Single-chain Form Research Articles

12538 Blocking Oxidized Phospholipids Improves Bone Mass In Mice By Transcriptional Reprogramming Of Osteoblastic Cells: Analysis At The Single Cell Level

Abstract Disclosure: M. Palmieri: None. I. Nookaew: None. M. Almeida: None. T.E. Joseph: None. C. O'Brien: None. E. Ambrogini: None. Oxidized phospholipids containing phosphatidylcholine (PC-OxPLs), present on oxidized low-density lipoproteins (OxLDL) and apoptotic cells, are pathogenic in many diseases, including osteoporosis. The E06 IgM antibody binds PC-OxPLs and blocks their adverse effects. In our studies, six-month-old female and male mice overexpressing a single chain (scFv) form of the antigen-binding domain of E06 IgM (E06-scFv) have high cancellous and cortical bone mass. In addition, E06-scFv transgenic mice are protected against high fat diet- and age-induced bone loss. E06-scFv increases osteoblast number, bone formation rate and reduces osteoblast apoptosis. To investigate the effect of E06-scFv on osteoblastic cells transcriptional reprogramming, we performed single cell RNA-sequencing (scRNA-seq) of mesenchymal cells from long bones of 6-months old WT and E06-scFv transgenic female and male mice. Based on the total of over 7,500 osteoblastic cells, we focused the analysis on 3 cell clusters, pre-osteoblasts, identified by high levels of osteopontin (Spp1), osteoblasts, identified by high expression of osteocalcin (Bglap) and adipo-CAR expressing the adipogenic marker Adipoq in addition to high expression of the stromal marker Cxcl12. In both males and female transgenic mice, the relative number osteoblasts increased [12.0 % in females and 23.7 % in males; conversely, the relative number of adipo-CAR cells decreased [18.2 % in females and 15.1 % in males] compared to WT. We performed the analysis of functional enrichment-based gene ontology by piano software package and found that, in the three clusters of both male and female transgenic mice, there was an upregulation of gene expression, with a range of 37-125 genes per cluster, related to extracellular matrix organization [p range 0.0003-3.95E-09], bone mineralization [p range 0.04-0.0022] and cell adhesion [p range 0.019-0.00008]. Consistent with these results, bulk RNA sequencing indicated that these ontology terms were downregulated in calvaria-derived osteoblasts treated with OxLDL [extracellular matrix organization p= 1.19E-13; bone mineralization p= 0.0018655; cell adhesion p= 2.73E-12]. Based on these results, we conclude that PC-OxPLs not only affect osteoblast number but also decrease osteoblast activity on a single cell basis. Presentation: 6/1/2024

8627 Viral Hormones: The Role Of Viral Insulin/IGF-1 Like Peptides In Host-Pathogen Interactions

Abstract Disclosure: A. Chuard: None. Viruses, encode proteins that are similar to host proteins to manipulate the host. Until our recent discovery of viral hormones, examples of viral mimicry were limited to immunomodulatory proteins and growth factors. We recently showed that six viruses in Iridoviridae family encode genes mimicking human insulin and IGF-1. We previously chemically synthesizing these viral insulin/IGF-1 like peptides (VILPs) and showed that VILPs can bind to human insulin and IGF-1 receptor and further stimulate post-receptor signaling. VILPs also stimulate glucose uptake and proliferation in mammalian cells and lower the blood glucose in mice. Originally, VILP-carrying viruses were isolated from fish; however, the role of VILPs in viral pathogenesis is not studied in depth. Here, we examined the effects of Grouper Iridovirus (GIV, one of the VILP carrying viruses) infection and GIV-VILP on host insulin/IGF signaling system using fish cells.To this end, we chemically synthesized GIV-VILP in its single chain (sc, IGF-1 like) and double chain (dc, insulin-like) forms. Stimulation of grouper kidney (GK) and AB9 zebrafish cells with these peptides, insulin and IGF-1, showed that IGF-1 was the most potent ligand in these cells indicating an abundance of IGF-1 receptor (IGF1R). Further, both forms of the VILPs were as potent as insulin in its activity on receptor phosphorylation and post-receptor signaling. Examining the viral kinetics, we also showed that GIV32 gene (encoding GIV VILP) is an early viral gene in both cell types. Virion analysis through mass spectrometry indicated that VILPs are not viral structural proteins. On the other hand, analysis of the supernatant from infected cells indicated that VILPs are secreted peptides during the viral cycle. Our results with supernatant transfer experiments from infected cells to uninfected cells support the same conclusion that VILPs are secreted. The secreted VILPs initiate autophosphorylation of IGF1R and/or insulin receptor (IR), consequently triggering downstream cellular signaling and metabolism. Through the use of specific inhibitors for each receptor, we illustrated that IR signaling is crucial for GIV viral replication. Surprisingly, inhibition of IGF1R increased viral replication and we are currently examining potential inhibitory effects of GIV VILP. Furthermore, VILPs appear to exert a biased effect on the AKT pathway compared to the Erk pathway. Furthermore, the inhibition of the AKT pathway significantly reduces GIV viral replication. In summary, our findings reveal that GIV-VILP is actively produced during infection and contributes to the viral replication. To our knowledge, VILPs are the first hormone-mimics characterized in viral replication. Elucidating the role of VILPs in host-pathogen interactions, we propose a novel viral pathogenesis mechanism where viruses mimic host hormones to manipulate the host's endocrine system. Presentation: 6/3/2024

A viral insulin-like peptide inhibits IGF-1 receptor phosphorylation and regulates IGF1R gene expression

ObjectiveThe insulin/IGF superfamily is conserved across vertebrates and invertebrates. Our team has identified five viruses containing genes encoding viral insulin/IGF-1 like peptides (VILPs) closely resembling human insulin and IGF-1. This study aims to characterize the impact of Mandarin fish ranavirus (MFRV) and Lymphocystis disease virus-Sa (LCDV-Sa) VILPs on the insulin/IGF system for the first time. MethodsWe chemically synthesized single chain (sc, IGF-1 like) and double chain (dc, insulin like) forms of MFRV and LCDV-Sa VILPs. Using cell lines overexpressing either human insulin receptor isoform A (IR-A), isoform B (IR-B) or IGF-1 receptor (IGF1R), and AML12 murine hepatocytes, we characterized receptor binding, insulin/IGF signaling. We further characterized the VILPs’ effects of proliferation and IGF1R and IR gene expression, and compared them to native ligands. Additionally, we performed insulin tolerance test in CB57BL/6 J mice to examine in vivo effects of VILPs on blood glucose levels. Finally, we employed cryo-electron microscopy (cryoEM) to analyze the structure of scMFRV-VILP in complex with the IGF1R ectodomain. ResultsVILPs can bind to human IR and IGF1R, stimulate receptor autophosphorylation and downstream signaling pathways. Notably, scMFRV-VILP exhibited a particularly strong affinity for IGF1R, with a mere 10-fold decrease compared to human IGF-1. At high concentrations, scMFRV-VILP selectively reduced IGF-1 stimulated IGF1R autophosphorylation and Erk phosphorylation (Ras/MAPK pathway), while leaving Akt phosphorylation (PI3K/Akt pathway) unaffected, indicating a potential biased inhibitory function. Prolonged exposure to MFRV-VILP led to a significant decrease in IGF1R gene expression in IGF1R overexpressing cells and AML12 hepatocytes. Furthermore, insulin tolerance test revealed scMFRV-VILP's sustained glucose-lowering effect compared to insulin and IGF-1. Finally, cryo-EM analysis revealed that scMFRV-VILP engages with IGF1R in a manner closely resembling IGF-1 binding, resulting in a highly analogous structure. ConclusionsThis study introduces MFRV and LCDV-Sa VILPs as novel members of the insulin/IGF superfamily. Particularly, scMFRV-VILP exhibits a biased inhibitory effect on IGF1R signaling at high concentrations, selectively inhibiting IGF-1 stimulated IGF1R autophosphorylation and Erk phosphorylation, without affecting Akt phosphorylation. In addition, MFRV-VILP specifically regulates IGF-1R gene expression and IGF1R protein levels without affecting IR. CryoEM analysis confirms that scMFRV-VILP’ binding to IGF1R is mirroring the interaction pattern observed with IGF-1. These findings offer valuable insights into IGF1R action and inhibition, suggesting potential applications in development of IGF1R specific inhibitors and advancing long-lasting insulins.

Structure of Single-Chain Nanoparticles under Crowding Conditions: A Random Phase Approximation Approach.

The conformation of poly(methyl methacrylate) (PMMA)-based single-chain nanoparticles (SCNPs) and their corresponding linear precursors in the presence of deuterated linear PMMA in deuterated dimethylformamide (DMF) solutions has been studied by small-angle neutron scattering (SANS). The SANS profiles were analyzed in terms of a three-component random phase approximation (RPA) model. The RPA approach described well the scattering profiles in dilute and crowded solutions. Considering all the contributions of the RPA leads to an accurate estimation of the single chain form factor parameters and the Flory-Huggins interaction parameter between PMMA and DMF. The value of the latter in the dilute regime indicates that the precursors and the SCNPs are in good solvent conditions, while in crowding conditions, the polymer becomes less soluble.

OR27-02 The Bone Anabolic Effect Of An Antibody Blocking Oxidized Phospholipids Is Associated With An Increase In Wnt10b In Osteoblasts.

Abstract Disclosure: M. Palmieri: None. I. Nookaew: None. M.J. Schuller-Almeida: None. H. Kim: None. T.E. Jospeh: None. S.C. Manolagas: None. C.A. O'Brien: None. E. Ambrogini: None. Oxidized phospholipids containing phosphatidylcholine (PC-OxPLs) are toxic molecules present on oxidized low-density lipoproteins (OxLDL) and apoptotic cells; and are pathogenic in many diseases, including osteoporosis. OxPLs decrease Wnt and BMP2 signaling, induce ferroptosis and stimulate pro-inflammatory cytokine production, in atherosclerosis and nonalcoholic steatohepatitis. The natural antibody E06 blocks PC-OxPLs. Transgenic expression of a single chain (scFv) form of the antigen-binding domain of E06 IgM (E06-scFv) increases bone mass in 6-month-old mice and attenuates high fat diet- and age-induced bone loss by increasing osteoblast number and bone formation rate and reducing osteoblast apoptosis. To elucidate the mechanism(s) mediating the bone anabolic effect of E06-scFv we performed bulk-RNA sequencing (bulk-RNA-seq) of vertebral bone and single cell RNA-sequencing (scRNA-seq) of mesenchymal and myeloid cells from long bones of WT and E06-scFv transgenic mice. Earlier bulk-RNA-seq performed in vertebral bone of 22- month-old females had shown that E06-scFv upregulates Wnt10b, but not other Wnt ligands, and the Wnt signaling pathway. We report now that scRNA-seq of mesenchymal cells isolated from 7-month-old female mice revealed that Wnt10b expression is increased by 3-fold (adj-P=3.04E-11) in pre-osteoblasts and osteoblasts from E06-scFv transgenic mice as compared to cells from WT mice. Wnt target genes were also upregulated. An integrated analysis of 9 published scRNA-seq datasets from bone resident mesenchymal and hematopoietic cell types revealed that the majority of Wnt10b in the bone microenvironment originates from cells of the osteoblast lineage. Further, OxLDL decreased Wnt10b and Wnt target genes in calvaria- or bone marrow-derived osteoblastic cell cultures. In contrast, OxLDL did not cause the ferroptosis associated upregulation of Chac1, Ptgs2, Slc7A11, and Acsl4, and the downregulation of Rgs4 in calvaria cells. In addition, no changes in the expression of these genes were detected in the scRNA-seq of mesenchymal cells from E06-scFv compared to WT mice. BMP signaling pathways were also unchanged in this scRNA-seq analysis. Finally, using bulk-RNA-seq of vertebral bone and scRNA-seq of myeloid cells, we could not detect changes in the expression of pro-inflammatory cytokines, such as Tnf-α, Il-6, Il1-α, Il-β, Il-4, Il-12, Lif, Il-23, interferon-α, and interferon-β, between WT and E06-scFv mice. Together, these findings indicate the PC-OxPLs decrease Wnt10b and E06-scFv increases Wnt10b in bone but has no effect on other pathways shown to be affected by OxPLs in other tissues. Thus, Wnt10b may be a critical mediator of the bone anabolic effects of E06-scFv. Presentation: Saturday, June 17, 2023

Development of C1q Affinity Chromatography for the Study of C1q-IgG Interactions.

The classical complement system represents a central effector mechanism of Abs initiated by the binding of C1q to target bound IgG. Human C1q contains six heterotrimeric globular head groups that mediate IgG interaction, resulting in an avidity-driven binding event involving multiple IgG molecules binding a single C1q. Accordingly, surface bound IgG molecules are thought to assemble into noncovalent hexameric rings for optimal binding to the six-headed C1q. To study the C1q-Fc interaction of various Abs and screen for altered C1q binding mutants, we developed, to our knowledge, a novel HPLC-based method. Employing a single-chain form of C1q representing one C1q head group, our HPLC methodology was able to detect the interaction between the single-chain monomeric form of C1q and various ligands. We show that, despite a narrow window of specific binding owing to the low affinity of the monomeric C1q-IgG interaction, this approach clearly distinguished between IgG subclasses with established C1q binding properties. IgG3 displayed the strongest binding, followed by IgG1, with IgG2 and IgG4 showing the weakest binding. Fc mutants known to have increased C1q binding through oligomerization or enhanced C1q interaction showed greatly increased column retention, and IgG glycovariants displayed a consistent trend of increasing retention upon increasing galactosylation and sialylation. Furthermore, the column retention of IgG isotypes and glycovariants matches both the cell surface recruitment of C1q and complement-mediated cytotoxicity induced by each variant on an anti-CD20 Ab backbone. This methodology therefore provides a valuable tool for testing IgG Ab (glyco)variants for C1q binding, with clear relevance for therapeutic Ab development.

Quantifying structure and dynamics of bound and bulk polymer in tailor-made rubber-silica nanocomposites

The dynamics of long polymer chains in the presence of nanoparticles have been investigated. The nanocomposites of interest were inspired by tire industry-like rubber materials and consisted of entangled polyisoprene linear chains mixed with chemically pre-treated silica nanoparticles. Combining rheology, dielectric spectroscopy, and neutron spin echo measurements, we measured the modification of the polymer chain dynamics from bulk state to high filler concentration over a broad range of time and length scales. We show that the end-to-end relaxation does not seem to be impacted, whereas the polymer dynamics is significantly slowed down at a very local scale in the presence of nano-fillers. In addition to this length scale dependent different dynamics, additional Neutron Spin Echo spectroscopy experiments and Small Angle Scattering on labelled polymer chains, irreversibly bound to the filler surface and re-dispersed in a fully deuterated matrix, revealed a negligible dynamical behavior of this particular population of localized chains in the rubber layer. The conformational statistics of these chains is that of self-avoiding walk train within a shell that is thinner than the size of the chain. To the best of our knowledge, this work is the first of its kind which measures the single chain form factor in the bound layer of chemically multi-linked chains to the filler surface.

RF04 | PSUN313 A Natural Antibody Against Oxidized Phospholipids Improves Glucose Metabolism in Aging Mice

Abstract Oxidative stress-induced lipid peroxidation generates oxidized phospholipids (OxPL), such as oxidized phosphatidylcholine, that are pro-inflammatory moieties present on oxidized low density lipoproteins and apoptotic cells. OxPL have been implicated in the pathogenesis of cardiovascular, neurological, autoimmune, liver, and lung diseases. It has been shown earlier that OxPL decrease bone formation. Natural antibodies (NAb) produced by B-1 lymphocytes, bind OxPL and prevent their inflammatory activity. The NAb E06 blocks oxidized phosphatidylcholine, and transgenic expression of a single chain (scFv) form of the antigen-binding domain of E06 IgM (E06-scFv) prevents many age-associated diseases, such as atherosclerosis, non-alcoholic steatohepatitis and age-related bone loss. OxPL are also implicated in the pathogenesis of the metabolic syndrome and diabetes by either causing insulin resistance, increasing macrophage infiltration and inflammation in white adipose tissue, decreasing insulin gene expression, and/or causing death of pancreatic beta cells through induction of oxidative stress and amyloid protein misfolding. Consistent with this evidence, NAb produced by B-1b lymphocytes are protective against obesity associated inflammation, glucose intolerance, and insulin resistance. In the work reported here, we have investigated the impact of oxidized phosphatidylcholine on glucose metabolism and age–related glucose intolerance. To this end, we aged chow-fed female and male C57BL/6 E06-scFv transgenic mice (and their WT littermates) up to 22 and 24 months. Both male and female E06-scFv transgenic mice accumulated less fat mass than WT littermates during aging, as determined by sequential body composition measurements by DXA. Intraperitoneal glucose tolerance test, performed at 10 and at 15 months of age, showed that glucose tolerance was greater in both male and female E06-ScFv transgenic mice than WT littermates and did not differ from the glucose tolerance of young mice. Insulin sensitivity, measured by insulin tolerance test, was increased in 4- and 10-month-old old transgenic females and in 15-month-old transgenic males. There was no difference in food consumption calculated over 5 days or the production of glucose after pyruvate infusion, indicating similar liver gluconeogenesis in both strains. Physical activity or energy expenditure, measured by indirect calorimetry in 11-month-old females, was indistinguishable between transgenic and WT mice. However, E06-scFv transgenic mice exhibited a significant increase in the respiratory exchange ratio, indicating that utilization of glucose was the main metabolic source under basal conditions. Collectively, these results show that blocking oxidized phosphatidylcholine improves age–related glucose intolerance in mice fed a normal diet; hence, OxPL are major culprits of the aging-associated glucose intolerance. Therefore, targeting OxPL with a neutralizing antibody represents a novel therapeutic intervention that may simultaneously ameliorate several age-associated diseases, including the metabolic syndrome and diabetes. Presentation: Saturday, June 11, 2022 1:42 p.m. - 1:47 p.m., Sunday, June 12, 2022 12:30 p.m. - 2:30 p.m.

Deletion of the scavenger receptor Scarb1 in osteoblast progenitors does not affect bone mass.

The scavenger receptor class B member 1 (SR-B1 or Scarb1) is a cell surface receptor for high density lipoproteins. It also binds oxidized low density lipoproteins and phosphocholine-containing oxidized phospholipids (PC-OxPL), which adversely affect bone homeostasis. Overexpression of a single chain form of the antigen-binding domain of E06 IgM-a natural antibody that recognizes PC-OxPL-increases trabecular and cortical bone mass in female and male mice by stimulating bone formation. We have previously reported that Scarb1 is the most abundant scavenger receptor for PC-OxPL in calvaria-derived osteoblastic cells. Additionally, bone marrow- and calvaria-derived osteoblasts from Scarb1 knockout mice (Scarb1 KO) are protected from the pro-apoptotic and anti-differentiating effects of OxPL. Previous skeletal analysis of Scarb1 KO mice has produced contradictory results, with some studies reporting elevated bone mass but another study reporting low bone mass. To clarify the role of Scarb1 in osteoblasts, we deleted Scarb1 specifically in cells of the osteoblast lineage using Osx1-Cre transgenic mice. We observed no difference in bone mineral density measured by DXA in either female or male Osx1-Cre;Scarb1fl/fl mice compared to wild type (WT), Osx1-Cre, or Scarb1fl/fl littermate controls. Additionally, microCT analysis of 6-month-old females and 7-month-old males did not detect any difference in trabecular or cortical bone mass between genotypes. These results indicate that expression of Scarb1 in cells of the osteoblast lineage does not play an important role in bone homeostasis and, therefore, it is not essential for the effects of PC-OxPL on these cells.

Two-Chains Tissue Plasminogen Activator Unifies Met and NMDA Receptor Signalling to Control Neuronal Survival.

Tissue-type plasminogen activator (tPA) plays roles in the development and the plasticity of the nervous system. Here, we demonstrate in neurons, that by opposition to the single chain form (sc-tPA), the two-chains form of tPA (tc-tPA) activates the MET receptor, leading to the recruitment of N-Methyl-d-Aspartate receptors (NMDARs) and to the endocytosis and proteasome-dependent degradation of NMDARs containing the GluN2B subunit. Accordingly, tc-tPA down-regulated GluN2B-NMDAR-driven signalling, a process prevented by blockers of HGFR/MET and mimicked by its agonists, leading to a modulation of neuronal death. Thus, our present study unmasks a new mechanism of action of tPA, with its two-chains form mediating a crosstalk between MET and the GluN2B subunit of NMDARs to control neuronal survival.

Structure and Dynamics of Highly Attractive Polymer Nanocomposites in the Semi-Dilute Regime: The Role of Interfacial Domains and Bridging Chains.

Detailed molecular dynamics (MD) simulations are employed to study how the presence of adsorbed domains and nanoparticle bridging chains affect the structural, conformational, thermodynamic, and dynamic properties of attractive polymer nanocomposite melts in the semi-dilute regime. As a model system we have chosen an unentangled poly(ethylene glycol) (PEG) matrix containing amorphous spherical silica nanoparticles with different diameters and at different concentrations. Emphasis is placed on properties such as the polymer mass density profile around nanoparticles, the compressibility of the system, the mean squared end-to-end distance of PEG chains, their orientational and diffusive dynamics, the single chain form factor, and the scattering functions. Our analysis reveals a significant impact of the adsorbed, interfacial polymer on the microscopic dynamic and conformational properties of the nanocomposite, especially under conditions favoring higher surface-to-volume ratios (e.g., for small nanoparticle sizes at fixed nanoparticle loading, or for higher silica concentrations). Simultaneously, adsorbed polymer chains adopt graft-like conformations, a feature that allows them to considerably extend away from the nanoparticle surface to form bridges with other nanoparticles. These bridges drive the formation of a nanoparticle network whose strength (number of tie chains per nanoparticle) increases substantially with increasing concentration of the polymer matrix in nanoparticles, or with decreasing nanoparticle size at fixed nanoparticle concentration. The presence of hydroxyl groups at the ends of PEG chains plays a key role in the formation of the network. If hydroxyl groups are substituted by methoxy ones, the simulations reveal that the number of bridging chains per nanoparticle decreases dramatically, thus the network formed is less dense and less strong mechanically, and has a smaller impact on the properties of the nanocomposite. Our simulations predict further that the isothermal compressibility and thermal expansion coefficient of PEG-silica nanocomposites are significantly lower than those of pure PEG, with their values decreasing practically linear with increasing concentration of the nanocomposite in nanoparticles.

Abstract 2826: BAG2 promotes tumorigenesis and metastasis by regulating the Cathepsin B cleavage in triple-negative breast cancer cells

Abstract Breast cancer is classified into clinical subtypes based upon receptor expression (ER, PR, Her2). Among the breast cancer subtypes, triple-negative breast cancer (TNBC) refers to a form of breast cancer which lacks expression of ER, PR and HER2. Most of breast cancer treatments can be targeted for cancers that express hormonal receptors or HER2, whereas traditional chemotherapy does not work in TNBCs. Therefore, TNBC remains a clinical challenge for discovery novel specific target biomarker because of no targeted therapies for TNBC. In this study, we show that BAG2 promotes cancer progression by regulating the dual function of Cathepsin B in TNBC cells. We found that BAG2 is highly expressed in basal-like breast cancer cells, where it was associated with recurrence or distant metastasis-free survival in patients. In loss-of-function experiment, BAG2 knockdown strongly decreased the ability of tumor formation and lung metastasis in vivo in TNBC cells. Mechanistically, BAG2 induces metastasis through regulation of secretion of pro-form Cathepsin B and prevents lysosome-mediated cell death through decrease of intracellular single-chain form of Cathepsin B into cytoplasm. Thus, our results suggest that BAG2 promotes tumorigenesis and metastasis through regulation of Cathepsin B cleavage in TNBC cells and may be possible as a novel specific target for treatment of TNBC. [This work was supported by National R&D Program for Cancer Control, Ministry for Health and Welfare, Republic of Korea (HA17C0037).] Citation Format: Kyung-Min Yang, Kyoungwha Pang, Akira Ooshima, Jihee Lee, Seong-jin Kim. BAG2 promotes tumorigenesis and metastasis by regulating the Cathepsin B cleavage in triple-negative breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2826.

Molecular Design and Characterization of a Novel, Vwf Independent Coagulation Factor VIII (LAFATE)

We have known for long that most FVIII is carried by vWF in the blood stream, and began to aware that vWF determines the fate of FVIII whatever modification we make on it for its prolonged circulation. Thus, the consideration that vWF binding property of FVIII is to be conserved is now facing change.The aim of this study is to find out a way to unbind FVIII from endogenous vWF but to stabilize it to generate a novel longer-acting coagulation FVIII. We took a way to shield three major LRP1 binding sites by conjugating PEG and at the same time vWF binding sites by connecting D'D3 domains, the FVIII binding domains of vWF, or its variants using a flexible amino acid linker. We constructed a stable single chain form of FVIII (scFVIII) and employed it as a basic FVIII backbone structure for doing so.We generated FVIII variants according to this strategy, and evaluated their pharmacokinetic property in hemophilia A mice. Unmodified rFVIII (Advate®) was used as the reference. The mice were intravenously injected with the reference and test articles at a dose of 125 IU/kg rFVIII, respectively, and blood was sampled 5 min to 72 h or further after administration. Citrated plasma was prepared and analyzed for FVIII activity (Coamatic®, Chromogenix). PK parameters were determined using non-compartmental modeling with Phoenix WinNonlin 6.4 (Pharsight).We first generated three different types of FVIII variants containing vWF D'D3 domains (D'D3 monomer, D'D3 dimer, D'D3-Fc dimer linked to C-terminus of C2 domain or N-terminus of A1 domain of FVIII), and then conjugated a 40kDa PEG to the B-domain of each variant site-specifically. In the subsequent pharmacokinetic study in hemophilia A mice, we observed significant prolongation of half-lives from 1.8- to 3.6-fold comparing with Advate®. The D'D3 monomer linked FVIII exhibited most prolonged half-life, suggesting that a D'D3 monomer is active enough to substantially attenuate binding of endogenous vWF to the chimera.Recently published electron microscopy and hydrogen-deuterium exchange analyses established the C1 domain as the major binding site for the vWF D'D3 domains on FVIII, and noted additional sites located within the a3 acidic peptide and the A3 and C2 domains of FVIII as being implicated in the FVIII-vWF interaction. In the following experiment, we aimed to find out optimal length of the amino acids linker that would secure easy access of D'D3 domains to A3 and C2 domains of FVIII. Considering the distance between these two domains measurable from the structural information of FVIII, we generated D'D3 monomer connected with 60, 120 and 300 amino acids linkers to scFVIII, respectively, PEGylated them and evaluated their pharmacokinetic properties in hemophilia A mice again. These molecules showed 2.9-, 3.5-, and 2.5-fold more prolonged half-lives than Advate®, respectively, and the 120 amino acids linker seemed optimal.Finally we conjugated several PEGs of different type and size, respectively, to the FVIII-D'D3 connected with this optimal linker, and evaluated their PK properties in hemophilia A mice in the next experiment. Consequently, we identified four different PEGs that support best performance, and named these compounds as LAFATE. LAFATE corrected impaired thrombin generation of the FVIII deficient plasma in a concentration-dependent manner, and stopped acute bleeding as effectively as Advate®, evaluated by the amount of hemoglobin loss for a given time period in a tail-clip bleeding model of hemophilia A mice. LAFATE has a good potential to bring in possible administration of a hemophilia A drug on weekly bases and therefore, warrants further investigation.Related Articles1. The tertiary structure and domain organization of coagulation factor VIII. Betty W. Shen et al., Blood, 20082. A von Willebrand factor fragment containing the D'D3 domains is sufficient to stabilize coagulation factor VIII in mice. Andrew Yee et al., Blood, 20143. von Willebrand factor biosynthesis, secretion, and clearance: connecting the far ends. Peter J. Lenting et al., Blood, 20154. Mapping the interaction between factor VIII and von Willebrand factor by electron microscopy and mass spectrometry. Po-Lin Chiu et al., Blood, 20155. Life in the shadow of a dominant partner: the FVIII-VWF association and its clinical implications for hemophilia A. Steven W. Pipe et al., Blood, 2016 DisclosuresJo:Mogam Institute for Biomedical Research: Employment. Oh:Mogam Institute for Biomedical Research: Employment. Lee:Mogam Institute for Biomedical Research: Employment. Oh:Green Cross Corporation: Employment. Ryu:Mogam Institute for Biomedical Research: Employment. Kim:Green Cross Corporation: Employment, Equity Ownership. Park:Green Cross Corporation: Employment. Kim:Green Cross Corporation: Employment. Jung:Green Cross Corporation: Employment, Equity Ownership.

Blocking Oxidized Phospholipids Attenuates the Age-Associated, but Not the Ovariectomy- or Unloading- Induced, Bone Loss in Mice

Oxidized phospholipids (OxPL), such as oxidized phosphatidylcholine, are generated by oxidative stress (OS)-induced lipid peroxidation. E06 IgM is a natural antibody that recognizes the phosphocholine (PC) moiety of OxPLs, but not native PLs. Generation of transgenic mice expressing a single chain (scFv) form of its antigen-binding domain, “E06-scFv” mice, protects against atherosclerosis, hepatic steatosis and high fat diet-induced loss of bone mass. In addition, E06-scFv increases cancellous and cortical bone mass in both male and female adult mice fed chow diet, by increasing bone formation. Age-related bone loss is associated with increased OS and lipid peroxidation, and is characterized by a reduction in osteoblast number and bone formation. Oxidative stress is involved also in the bone loss caused by sex-steroid deficiency and elevated OS markers are found in unloading-induced bone loss, raising the possibility that an increase of OxPLs induced by OS might be contributing to the pathogenesis of these conditions as well. We aged homozygous E06-scFv transgenic female and male mice and their wild-type littermates up to 22 and 24 months respectively. Serial DXA BMD every 3 months showed that overexpression of E06-scFv attenuated the age-associated bone loss in both sexes. In addition, male and female E06-scFv transgenic mice also accumulated less fat mass than WT littermates during aging. Micro-CT analysis revealed that E06-scFv attenuated the age-associated decline in cancellous, but not cortical, bone mass. The histological analysis of the vertebrae indicated that the aged E06-scFv transgenic mice had increased osteoblasts and decreased osteoclasts compared to the WT mice. To investigate whether the beneficial effect of the E06-scFv could be seen after ovariectomy, 4.5 month old E06-scFv homozygous females and WT controls were ovariectomized (OVX). DXA and micro-CT measurements 6 weeks post- surgery indicated that, unlike aging, E06-scFv did not protect against OVX-induced bone loss in either the cancellous or the cortical compartment. Lastly, we tail-suspended 5.5 month old male mice and sacrificed them 21 days later. E06-scFv transgenic mice had similar cortical bone loss compared to WT mice. In conclusion, the E06-scFV transgene attenuates the age-associated cancellous bone loss in both female and male mice, but has no effect on the OVX- or unloading-induced bone loss. These results fully support our hypothesis that an increase in PC-OxPLs with age, caused at least in part by a decrease in natural anti-PC antibodies, contributes to the age-associated bone loss. This evidence provides proof of concept that blocking PC-OxPLs represents a therapeutic approach to countering the increase of PC-OxPLs with age and their adverse effects on age-related bone loss as well as atherosclerosis and NASH. It also confirms that the mechanisms of cancellous and cortical bone loss are distinct.

Characterization of Viral Insulin-Like Peptides Reveals Unique White Adipose Tissue Specific Characteristics

The members of the insulin superfamily are well conserved across the evolution tree. We recently showed that four viruses in the Iridoviridae family possess genes that share high similarity with human insulin and IGF-1. By chemically synthesizing single chain (sc, IGF-1 like) forms of these viral insulin/IGF-1 like peptides (VILPs), we previously showed that sc VILPs have insulin/IGF properties in vitro and in vivo. However, characteristics of double chain (dc, insulin-like) VILPs remain unknown. In this study, we characterized dc forms of VILPs for Grouper iridovirus (GIV), Singapore grouper iridovirus (SGIV) and Lymphocystis disease virus-1 (LCDV-1). We showed that GIV and SGIV dcVILPs bind to both isoforms of human insulin receptor (IR-A, IR-B) and they bind to IGF-1R with a higher affinity than human insulin. These dcVILPs stimulate receptor phosphorylation and post-receptor signaling in vitro and in vivo. LCDV-1 dcVILP stimulated a weak response in in vitro signaling experiments, although we could not determine binding competition. Both GIV and SGIV dcVILPs stimulated glucose uptake in mice. In vivo infusion experiments in awake mice revealed that while insulin (2.5 mU/kg/min) and GIV dcVILP (125 mU/kg/min) stimulate a comparable glucose uptake in heart, skeletal muscle and brown adipose tissue, GIV dcVILP stimulates ~2 fold higher glucose uptake in white adipose tissue (WAT) compared to insulin. This is due to increased Akt phosphorylation and glucose transporter type 4 (GLUT4) expression compared to insulin specifically in WAT. Taken together, these results show that dc GIV and SGIV dcVILPs are active members of the insulin superfamily with unique characteristics. This observation evokes questions about their potential roles in human disease including diabetes and cancer. Elucidating the mechanism of tissue specificity for GIV dcVILP will help us to better understand insulin action and design new analogues that specifically target the tissues.

A Neutralizing Antibody Targeting Oxidized Phospholipids Promotes Bone Anabolism in Chow-Fed Young Adult Mice.

ABSTRACTOxidized phospholipids containing phosphocholine (OxPL) are pro‐inflammatory lipid peroxidation products that bind to scavenger receptors (SRs), such as Scarb1, and toll‐like receptors (TLRs). Excessive OxPL, as found in oxidized low‐density lipoprotein (OxLDL), overwhelm these defense mechanisms and become pathogenic in atherosclerosis, nonalcoholic steatohepatitis (NASH), and osteoporosis. We previously reported that the innate IgM natural antibody E06 binds to OxPL and neutralizes their deleterious effects; expression of the single‐chain (scFv) form of the antigen‐binding domain of E06 (E06‐scFv) as a transgene increases trabecular bone in male mice. We show herein that E06‐scFv increases trabecular and cortical bone in female and male mice by increasing bone formation and decreasing osteoblast apoptosis in vivo. Homozygous E06‐scFv mice have higher bone mass than hemizygous, showing a dose effect of the transgene. To investigate how OxPL restrain bone formation under physiologic conditions, we measured the levels of SRs and TLRs that bind OxPL. We found that osteoblastic cells primarily express Scarb1. Moreover, OxLDL‐induced apoptosis and reduced differentiation were prevented in bone marrow–derived or calvaria‐derived osteoblasts from Scarb1 knockout mice. Because Scarb1‐deficient mice are reported to have high bone mass, our results suggest that E06 may promote bone anabolism in healthy young mice, at least in part, by neutralizing OxPL, which in turn promote Scarb1‐mediated apoptosis of osteoblasts or osteoblast precursors. © 2020 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR)..

Environmental applications of a biodegradable cysteine-based surfactant

An improved method has been developed for the efficient synthesis of octanoyl-cysteine in single-chain form (N-octanoyl-cys) which operates as a surfactant over a wide pH range, is easily decomposed into natural products and has a high product yield. The compound offers an environmentally acceptable agent for the adsorption of a range of heavy metals from contaminated waters/soils, and it could also be used in general household detergents or personnel-cleaner formulations, and even in toothpastes. The surfactant was used as a co-surfactant for flotation of perfluorooctanoic acid (PFOA), a per/poly-fluoroalkyl substance (PFAS). The new surfactant produced significant foaming and removed 70% of the PFOA after 30min of foam fractionation. The compound is also potentially useful in facilitating the release of PFAS compounds; these are negatively charged and often bound to charged particles in sand, clay, and humic-acid-coated materials and microorganisms via bridging multivalent ions, such as Ca2+, Mg2+, Al3+ and Fe3+, as well as a range of other heavy-metal ions present in soil to varying degrees. In soils (and also in contaminated water), the common chelating agent EDTA is often used to encapsulate these ions (e.g. Ca2+, the dominant ion in soil) at moderately high pH to aid in the release of the bound PFAS compounds. However, it would be more environmentally acceptable to use this new biodegradable surfactant, which could combine chelation with foam-fractionation separation of surface-active (e.g. PFAS) components in soils.

Structural optimization of a TNFR1-selective antagonistic TNFα mutant to create new-modality TNF-regulating biologics

Excessive activation of the proinflammatory cytokine tumor necrosis factor-α (TNFα) is a major cause of autoimmune diseases, including rheumatoid arthritis. TNFα induces immune responses via TNF receptor 1 (TNFR1) and TNFR2. Signaling via TNFR1 induces proinflammatory responses, whereas TNFR2 signaling is suggested to suppress the pathophysiology of inflammatory diseases. Therefore, selective inhibition of TNFR1 signaling and preservation of TNFR2 signaling activities may be beneficial for managing autoimmune diseases. To this end, we developed a TNFR1-selective, antagonistic TNFα mutant (R1antTNF). Here, we developed an R1antTNF derivative, scR1antTNF-Fc, which represents a single-chain form of trimeric R1antTNF with a human IgG-Fc domain. scR1antTNF-Fc had properties similar to those of R1antTNF, including TNFR1-selective binding avidity, TNFR1 antagonistic activity, and thermal stability, and had a significantly extended plasma t1/2in vivo In a murine rheumatoid arthritis model, scR1antTNF-Fc and 40-kDa PEG-scR1antTNF (a previously reported PEGylated form) delayed the onset of collagen-induced arthritis, suppressed arthritis progression in mice, and required a reduced frequency of administration. Interestingly, with these biologic treatments, we observed an increased ratio of regulatory T cells to conventional T cells in lymph nodes compared with etanercept, a commonly used TNF inhibitor. Therefore, scR1antTNF-Fc and 40-kDa PEG-scR1antTNF indirectly induced immunosuppression. These results suggest that selective TNFR1 inhibition benefits the management of autoimmune diseases and that R1antTNF derivatives hold promise as new-modality TNF-regulating biologics.

SUN-346 A Natural Antibody Against Oxidized Phospholipids Attenuates Age-Related Bone Loss and Prevents Age-Related Glucose Intolerance in Mice

Lipid peroxidation produces oxidized phospholipids (OxPL) such as oxidized phosphatidylcholine. These compounds react with amino groups of proteins and lipids to form adducts called oxidation specific epitopes (OSEs), which are proinflammatory moieties present on oxidized low density lipoproteins and on apoptotic cells and, unless removed, cause extensive cell damage. Natural antibodies (NAb) produced by B-1 lymphocytes, bind OxPL and prevent their inflammatory activity. E06 is a NAb that recognizes the phosphocholine moiety of OxPL. We previously showed that transgenic expression of a single chain (scFv) form of the antigen-binding domain of E06 IgM (E06-scFv) increases cancellous and cortical bone mass in both male and female mice by increasing bone formation. Age-related bone loss is characterized by a decline in osteoblast number and bone formation, associated with increased oxidative stress and lipid peroxidation. These findings, together with the evidence that serum anti-OxPL IgM titers decrease with age, suggest that increased OxPL formation and decreased anti-OxPL antibodies may contribute to age-related bone loss. Like humans, mice exhibit an age-dependent worsening in glucose tolerance, mainly due to alteration in body composition and increased fat tissue. Chronic low grade inflammation and oxidative stress are associated with development of diabetes mellitus and B-1 lymphocytes have been shown to be protective against obesity associated inflammation, glucose intolerance, and insulin resistance. We tested the hypothesis that overexpression of E06-scFv could attenuate age-related bone loss and glucose intolerance. Serial BMD measurements by DXA of both female and male C57BL/6 E06-scFv transgenic mice (and their WT littermates) up to 22 and 24 months, respectively, showed that E06-scFv attenuated age-related bone loss at the spine and femur in both sexes. As revealed by microCT analysis, this effect was due to the attenuation of the age-associated decline in cancellous bone in both sexes. Additionally, both male and female E06-scFv transgenic mice accumulated less fat mass than WT littermates during aging. Intraperitoneal glucose tolerance test, at 15 months of age, revealed that glucose tolerance was greater in both male and female E06-ScFv mice than in respective WT littermates and did not differ from the glucose tolerance of young mice, indicating that E06-scFv improves glucose metabolism. These data suggest that OxPL impair both age-related bone loss and age-related glucose intolerance. Therefore, targeting OxPL with a neutralizing antibody such as E06, represents a prototypic therapeutic intervention that may simultaneously ameliorate important age-associated diseases.

Purification, cDNA cloning and characterization of Kunitz-type protease inhibitors from Apios americana tubers.

Two kinds of Kunitz-type protease inhibitors, AKPI1 and AKPI2, were purified from Apios americana tubers by four steps of column chromatographies and their cDNA cloning was performed. AKPI1 cDNA consist of 809 nucleotides, and the matured protein had 190 amino acids with 20,594 Da. AKPI2 cDNA consist of 794 nucleotides, and the matured protein had 177 amino acids with 19,336 Da. P1 site of AKPI2 was Leu88, suggested the target enzyme was chymotrypsin. On the other hand, Gly85-Ile86-Ser87 was positioned around P1 site of AKTI1. Sequence analysis suggested that two forms (single-chain and two-chain form) of AKPI2 protein were present in the tubers. Recombinant AKPI2 expressed by E.coli system showed inhibitory activity toward serine proteases and heat stability. The Ki values toward chymotrypsin and trypsin were 4 × 10-7 M and 6 × 10-6 M, respectively.Abbreviations: AAL: Apios americana lectin; AATI: Apios americana Bowman-Birk type trypsin inhibitor; ACE: angiotensin-converting enzyme; IPTG: isopropyl-β-D-thio-galactopyranoside; Ki: inhibition constant; KPIs: Kunitz-type protease inhibitors; L-BAPA: Benzoyl-L-arginine p-nitroanilide monohydrochloride; L-BTPA: Benzoyl-L-tyrosine p-nitroanilide; PFLNA: Pyr-Phe-Leu-p-nitroanilide; RP-HPLC: reverse-phase high-performance liquid chromatography; RT-PCR: reverse transcription-polymerase chain reaction; SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SLIC: sequence and ligation independent cloning; STANA: N-Succinyl-Ala-Ala-Ala-p-nitroanilide; SHR: spontaneously hypertensive rats; TFA: trifluoroacetic acid; UTR: untranslated region.