Large Glycoprotein Research Articles

Thrombospondin-I is a critical modulator in non-alcoholic steatohepatitis (NASH).

Non-alcoholic fatty liver disease (NAFLD) is a progressive liver disease characterized by dysregulated lipid metabolism and chronic inflammation ultimately resulting in fibrosis. Untreated, NAFLD may progress to non-alcoholic steatohepatitis (NASH), cirrhosis and death. However, currently there are no FDA approved therapies that treat NAFLD/NASH. Thrombospondin-I (TSP-1) is a large glycoprotein in the extracellular matrix that regulates numerous cellular pathways including transforming growth factor beta 1 (TGF-β1) activation, angiogenesis, inflammation and cellular adhesion. Increased expression of TSP-1 has been reported in various liver diseases; however, its role in NAFLD/NASH is not well understood. We first examined TSP-1 modulation in hepatic stellate cell activation, a critical initiating step in hepatic fibrosis. Knockdown or inhibition of TSP-1 attenuated HSC activation measured by alpha smooth muscle actin (α-SMA) and Collagen I expression. To investigate the impact of TSP-1 modulation in context of NAFLD/NASH, we examined the effect of TSP-1 deficiency in the choline deficient L-amino acid defined high fat diet (CDAHFD) model of NASH in mice by assessing total body and liver weight, serum liver enzyme levels, serum lipid levels, liver steatosis, liver fibrosis and liver gene expression in wild type (WT) and TSP-1 null mice. CDAHFD fed mice, regardless of genotype, developed phenotypes of NASH, including significant increase in liver weight and liver enzymes, steatosis and fibrosis. However, in comparison to WT, CDAHFD-fed TSP-1 deficient mice were protected against numerous NASH phenotypes. TSP-1 null mice exhibited a decrease in serum lipid levels, inflammation markers and hepatic fibrosis. RNA-seq based transcriptomic profiles from the liver of CDAHFD fed mice determined that both WT and TSP-1 null mice exhibited similar gene expression signatures following CDAHFD, similar to biophysical and histological assessment comparison. Comparison of transcriptomic profiles based on genotype suggested that peroxisome proliferator activated receptor alpha (PPARα) pathway and amino acid metabolism pathways are differentially expressed in TSP-1 null mice. Activation of PPARα pathway was supported by observed decrease in serum lipid levels. Our findings provide important insights into the role of TSP-1 in context of NAFLD/NASH and TSP-1 may be a target of interest to develop anti-fibrotic therapeutics for NAFLD/NASH.

Lubricin (PRG4) reduces fouling susceptibility and improves sensitivity of carbon-based electrodes

In order to overcome the problems of reliability and accuracy often associated with electrochemical sensing in bioassays due to electrode surface fouling, we present an innovative antifouling surface coating based on lubricin (LUB). LUB is a large glycoprotein found in the articular joints of mammals with the ability to self-assemble on surfaces of different nature, creating an antiadhesive brush layer. This work demonstrates the size selective and antiadhesive properties of LUB coatings which prevent large biomolecules from adhering onto the electrode surface while simultaneously allowing smaller electroactive species to pass through the layer unimpeded. Electrochemical and surface characterisation experiments were performed using glassy carbon (GC), carbon black SPEs (CB), and reduced graphene oxide SPEs (rGO) in order to evaluate the antifouling capabilities of LUB coatings in solutions containing bovine serum albumin (BSA) and human saliva. The LUB coatings have been shown to exhibit outstanding antiadhesive properties, size selectivity, self-assembly as well as providing excellent electrochemistry in these highly fouling solutions.

IL-33/ST2 axis promotes glioblastoma cell invasion by accumulating tenascin-C

Tenascin-C (TNC), a very large multimeric glycoprotein, is overexpressed in human glioblastomas, leading to a highly motile and invasive phenotype of glioma cells. However, the regulation of TNC expression in glioma has remained unclear until now. Our data suggest that interleukin-33 (IL-33) may promote the accumulation of TNC protein by autocrine or paracrine modes of action in glioma. In the present study, the expression levels of TNC, IL-33, and ST2 were measured in glioma tissue specimens, and the impact of altered IL-33 expression on TNC was investigated in vitro and in vivo. In contrast with control treatment, IL-33 treatment increased TNC expression, and knockdown of IL-33 attenuated TNC expression in glioma cells. Furthermore, IL-33 induced the activation of nuclear factor κB (NF-κB) and increased the expression of TNC in U251 cells. In addition, blockage of the IL-33-ST2-NFκB pathway resulted in downregulation of TNC production. IL-33 promoted glioma cell invasion by stimulating the secretion of TNC. Similarly, knockdown of TNC inhibited the invasiveness of glioma cells. These findings provide a novel perspective on the role of the IL-33/NF-κB/TNC signalling pathway in supporting cancer progression. Thus, targeting the IL-33/NF-κB/TNC signalling pathway may be a useful therapeutic approach in glioma.

Dynamics, nanomechanics and signal transduction in reelin repeats

Reelin is a large glycoprotein controlling brain development and cell adhesion. It regulates the positioning of neurons, as well as neurotransmission and memory formation. Perturbations in reelin signaling are linked to psychiatric disorders. Reelin participates in signal transduction by binding to the lipoprotein receptors VLDLR and ApoER2 through its central region. This part is rich in repeating BNR-EGF-BNR modules. We used standard molecular dynamics, steered molecular dynamics, and perturbation response scanning computational methods to characterize unique dynamical properties of reelin modules involved in signaling. Each module has specific sensors and effectors arranged in a similar topology. In the modules studied, disulfide bridges play a protective role, probably making both selective binding and protease activity of reelin possible. Results of single reelin molecule stretching by atomic force microscopy provide the first data on the mechanical stability of individual reelin domains. The forces required for partial unfolding of the modules studied are below 60 pN.

Increased Release of Soluble MD-2 in Sickle Cell Disease and Its Role in Pro-Inflammatory Signaling in Endothelial Cells

Sickle cell disease (SCD) is the most common hemoglobinopathy worldwide, resulting from a mutation in the beta globin gene. SCD has significant pathophysiological consequences -- hemolysis, inflammation, oxidative stress, hypercoagulability, endothelial dysfunction and painful vaso-occlusive crises. The latter can be precipitated by infection or other metabolic stressors. Hemolysis chronically exposes endothelial cells, leukocytes, and platelets to hemoglobin and heme that promote pro-inflammatory and prothrombotic phenotypes. We previously demonstrated that toll-like receptor 4 (TLR4) signaling is required for microvascular stasis induced by hemoglobin, heme, or lipopolysaccharide (LPS) in sickle mice. MD-2 is a glycoprotein, co-expressed with TLR4 at the surface of various cell types, principally myeloid and endothelial lineages. MD-2 also exists as a soluble plasma protein (sMD-2), mainly as a large disulfide-bound multimeric glycoprotein, as well as oligomers and monomers. sMD-2 binds LPS and confers TLR4 sensitivity to LPS . A marked increase in sMD-2 has been reported in plasma from patients with sepsis and rheumatoid arthritis. sMD-2 in SCD plasma has not been studied. Since SCD has a pro-inflammatory phenotype, we hypothesized that sMD-2 is increased in SCD plasma and promotes pro-inflammatory signaling of endothelial cells. We assessed plasma levels of sMD-2 by Western blot and found that sMD-2 was increased 1.7-fold in SS human plasma (n=8) compared to healthy AA plasma (p<0.05, n=7). In mice, plasma sMD-2 was increased 7.6-fold in Townes-SS sickle mice (n=9) compared to control Townes-AA mice (p<0.0002, n=7). In contrast, plasma CD14, another required component of LPS-TLR4 signaling, was not significantly different in SS humans (n=8) and SS mice (n=9) compared to AA controls (p<0.05). The liver is one potential source of sMD-2 in plasma. In mice, hepatic MD-2 mRNA was increased 2.1-fold in SS compared to AA (p<0.05, n=6). Activated vascular endothelium is another potential source and target of sMD-2 in plasma. It has been reported by other groups and confirmed by us that LPS induces sMD-2 secretion by human umbilical vein endothelial cells (HUVEC). To determine whether heme can induce sMD-2 secretion from endothelial cells, we treated HUVEC with heme (0-30 μM) for 18 hours and found heme increased sMD-2 in media in a dose-responsive manner. To determine if sMD-2 in plasma could activate TLR4 signaling in endothelial cells, we incubated HUVEC with 2% SS or AA human plasma for 18 hours and measured IL-8 in the media by ELISA. Media IL-8 concentration was 2.6-fold higher in HUVEC incubated with SS plasma compared to AA plasma (p<0.02, n=4). Tak242, a TLR4 signaling inhibitor, blocked IL-8 secretion by HUVEC + SS plasma. Since heme has been shown to activate TLR4 signaling, we examined whether heme could bind to sMD-2 in plasma using a heme-agarose pull-down assay. Human plasma was incubated with heme-agarose to pull down heme binding proteins, followed by Western blot for sMD-2 protein in the pellet. The blot confirmed that sMD-2 in plasma bound specifically to heme. When sMD-2 was removed from SS plasma using an anti-MD-2 affinity column, the sMD-2-depleted plasma reduced IL-8 secretion by HUVEC by 34.3% (p<0.002, n=4). Furthermore, when the high-affinity heme-binding protein hemopexin (10 μM) was added to SS plasma, IL-8 secretion by HUVEC was reduced by 31.6% (p<0.01, n=7). Next, we made recombinant human sMD-2 in CHO cells with protein-free ProCHO medium. UV/Vis absorption spectra (250-600 nm) and heme-agarose pull-down assays found there was heme bound to recombinant sMD-2 in the ProCHO medium. When recombinant sMD-2-heme was added to human AA plasma and incubated with HUVEC, IL-8 secretion increased 2.2-fold (p<0.004, n=3). TLR4 inhibitor Tak242 blocked this increase in IL-8 secretion. When hemopexin was added to the recombinant sMD-2-heme before adding it to AA plasma, IL-8 production was reduced 38% compared to non-hemopexin treated (p<0.01, n=7). In conclusion, these data indicate that sMD-2 is increased in SCD plasma, binds heme, and can stimulate endothelial cell IL-8 production through a TLR4-dependent mechanism. We speculate that sMD-2 bound to heme might play an important role in pro-inflammatory signaling by endothelium in SCD. Disclosures Belcher: Mitobridge, an Astellas Company: Consultancy, Research Funding. Vercellotti:Mitobridge, an Astellas Company: Consultancy, Research Funding.

ADAMTS13, an Anti-thrombotic Protein: Evidence Outside of Thrombotic Thrombocytopenic Purpura

von Willebrand factor (VWF) is a large multimeric plasma glycoprotein that plays a crucial role in hemostasis and thrombosis. VWF recruits platelets at sites of vascular injury by acting as a molecular bridge between circulating platelets and the site of injured or activated blood vessels. Biosynthesis of VWF is restricted to endothelial cells and megakaryocytes. Endothelial VWF is constitutively secreted into plasma and subendothelium, or is stored as "ultra-large" (UL)-VWF multimers in endothelial Weibel-Palade bodies. VWF produced in megakaryocytes is packaged as UL-VWF in the a-granules of platelets. VWF stored in endothelial and platelet storage organelles is secreted in a regulated process in response to stimulation by secretagogues. Absence or dysfunction of VWF results in bleeding symptoms, as observed in patients with von Willebrand disease. An abnormally high activity of VWF can lead to thrombotic events. Interestingly, the activity of VWF is determined by the size of its multimers and UL-VWF can spontaneously form platelet aggregates. An important regulator of VWF size is the metalloprotease ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13), which digests large thrombogenic VWF molecules into smaller, less reactive multimers via cleavage of the Y1605-M1606 bond in the VWF A2 domain. ADAMTS13 is mainly synthesized in the liver by hepatic stellate cells, but other sites of synthesis, including renal podocytes, tubular epithelial cell, platelets and endothelial cells, have also been described. ADAMTS13 is released as an active enzyme into the circulation with no physiological inhibitors. Reduced or absent ADAMTS13 activity causes the microangiopathic disorder thrombotic thrombocytopenic purpura (TTP), characterized by VWF and platelet-rich microthrombi that cause multiple organ failure and even death when left untreated. Besides its clear role in the pathophysiology of TTP, the anti-thrombotic and even anti-inflammatory properties of ADAMTS13 have also become apparent in various other thrombotic conditions. High VWF levels and low ADAMTS13 levels are associated with increased risk or even worse outcome of cardiovascular disease, including ischemic stroke and myocardial infarction. Preclinical studies in mouse models showed the beneficial effect of ADAMTS13 in both cerebral and myocardial ischemia/ reperfusion injury by decreasing both thrombosis and inflammation. In addition, ADAMTS13 was shown to also exert a direct thrombolytic effect on VWF-rich thrombi. In a mouse model of ischemic stroke, this thrombolytic activity resulted in efficient lysis of intracranial thrombi that were resistant to standard treatment with tissue plasminogen activator. Hence, ADAMTS13, as a therapeutic agent, could become an interesting avenue, not only to manage TTP, but also to treat other thrombotic complications. Disclosures De Meyer: Fonds voor Wetenschappelijk Onderzoek: Research Funding; KU Leuven: Employment, Research Funding; Ablynx: Consultancy, Research Funding; Cerenovus: Membership on an entity's Board of Directors or advisory committees; WhiteSwell: Consultancy.

Minor Chemistry Changes Alter Surface Hydration to Control Fibronectin Adsorption and Assembly into Nanofibrils

AbstractFibronectin (FN) is a large glycoprotein which links and transmits signals between the cell's cytoskeleton and the extracellular matrix. FN organization into fibrils and then fibrillogenesis can be induced with the right substrate, such as poly(ethyl acrylate) (PEA), on which FN becomes extended. Interestingly, the almost identical polymer poly(methyl acrylate) (PMA), which has one less methylene bridge (─CH2─), does not cause fibrillogenesis. To investigate the difference in FN behavior on PEA and PMA, the two substrates are modeled using ethyl acrylate (EA) and methyl acrylate (MA) functionalized self‐assembled monolayers (SAMs). It is confirmed experimentally that the EA and MA SAMs exhibit a similar behavior in vitro to the polymers in terms of fibronectin fibrillogenesis, domain exposure, and cell adhesion. All‐atom molecular dynamics simulations of the FNIII 9‐10 domains interacting with each SAM show the adsorption of these two domains on EA SAMs and no adsorption on MA SAMs. Consistently, the experiments show that FN fibrillogenesis takes place on EA SAMs but not on MA SAMs. It is found that the extra methylene group in the EA headgroup leads to more motion within the headgroup that results in a markedly less dense hydration layer, which facilitates FN fibrillogenesis.

Evidence for the Misfolding of the A1 Domain within Multimeric von Willebrand Factor in Type 2 von Willebrand Disease

Von Willebrand factor (VWF), an exceptionally large multimeric plasma glycoprotein, functions to initiate coagulation by agglutinating platelets in the blood stream to sites of vascular injury. This primary hemostatic function is perturbed in type 2 dysfunctional subtypes of von Willebrand disease (VWD) by mutations that alter the structure and function of the platelet GPIbα adhesive VWF A1 domains. The resulting amino acid substitutions cause local disorder and misfold the native structure of the isolated platelet GPIbα–adhesive A1 domain of VWF in both gain-of-function (type 2B) and loss-of-function (type 2M) phenotypes. These structural effects have not been explicitly observed in A1 domains of VWF multimers native to blood plasma. New mass spectrometry strategies are applied to resolve the structural effects of 2B and 2M mutations in VWF to verify the presence of A1 domain structural disorder in multimeric VWF harboring type 2 VWD mutations. Limited trypsinolysis mass spectrometry (LTMS) and hydrogen-deuterium exchange mass spectrometry (HXMS) are applied to wild-type and VWD variants of the single A1, A2, and A3 domains, an A1A2A3 tridomain fragment of VWF, plasmin-cleaved dimers of VWF, multimeric recombinant VWF, and normal VWF plasma concentrates. Comparatively, these methods show that mutations known to misfold the isolated A1 domain increase the rate of trypsinolysis and the extent of hydrogen-deuterium exchange in local secondary structures of A1 within multimeric VWF. VWD mutation effects are localized to the A1 domain without appreciably affecting the structure and dynamics of other VWF domains. The intrinsic dynamics of A1 observed in recombinant fragments of VWF are conserved in plasma-derived VWF. These studies reveal that structural disorder does occur in VWD variants of the A1 domain within multimeric VWF and provides strong support for VWF misfolding as a result of some, but not all, type 2 VWD variants.

A mechano-reactive coarse-grained model of the blood-clotting agent von Willebrand factor.

The von Willebrand Factor (vWF) is a large blood glycoprotein that aids in hemostasis. Within each vWF monomer, the A2 domain hosts a cleavage site for enzyme ADAMTS13, which regulates the size of vWF multimers. This cleavage site can only be exposed when an A2 domain unfolds, and the unfolding reaction energy landscape is highly sensitive to the force conditions on the domain. Based on previous optical tweezer experimental results, we advance here a new activated A2 monomer model (AA2MM) for coarse-grained modeling of vWF that accurately represents the force-based probabilistic change between the unfolded/refolded states. A system of springs is employed to mimic the complex mechanical response of vWF monomers subject to pulling forces. AA2MM was validated by comparing monomer scale simulation results to data from prior pulling experiments on vWF monomer fragments. The model was further validated by comparing multimer scale Brownian dynamics simulation results to experiments using microfluidic chamber microscopy to visualize tethered vWF proteins subject to flow. The A2 domain unfolding reaction was studied in bulk flow simulations (pure shear and elongation flow), giving evidence that elongational flow drives the vWF size regulation process in blood. The mechanoreactive, coarse-grained AA2MM accurately describes the complex mechanical coupling between human blood flow conditions and vWF protein reactivity.

Parallel Comparison of Cancer Antigen 15.3 and Cancer Antigen 27.29 in Breast Cancer

Abstract Objectives Mucins are large and complex glycoproteins expressed by several epithelial tissues. The expression of MUC1 mucin is upregulated in breast cancer and the protein is released in high amounts in patients’ blood. Cancer antigens (CA) 15.3 and CA 27.29, which measure different epitopes of MUC1 protein, are useful biomarkers for postoperative surveillance and therapy monitoring of advanced breast cancer. The aim of this study was to compare CA 15.3 assay and CA 27.29 assay by using paired patient samples. Methods The study was performed in 523 patient samples. CA 15.3 was measured by a sandwich assay on Beckman Coulter UniCel® DxI 800. CA 27. 29 was measured by a competitive immunoassay on Siemens ADVIA Centaur System. Results On Deming regression, the slope was 0.310 with an intercept of 8.6 and correlation coefficient of 0.951. The mean bias between the two assays was –39.2 (–56.6%). The agreement of the two methods was 89% (n = 467), among which, 12% (n = 62) were positive for both CA 15.3 and CA 27.29 (above the upper limit of the reference range), and 77% (n = 405) were negative for both assays (within the reference range). The disagreement was 11% (n = 56), which showed positive CA 27.29 and negative CA 15.3. The correlation coefficient between CA 15.3 and CA 27.29 is 0.9085 when both are positive and 0.6315 when both are negative. The bias is –238.9 (–84.7%) when both are positive and –8.5 (–52.4%) when both are negative. Paired t tests showed significant difference (P < .001) between the CA 15.3 and CA 27.29 in both positive samples and both negative samples. Conclusion In summary, there is a good correlation between CA 15.3 tested on Beckman Coulter UniCel® DxI 800 and CA 27.29 on ADVIA Centaur System. However, with the negative bias, they should not be used interchangeably.

The scavenger receptor SCARA5 is an endocytic receptor for von Willebrand factor expressed by littoral cells in the human spleen

BackgroundScavenger receptors play a significant role in clearing aged proteins from the plasma, including the large glycoprotein coagulation factors von Willebrand factor (VWF) and factor VIII (FVIII). A large genome‐wide association study meta‐analysis has identified genetic variants in the gene SCARA5, which encodes the class A scavenger receptor SCARA5, as being associated with plasma levels of VWF and FVIII. ObjectivesThe ability of SCARA5 to regulate the clearance of VWF‐FVIII was characterized. MethodsVWF‐FVIII interactions with SCARA5 were evaluated by solid phase binding assays and in vitro cell based assays. The influence of SCARA5 deficiency on VWF:Ag and half‐life was assessed in a murine model. The expression pattern of SCARA5 and its colocalization with VWF was evaluated in human tissues. ResultsVWF and the VWF‐FVIII complex bound to human recombinant SCARA5 in a dose‐ and calcium‐dependent manner. SCARA5 expressing HEK 293T cells bound and internalized VWF and the VWF‐FVIII complex into early endosomes. In vivo, SCARA5 deficiency had a modest influence on the half‐life of human VWF. mRNA analysis and immunohistochemistry determined that human SCARA5 is expressed in kidney podocytes and the red pulp, white pulp, and marginal zone of the spleen. VWF was found to colocalize with SCARA5 expressed by littoral cells lining the red pulp of the human spleen. ConclusionsSCARA5 is an adhesive and endocytic receptor for VWF. In human tissues, SCARA5 is expressed by kidney podocytes and splenic littoral endothelial cells. SCARA5 may have a modest influence on VWF clearance in humans.

S3440P Substitution in C-Terminal Region of Human Reelin Dramatically Impairs Secretion of Reelin from HEK 293T cells

Reelin is a large extracellular glycoprotein secreted by Cajal-Retzius cells and has a main role during brain development, especially in neuronal migration. Reelin is comprised of N-terminal F-Spondin like domain, eight tandem repeats, and a highly conserved basic C-Terminal Region (CTR). The CTR main role in the secretion of Reelin has been investigated by advertently inducing deletion in whole or a part of this region; however, the role of CTR point mutations on the secretion of Reelin is shrouded in mystery. In this study, we performed experimental analyses on a sub-region of Human Reelin containing 5th and 6th repeats (R5-R6), a part of 8th repeat and the CTR which were amplified from cDNA of K562 and HEPG2(HepatocellularG2) cells and cloned into a mammalian expressional plasmid (pVP22/myc-His). Bioinformatics investigation was performed on the CTR at both level of nucleotide and amino acid as well as mutant type. Random mutagenesis by error-prone PCR method was utilized to induce mutation in the CTR. The secretion efficiency of recombinant wild-type and mutant Reelin constructs compared in cell lysate and supernatant isolated from the transiently transfected HEK 293T cells using 6XHistag ELISA method. In-vitro study demonstrated that the CTR alteration (S3440P) leads to impairment of Reelin secretion even after overexpression. Our results indicate that S3440P substitution is the highly conserved structure of the CTR has an important effect on Reelin secretion.

Bacterial pathogens under high-tension: Staphylococcus aureus adhesion to von Willebrand factor is activated by force.

Attachment of Staphylococcus aureus to platelets and endothelial cells involves binding of bacterial cell surface protein A (SpA) to the large plasma glycoprotein von Willebrand factor (vWF). SpA-mediated bacterial adhesion to vWF is controlled by fluid shear stress, yet little is currently known about the underlying molecular mechanism. In a recent publication, we showed that the SpA-vWF interaction is tightly regulated by mechanical force. By means of single-molecule pulling experiments, we found that the SpA-vWF bond is extremely strong, being able to resist forces which largely outperform the strength of typical receptor-ligand bonds. In line with flow experiments, strong adhesion is activated by mechanical tension. These results suggest that force induces conformational changes in the vWF molecule, from a globular to an extended state, leading to the exposure of cryptic binding sites to which SpA strongly binds. This force-sensitive mechanism may largely contribute to help S. aureus bacteria to resist shear stress of flowing blood during infection.

Roles of Reelin/Disabled1 pathway on functional recovery of hemiplegic mice after neural cell transplantation; Reelin promotes migration toward motor cortex and maturation to motoneurons of neural grafts

Reelin is a large glycoprotein which regulates central nervous system (CNS) development. Dysfunctions of Reelin were reported on certain neuropsychiatric diseases. We examined involvement of Reelin pathway in functional recovery of hemiplegic mice after neural transplantation.Reelin was expressed 1 day after cryogenic injury of right motor cortex. We transplanted neural stem/progenitor cells (NSPCs) from wild-type mice into ipsilateral striatum of hemiplegic mice. The grafts migrated from the striatum and reached the injured cortex 14 days after transplantation. The transplantation significantly improved their motor functions (P < .05).The NSPCs migrating toward the cortex expressed Reelin receptors, Apoer and Vldlr, and phosphorylated Disabled1 (Dab1), a downstream signaling molecule of Reelin. The grafts expressed Ncadherin and active form of Integrin β1, both of which were known to become active with Reelin stimulation. At day 28, the grafts expressed Ctip2, Crim1, Foxp2, and Fezf2, all of which were forebrain motoneuron associated markers, and Nfm and Synapsin1 on the damaged cortex.We then transplanted NSPCs of yotari mice (yot/yot genotype) having nonfunctional Dab1 by a mutation of its gene. Majority of the grafts from yotari mice (>80%) did not migrate and thus remained at the striatum. The grafts did not express the forebrain motoneuron associated markers nor the cell adhesion molecules including Ncadherin and active Integrin β1.Reelin pathway was involved in graft migration by regulating certain adhesion molecules and in their differentiation to functional motoneurons accompanying synapse formation. We suggested involvement of Reelin pathway for neural regeneration and functional recovery of hemiplegic mice in adulthood after neural transplantation.

Reelin is modulated by diet-induced obesity and has direct actions on arcuate proopiomelanocortin neurons

ObjectiveReelin (RELN) is a large glycoprotein involved in synapse maturation and neuronal organization throughout development. Deficits in RELN signaling contribute to multiple psychological disorders, such as autism spectrum disorder, schizophrenia, and bipolar disorder. Nutritional stress alters RELN expression in brain regions associated with these disorders; however, the involvement of RELN in the neural circuits involved in energy metabolism is unknown. The RELN receptors apolipoprotein E receptor 2 (ApoER2) and very low-density lipoprotein receptor (VLDLR) are involved in lipid metabolism and expressed in the hypothalamus. Here we explored the involvement of RELN in hypothalamic signaling and the impact of diet-induced obesity (DIO) on this system. MethodsAdult male mice were fed a chow diet or maintained on a high-fat diet (HFD) for 12–16 weeks. HFD-fed DIO mice exhibited decreased ApoER2 and VLDLR expression and increased RELN protein in the hypothalamus. Electrophysiology was used to determine the mechanism by which the central fragment of RELN (CF-RELN) acts on arcuate nucleus (ARH) satiety-promoting proopiomelanocortin (POMC) neurons and the impact of DIO on this circuitry. ResultsCF-RELN exhibited heterogeneous presynaptic actions on inhibitory inputs onto ARH-POMC-EGFP neurons and consistent postsynaptic actions. Additionally, central administration of CF-RELN caused a significant increase in ARH c-Fos expression and an acute decrease in food intake and body weight. ConclusionsWe conclude that RELN signaling is modulated by diet, that RELN is involved in synaptic signaling onto ARH-POMC neurons, and that altering central CF-RELN levels can impact food intake and body weight.

Structural biology of laminins.

Laminins are large cell-adhesive glycoproteins that are required for the formation and function of basement membranes in all animals. Structural studies by electron microscopy in the early 1980s revealed a cross-shaped molecule, which subsequently was shown to consist of three distinct polypeptide chains. Crystallographic studies since the mid-1990s have added atomic detail to all parts of the laminin heterotrimer. The three short arms of the cross are made up of continuous arrays of disulphide-rich domains. The globular domains at the tips of the short arms mediate laminin polymerization; the surface regions involved in this process have been identified by structure-based mutagenesis. The long arm of the cross is an α-helical coiled coil of all three chains, terminating in a cell-adhesive globular region. The molecular basis of cell adhesion to laminins has been revealed by recent structures of heterotrimeric integrin-binding fragments and of a laminin fragment bound to the carbohydrate modification of dystroglycan. The structural characterization of the laminin molecule is essentially complete, but we still have to find ways of imaging native laminin polymers at molecular resolution.

Alteration in GPIIb/IIIa Binding of VWD-Associated von Willebrand Factor Variants with C-Terminal Missense Mutations.

The platelet receptor glycoprotein (GP) IIb/IIIa, formed by integrins αIIb and β3, plays an important role in platelet adhesion and aggregation. Its major binding site is the arginine-glycine-aspartic acid (RGD) sequence present in several adhesive proteins. Upon platelet activation, inside-out signaling activates the complex permitting binding to RGD motif containing proteins, such as von Willebrand factor (VWF). VWF is a large multidomain plasma GP essential to primary hemostasis, which can directly interact with platelets because it exhibits binding sites for GPIbα and GPIIb/IIIa in its A1 and C4 domain, respectively. A vast variety of VWF variants have been identified in which domain-specific mutations affect distinct functions of VWF but reduced GPIIb/IIIa binding has barely been studied so far. Here, we strived to investigate the influence of C domain mutations, which have been identified in patients diagnosed with von Willebrand disease (VWD), on VWF-GPIIb/IIIa interaction. To determine binding to membrane-incorporated GPIIb/IIIa in the absence of GPIbα, we developed and validated a cell-based binding assay which uses HEK293 cells stably expressing a constitutively active form of the GPIIb/IIIa receptor complex on their plasma membrane. By employing this assay, we measured GPIIb/IIIa binding of 14 VWF C domain mutants identified in VWD patients. Mutants p.Cys2257Arg, p.Gly2441Cys, p.Cys2477Tyr, and p.Pro2722Ala exhibited significantly reduced binding. Summarizing, we have developed a useful research tool to specifically investigate GPIIb/IIIa interaction with its protein binding partners and identified four VWF variants that exhibit impaired GPIIb/IIIa binding. At least in the homozygous state, this defect could contribute to the VWD phenotype.

Fetal exposure to high levels of maternal glucocorticoids alters reelin signaling in the prefrontal cortex of rat pups

Maternal stress (MS) is associated with various neuropsychiatric disorders and cognitive impairment in the offspring. However, it is unclear how early life stress alters the pup’s brain development and how it contributes to the pathology of neuropsychiatric disorders later in life. Reelin is a large extracellular matrix glycoprotein that plays essential roles in early brain development such as neural migration, synaptic development, and maturation. Dysregulation of reelin and its signaling proteins is associated with the emergence of neuropsychiatric disorders in adulthood. This study examined the effect of repeated maternal Carbenoxolone (CBX) injection during late gestation on reelin signaling in the prefrontal cortex (PFC) of rat pups. CBX is a selective 11β-HSD2 enzyme inhibitor that promotes the direct transfer of maternal corticosteroids (CORT) to the fetus. Therefore, treatment with CBX can mimic the animal model of early life exposure to high levels of maternal stress hormone. In this study, pregnant rats were injected daily with either saline or CBX during gestation day (GD) 14–21, and the levels of reelin and its signaling proteins were examined in the PFC of rat pups at different postnatal age from P0-P21. The main result of this study is the repeated maternal CBX injections during GD14-21 acutely increase reln mRNA and protein expression in the PFC of rat pups at birth (P0) and follow by a significant decrease during P7-P14. The treatment also causes long term decreases in the amount of VLDLR and Dab1 which are the downstream signaling proteins for the reelin pathway, at least until P21. Our results indicated that fetal exposure to high levels of maternal CORT interferes with reelin signaling which might have profound effects on cortical development associated with neuropsychiatric disorders later in life.

Acquired Von Willebrand Syndrome (AVWS) in cardiovascular disease: a state of the art review for clinicians.

Von Willebrand Factor (vWF) is a large glycoprotein with a broad range of physiological and pathological functions in health and disease. While vWF is critical for normal hemostasis, vascular integrity and repair, quantitative and qualitative abnormalities in the molecule can predispose to serious bleeding and thrombosis. The heritable form of von Willebrand Disease was first described nearly a century ago, but more recently, recognition of an acquired condition known as acquired von Willebrand Syndrome (AVWF) has emerged in persons with hematological, endocrine and cardiovascular diseases, disorders and conditions. An in-depth understanding of the causes, diagnostic approach and management of AVWS is important for practicing clinicians.

A Review of the Type-1 Fibrillinopathies: Pathophysiology, Diagnosis and Novel Therapeutic Strategies

Type-1 fibrillinopathies are a family of connective tissue disorders with major clinical manifestations in the skeletal, ocular and cardiovascular systems. The type-1 fibrillinopathies are caused by mutations in the fibrillin-1 gene (FBN1), which encodes fibrillin-1, a large glycoprotein and a major component of the extracellular matrix microfibrils, providing both structural and regulatory support to connective tissues. The type-1 fibrillinopathies have been associated with over 1800 unique mutations within the FBN1 and demonstrate a wide range of phenotypic variability. This, in conjunction with a number of other factors has impacted on the identification of genotypephenotype correlations, pathogenesis and diagnostic tests for this family of diseases, leaving many open-ended theories. Current standard of care relies heavily on surgical intervention and lifelong use of β-blockers to slow disease progression, with research focused heavily on antagonism of transforming growth factor β, which is known to be dysregulated in patients with FBN1 mutations. Antisense oligonucleotides present a novel therapeutic strategy for the type-1 fibrillinopathies, by mediating the alteration of exon arrangement of both the normal and disease-causing mRNA transcripts, to re-establish the periodicity of fibrillin-1. The induced proteins, while internally truncated, should be homologous and thus be able to form multimer units. This treatment alone or in association with isoform switching, TGF-β antagonism or enhanced/inhibited protein degradation could facilitate the assembly of fibrillin-1 monomers into multimers and consequently a decrease in phenotypic severity. This review presents a basic overview of the past and current knowledge about the spectrum of type-1 fibrillinopathies with a particular focus on Marfan syndrome, as well as presenting novel potential therapeutic strategies.