching vasculature, VEGF expression around the angiogenic front is reduced in those mouse retinas. Conversely, VEGF expression was enhanced in the hyper-branching vasculature in the mouse retina. Our results indicate the importance of detailed flow analysis in evaluating the vascular transport properties of branching morphology of the blood vessels. Author SummaryBlood vessels are important for the transport of various substances, such as oxygen, nutrients, and cells, to the entire body. Control of blood vessel formation is thought to be important in health and disease. In the last decades, various factors which regulate blood vessel branching morphology have been identified. Gene modification of some of these identified factors results in hyper-branching of the vasculature while others cause hypo-branching of the vessel. Given the importance of the transport property of the blood vessel, it is important to examine the effect of these identified factors on the transport property of the affected vascular morphology. In line with these facts, we reconstruct 3D vessel structures from 2D confocal microscopy images. We then simulate blood flow in the structures numerically. Interestingly, our results suggest vessel network complexity negatively affects the blood perfusion efficiency and tissue oxygenation during angiogenesis. Thus, our results highlight the importance of flow analysis considering the detailed 3D branching pattern of the vascular network to quantitatively evaluate its transport properties.

developmental biology↗