Ectodomain Fragment Research Articles

Production and characterization of single-chain variable fragment antibodies targeting the breast cancer tumor marker nectin-4.

Nectin-4 is a novel biomarker overexpressed in various types of cancer, including breast cancer, in which it has been associated with poor prognosis. Current literature suggests that nectin-4 has a role in cancer progression and may have prognostic and therapeutic implications. The present study aims to produce nectin-4-specific single-chain variable fragment (scFv) antibodies and evaluate their applications in breast cancer cell lines and clinical specimens. We generated recombinant nectin-4 ectodomain fragments as immunogens to immunize chickens and the chickens' immunoglobulin genes were amplified for construction of anti-nectin-4 scFv libraries using phage display. The binding capacities of the selected clones were evaluated with the recombinant nectin-4 fragments, breast cancer cell lines, and paraffin-embedded tissue sections using various laboratory approaches. The binding affinity and in silico docking profile were also characterized. We have selected two clones (S21 and L4) from the libraries with superior binding capacity. S21 yielded higher signals when used as the primry antibody for western blot analysis and flow cytometry, whereas clone L4 generated cleaner and stronger signals in immunofluorescence and immunohistochemistry staining. In addition, both scFvs could diminish attachment-free cell aggregation of nectin-4-positive breast cancer cells. As results from ELISA indicated that L4 bound more efficiently to fixed nectin-4 ectodomain, molecular docking analysis was further performed and demonstrated that L4 possesses multiple polar contacts with nectin-4 and diversity in interacting residues. Overall, the nectin-4-specific scFvs could recognize nectin-4 expressed by breast cancer cells and have the merit of being further explored for potential diagnostic and therapeutic applications.

Diabetic Endothelial Cell Glycogen Synthase Kinase 3β Activation Induces VCAM1 Ectodomain Shedding.

Soluble cell adhesion molecules (sCAMs) are secreted ectodomain fragments of surface adhesion molecules, ICAM1 and VCAM1. sCAMs have diverse immune functions beyond their primary function, impacting immune cell recruitment and activation. Elevated sVCAM1 levels have been found to be associated with poor cardiovascular disease (CVD) outcomes, supporting VCAM1's role as a potential diagnostic marker and therapeutic target. Inhibiting sVCAM1's release or its interaction with immune cells could offer cardioprotection in conditions such as diabetes. Membrane-bound surface adhesion molecules are widely expressed in a wide variety of cell types with higher expression in endothelial cells (ECs). Still, the source of sCAMs in the circulation is not clear. Hypothesizing that endothelial cells (ECs) could be a potential source of sCAMs, this study investigated whether dysfunctional EC signaling mechanisms during diabetes cause VCAM1 ectodomain shedding. Our results from samples from an inducible diabetic mouse model revealed increased sVCAM1 plasma levels in diabetes. Protein analysis indicated upregulated VCAM1 expression and metalloproteases ADAM10 and ADAM17 in diabetic ECs. ADAMs are known for proteolytic cleavage of adhesion molecules, contributing to inflammation. GSK3β, implicated in EC VCAM1 expression, was found to be activated in diabetic ECs. GSK3β activation in control ECs increased ADAM10/17 and VCAM1. A GSK3β inhibitor reduced active GSK3β and VCAM1 ectodomain shedding. These findings suggest diabetic ECs with elevated GSK3β activity led to VCAM1 upregulation and ADAM10/17-mediated sVCAM1 shedding. This mechanism underscores the potential therapeutic role of GSK3β inhibition in reducing the levels of circulating sVCAM1. The complex roles of sCAMs extend well beyond CVD. Thus, unraveling the intricate involvement of sCAMs in the initiation and progression of vascular disease, particularly in diabetes, holds significant therapeutic potential.

Neutrophil elastase decreases SARS-CoV-2 spike protein binding to human bronchial epithelia by clipping ACE-2 ectodomain from the epithelial surface

Patients with cystic fibrosis (CF) have decreased severity of SARS-CoV-2 infections, but the underlying cause is unknown. Patients with CF have high levels of neutrophil elastase (NE) in the airway. We examined whether respiratory epithelial angiotensin converting enzyme 2 (ACE-2), the receptor for the SARS-CoV-2 spike protein, is a proteolytic target of NE. Soluble ACE-2 (sACE-2) levels were quantified by ELISA in airway secretions and serum from patients with and without CF, and the association between sACE-2 levels and NE activity levels was evaluated in CF sputum. We determined that NE activity was directly correlated with increased ACE-2 in CF sputum. Additionally, primary human bronchial epithelial (HBE) cells, exposed to NE or control vehicle, were evaluated by western analysis for the release of cleaved ACE-2 ectodomain fragment into conditioned media, and by flow cytometry for the loss of cell surface ACE-2, its impact on SARS-CoV-2 spike protein binding. We found that NE treatment released ACE-2 ectodomain fragment from HBE and decreased spike protein binding to HBE. Furthermore, we performed NE treatment of recombinant ACE-2-Fc tagged protein in vitro to assess whether NE was sufficient to cleave recombinant ACE-2-Fc protein. Proteomic analysis identified, specific NE cleavage sites in the ACE-2 ectodomain that would result in loss of the putative N-terminal spike binding domain. Collectively, data support that NE plays a disruptive role in SARS-CoV-2 infection by catalyzing ACE-2 ectodomain shedding from the airway epithelia. This mechanism may reduce SARS-CoV-2 virus binding to respiratory epithelial cells and decrease severity of COVID19 infection.

Chitosan-based formulation of hemagglutinin antigens for oculo-nasal booster vaccination of chickens against influenza viruses

Avian influenza viruses (AIVs) and especially highly pathogenic (HP) AIVs of the H5 and H7 subtypes are of both veterinary and public health concern worldwide. In response to the demand for effective vaccines against H5N1 HPAIVs, we produced recombinant protein based on hemagglutinin (HA), a protective viral antigen. A fragment of the HA ectodomain, with a multibasic cleavage site deletion, was expressed in Escherichia coli, refolded, and chromatographically purified from inclusion bodies. Finally, the protein was formulated in Tris-HCl buffer of pH 8.0 or PBS of pH 7.4 to obtain antigens denoted rH5–1 and rH5–2, respectively. The systemic prime and boost immunizations proved that rH5–1 adsorbed to aluminum hydroxide induces anti-H5 HA neutralizing antibodies and protective immune responses against H5N1 HPAIVs in chickens. The present studies were aimed at stimulating immune responses via the mucosal routes using the systemic prime-mucosal boost strategy. Efficacy trials were performed in commercial layer chickens. For systemic and mucosal immunizations, H5 HA antigens were adjuvanted with aluminum hydroxide and chitosan glutamate, respectively. The first dose of rH5–2 was administered subcutaneously, while its second dose was administered subcutaneously, intraocularly, oculo-nasally, or intranasally. rH5–1 was delivered to the subcutaneously primed chickens by the intranasal route. Post-vaccination sera were analyzed for anti-H5 HA antibodies, using homologous ELISA and heterologous FluAC H5 and hemagglutination inhibition tests. Intraocularly and oculo-nasally delivered rH5–2 mixed with chitosan glutamate was capable of stimulating anti-H5 HA IgY antibody responses in the subcutaneously primed chickens; however, it was ineffective when administered by the intranasal route. Efficient intranasal boosting was achieved using rH5–1. The enhanced production of antigen-specific antibodies was reflected in the development of H5-subtype specific and hemagglutination inhibiting antibodies. Conclusively, the subcutaneous prime and oculo-nasal boost vaccination is proposed as the target strategy for future optimization.

Sulfated Glycoprotein Synthesis.

Chemical protein synthesis has achieved tremendous progress in the past decades. With the development of chemical ligation as powerful tools, the scope of synthetic protein is greatly expanded. Proteoglycans are a class of sulfated glycoproteins widely distributed on the cell surface and in the extracellular matrix, which are extensively engaged in cellular communication events. Consisting of protein backbone and glycosaminoglycan(s) side chain, proteoglycans are highly complex and heterogeneous in nature. Chemical synthesis provides facile and reliable approach to these molecules, with defined glycan structure and sulfation pattern. One remaining problem is that the acid-labile sulfates could hardly survive during the typical solid phase peptide synthesis (SPPS) process. In this chapter, strategic design of a "glycopeptide cassette" for the preparation of sulfated glycoprotein is described. In particular, we provide protocols for the chemical synthesis of ectodomain fragment (23-120) of sulfated glycoprotein syndecan-1.

Complement Receptor 3 Forms a Compact High-Affinity Complex with iC3b

Complement receptor 3 (CR3, also known as Mac-1, integrin αMβ2, or CD11b/CD18) is expressed on a subset of myeloid and certain activated lymphoid cells. CR3 is essential for the phagocytosis of complement-opsonized particles such as pathogens and apoptotic or necrotic cells opsonized with the complement fragment iC3b and, to a lesser extent, C3dg. Although the interaction between the iC3b thioester domain and the ligand binding CR3 αM I-domain is structurally and functionally well characterized, the nature of additional CR3-iC3b interactions required for phagocytosis of complement-opsonized objects remains obscure. In this study, we analyzed the interaction between iC3b and the 150-kDa headpiece fragment of the CR3 ectodomain. Surface plasmon resonance experiments demonstrated a 30 nM affinity of the CR3 headpiece for iC3b compared with 515 nM for the iC3b thioester domain, whereas experiments monitoring binding of iC3b to CR3-expressing cells suggested an affinity of 50 nM for the CR3-iC3b interaction. Small angle x-ray scattering analysis revealed that iC3b adopts an extended but preferred conformation in solution. Upon interaction with CR3, iC3b rearranges to form a compact receptor-ligand complex. Overall, the data suggest that the iC3b-CR3 interaction is of high affinity and relies on minor contacts formed between CR3 and regions outside the iC3b thioester domain. Our results rationalize the more efficient phagocytosis elicited by iC3b than by C3dg and pave the way for the development of specific therapeutics for the treatment of inflammatory and neurodegenerative diseases that do not interfere with the recognition of noncomplement CR3 ligands.

Abstract P-14: Molecular Modeling of the Transmembrane Domain of the SARS Cov-2 S-Protein and its Interaction with the Membrane

Background: The spike glycoprotein of SARS-coronavirus mediates the early events leading to infection of cells, including fusion of the viral and cellular membranes. The spike is a type I membrane glycoprotein that possesses a conserved transmembrane anchor and an unusual cysteine-rich domain that bridges the putative junction of the anchor and the cytoplasmic tail. In this study, we examined the role of these carboxyl-terminal domains in S-protein interaction with membrane. Methods: Structural model of the trimeric TM domain and adjacent fragments of ecto- and endo domains (residues 1157-1256) of the S-protein was built by homology basing on the solution structure of the SARS-coronavirus S-protein HR2 domain (pdb-code 2fxp), the structure of the transmembrane domain of HIV-1 gp41 in bicelle (5jyn), and assumption of generally coiled-coil fold of the considered domain. C-terminus of the domain was left unstructured but fully palmitoylated. Molecular dynamics simulation in heterogeneous lipid bilayer was prepared with CHARMM-GUI and performed with Gromacs during 100 ns. Results: 1. Ectodomain fragment (residues 1157-1212) demonstrates a tilt by the angle of 40-60 degrees from the axis of the TM domain (residues 1213-1237). This tilt is facilitated by glycine residues in position 1204. 2. Cholesterol molecules of the bottom layer tend to localize around protein due to interaction with palmitoyl tails while lipids in the upper layer do not show such tendency. Conclusion: Performed molecular simulations show that both palmitoylation and a large cluster of aromatic residues provide high stability of the S-protein TM domain.

Proteolytic Shedding of Human Colony-Stimulating Factor 1 Receptor and its implication.

Both Colony‐stimulating factor 1 receptor (CSF1R) and triggering receptor expressed on myeloid cells‐2 (TREM2) are trans‐membrane receptors and are expressed in the brain primarily by microglia. Mutations in these two microglia‐expressed genes associated with neurodegenerative disease have recently been grouped under the term “microgliopathy”. Several literatures have indicated that CSF1R and TREM2 encounters a stepwise shedding and TREM2 variants impair or accelerate the processing. However, whether CSF1R variant affects the shedding of CSF1R remains elusive. Here, plasmids containing human CSF1R or TREM2 were transiently transfected into the human embryonic kidney (HEK) 293T cells. Using Western Blot and/or ELISA assay, we demonstrated that, similar to those of TREM2, an N‐terminal fragment (NTF) shedding of CSF1R ectodomain and a subsequent C‐terminal fragment (CTF) of CSF1R intra‐membrane were generated by a disintegrin and metalloprotease (ADAM) family member and by γ‐secretase, respectively. And the shedding was inhibited by treatment with Batimastat, an ADAM inhibitor, or DAPT or compound E, a γ‐secretase inhibitor. Importantly, we show that the cleaved fragments, both extracellular domain and intracellular domain of a common disease associated I794T variant, were decreased significantly. Together, our studies demonstrate a stepwise approach of human CSF1R cleavage and contribute to understand the pathogenicity of CSF1R I794T variant in adult‐onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP). These studies also suggest that the cleaved ectodomain fragment released from CSF1R may be proposed as a diagnostic biomarker for ALSP.

Angiotensin-converting Enzyme 2–containing Small Extracellular Vesicles and Exomeres Bind the Severe Acute Respiratory Syndrome Coronavirus 2 Spike Protein

Angiotensin-converting Enzyme 2–containing Small Extracellular Vesicles and Exomeres Bind the Severe Acute Respiratory Syndrome Coronavirus 2 Spike Protein

Amyloid precursor protein glycosylation is altered in the brain of patients with Alzheimer\u2019s disease

BackgroundThe amyloid precursor protein (APP) is a transmembrane glycoprotein that undergoes alternative proteolytic processing. Its processing through the amyloidogenic pathway originates a large sAPPβ ectodomain fragment and the β-amyloid peptide, while non-amyloidogenic processing generates sAPPα and shorter non-fibrillar fragments. Hence, measuring sAPPα and sAPPβ has been proposed as a means to identify imbalances between the amyloidogenic/non-amyloidogenic pathways in the brain of Alzheimer’s disease (AD) patients. However, to date, no consistent changes in these proteolytic fragments have been identified in either the brain or cerebrospinal fluid of AD individuals.MethodsIn frontal cortex homogenates from AD patients (n = 7) and non-demented controls (NDC; n = 7), the expression of total APP mRNA and that of the APP isoforms generated by alternative splicing, APP695 and APP containing the Kunitz protease inhibitor (KPI), was analyzed by qRT-PCR using TaqMan and SYBR Green probes. The balance between the amyloidogenic/non-amyloidogenic pathways was examined in western blots estimating the sAPPα and sAPPβ fragments and their membrane-tethered C-terminal fragments CTFα and CTFβ. CHO-PS70 cells, stably over-expressing wild-type human APP, served to evaluate whether Aβ42 peptide treatment results in altered APP glycosylation. We determined the glycosylation pattern of sAPPα and sAPPβ in brain extracts and CHO-PS70 culture media by lectin-binding assays.ResultsIn the cortex of AD patients, we detected an increase in total APP mRNA relative to the controls, due to an increase in both the APP695 and APP-KPI variants. However, the sAPPα or sAPPβ protein levels remained unchanged, as did those of CTFα and CTFβ. We studied the glycosylation of the brain sAPPα and sAPPβ using lectins and pan-specific antibodies to discriminate between the fragments originated from neuronal APP695 and glial/KPI variants. Lectin binding identified differences in the glycosylation of sAPPβ species derived from the APP695 and APP-KPI variants, probably reflecting their distinct cellular origin. Moreover, the lectin-binding pattern differed in the sAPPα and sAPPβ originated from all the variants. Finally, when the lectin-binding pattern was compared between AD and NDC groups, significant differences were evident in sAPPα glycosylation. Lectin binding of the soluble sAPPα and sAPPβ from CHO-PS70 cells were also altered in cells treated with the Aβ peptide.ConclusionOur analysis of the lectin binding to sAPPα and sAPPβ suggests that glycosylation dictates the proteolytic pathway for APP processing. Differences between the demented and controls indicate that changes in glycosylation may influence the generation of the different APP fragments and, consequently, the pathological progression of AD.

Loss of GPVI and GPIbα contributes to trauma-induced platelet dysfunction in severely injured patients

AbstractTrauma-induced coagulopathy (TIC) is a complex, multifactorial failure of hemostasis that occurs in 25% of severely injured patients and results in a fourfold higher mortality. However, the role of platelets in this state remains poorly understood. We set out to identify molecular changes that may underpin platelet dysfunction after major injury and to determine how they relate to coagulopathy and outcome. We performed a range of hemostatic and platelet-specific studies in blood samples obtained from critically injured patients within 2 hours of injury and collected prospective data on patient characteristics and clinical outcomes. We observed that, although platelet counts were preserved above critical levels, circulating platelets sampled from trauma patients exhibited a profoundly reduced response to both collagen and the selective glycoprotein VI (GPVI) agonist collagen-related peptide, compared with those from healthy volunteers. These responses correlated closely with overall clot strength and mortality. Surface expression of the collagen receptors GPIbα and GPVI was reduced on circulating platelets in trauma patients, with increased levels of the shed ectodomain fragment of GPVI detectable in plasma. Levels of shed GPVI were highest in patients with more severe injuries and TIC. Collectively, these observations demonstrate that platelets experience a loss of GPVI and GPIbα after severe injury and translate into a reduction in the responsiveness of platelets during active hemorrhage. In turn, they are associated with reduced hemostatic competence and increased mortality. Targeting proteolytic shedding of platelet receptors is a potential therapeutic strategy for maintaining hemostatic competence in bleeding and improving the efficacy of platelet transfusions.

Magnetic Bead-Immobilized Mammalian Cells Are Effective Targets to Enrich Ligand-Displaying Yeast.

Yeast surface display empowers selection of protein binding ligands, typically using recombinant soluble antigens. However, ectodomain fragments of transmembrane targets may fail to recapitulate their true, membrane-bound form. Direct selections against adhered mammalian cells empower enrichment of genuine binders yet benefit from high target expression, robustly adherent mammalian cells, and nanomolar affinity ligands. This study evaluates a modified format with mammalian cells immobilized to magnetic beads; yeast-displayed fibronectin domain and affibody ligands of known affinities and cells with expression ranges of epidermal growth factor receptor (EGFR) and CD276 elucidate important parameters to ligand enrichment and yield in cell suspension panning with comparison to adherent panning. Cell suspension panning is hindered by significant background of nondisplaying yeast but exhibits yield advantages in model EGFR systems for a high affinity (KD = 2 nM) binder on cells with both high (106 per cell) target expression (9.6 ± 0.6% vs 3.2 ± 0.4%, p < 0.0001) and mid (105) target expression (2.3 ± 0.5% vs 0.41 ± 0.09%, p = 0.0008), as well as for a low affinity (KD > 600 nM) binder on high target expression cells (2.0 ± 0.5% vs 0.017 ± 0.005%; p = 0.001). Significant enrichment was observed for all EGFR systems except the low-affinity, high expression system. The CD276 system failed to provide significant enrichment, indicating that this technique may not be suitable for all targets. Collectively, this study highlights new approaches that yield successful enrichment of yeast-displayed ligands via panning on immobilized mammalian cells.

Prime-Boost Vaccination With a Novel Hemagglutinin Protein Produced in Bacteria Induces Neutralizing Antibody Responses Against H5-Subtype Influenza Viruses in Commercial Chickens.

The highly pathogenic (HP) avian influenza virus (AIV), H5N1 and reassortant H5-subtype HPAIVs, H5N2, H5N6, and H5N8, cause high mortality in domestic birds, resulting in economic losses in the poultry industry. H5N1 and H5N6 also pose significant public health risks and H5N1 viruses are a permanent pandemic threat. To control HPAIVs, eukaryotic expression systems have traditionally been exploited to produce vaccines based on hemagglutinin (HA), a protective viral antigen. In contrast, we used a bacterial expression system to produce vaccine targeting the HA protein. A fragment of the HA ectodomain from H5N1, with a multibasic cleavage site deletion, was expressed in Escherichia coli, refolded, and chromatographically purified from inclusion bodies. The resulting antigen, rH5-E. coli, was validated in terms of conformational integrity and oligomerization status. Previously, the protective efficacy of rH5-E. coli adjuvanted with aluminum hydroxide, has been positively verified by challenging the specific pathogen-free layer chickens with homologous and heterologous H5N1 HPAIVs. Protection was provided primarily by the H5 subtype-specific antibodies, as detected in the FluAC H5 test. The present studies were conducted to assess the performance of alum-adjuvanted rH5-E. coli in commercial birds. Broiler chickens were vaccinated twice with 25 μg of rH5-E. coli at 2- and 4-week intervals, while the layer chickens were vaccinated with 5- to 25-μg antigen doses at 4- and 6-week intervals. Post-vaccination sera were analyzed for anti-H5 HA antibodies, using homologous ELISA and heterologous FluAC H5 and hemagglutination inhibition (HI) tests. Prime-boost immunizations with rH5-E. coli elicited H5 HA-specific antibodies in all the chickens tested. Two antigen doses administered at 4- or 6-week intervals were sufficient to induce neutralizing antibodies against H5-subtype HAs; however, they were ineffective when applied with a 2-week delay. In the layers, 80% to 100% of individuals developed antibodies that were active in the FluAC H5 and/or HI tests. A dose-sparing effect was seen when using the longer prime-boost interval. In the broiler chickens, 62.5% positivity was achieved in the FluAC H5 and/or HI tests. The trials confirmed the vaccine potential of rH5-E. coli and provided indications for anti-influenza vaccination with respect to the chicken type and immunization scheme.

Ectopic expression of L1CAM ectodomain alters differentiation and motility, but not proliferation, of human neural progenitor cells

Adult human neural progenitor and stem cells have been implicated as a potential source of brain cancer causing cells, but specific events that might cause cells to progress towards a transformed phenotype remain unclear. The L1CAM (L1) cell adhesion/recognition molecule is expressed abnormally by human glioma cancer cells and is released as a large extracellular ectodomain fragment, which stimulates cell motility and proliferation. This study investigates the effects of ectopic overexpression of the L1 long ectodomain (L1LE; ˜180 kDa) on the motility, proliferation, and differentiation of human neural progenitor cells (HNPs). L1LE was ectopically expressed in HNPs using a lentiviral vector. Surprisingly, overexpression of L1LE resulted in reduced HNP motility in vitro, in stark contrast to the effects on glioma and other cancer cell types. L1LE overexpression resulted in a variable degree of maintenance of HNP proliferation in media without added growth factors but did not increase proliferation. In monolayer culture, HNPs expressed a variety of differentiation markers. L1LE overexpression resulted in loss of glutamine synthetase (GS) and β3-tubulin expression in normal HNP media, and reduced vimentin and increased GS expression in the absence of added growth factors. When co-cultured with chick embryonic brain cell aggregates, HNPs show increased differentiation potential. Some HNPs expressed p-neurofilaments and oligodendrocytic O4, indicating differentiation beyond that in monolayer culture. Most HNP-L1LE cells lost their vimentin and GFAP (glial fibrillary acidic protein) staining, and many cells were positive for astrocytic GS. However, these cells rarely were positive for neuronal markers β3-tubulin or p-neurofilaments, and few HNP oligodendrocyte progenitors were found. These results suggest that unlike for glioma cells, L1LE does not increase HNP cell motility, but rather decreases motility and influences the differentiation of normal brain progenitor cells. Therefore, the effect of L1LE on increasing motility and proliferation appears to be limited to already transformed cells.

Urinary Cell Adhesion Molecule 1 Is a Novel Biomarker That Links Tubulointerstitial Damage to Glomerular Filtration Rates in Chronic Kidney Disease.

Cell adhesion molecule 1 (CADM1) is an immunoglobulin superfamily member strongly expressed on renal tubular epithelia in the urinary tract. Enzymatic cleavage of its ectodomain increases in chronic kidney disease (CKD), and is assumed to contribute to tubulointerstitial lesion formation. Because the cleaved ectodomain fragments are likely to be released into the urine, a sandwich enzyme-linked immunosorbent assay (ELISA) system for urinary CADM1 was developed using two anti-ectodomain antibodies. Urinary CADM1 concentrations in patients with CKD based on various forms of glomerulonephritis and nephropathy (n = 127) were measured. A total of 44 patients (35%) had elevated CADM1 concentrations over the normal upper limit (362 pg/mL), with a mean of 1,727 pg/mL. Renal biopsy specimens of all patients were pathologically scored for tubulointerstitial lesions using epithelial degeneration, interstitial inflammation, and fibrosis. There were no correlations between urinary CADM1 concentrations and pathological scores or any widely used renal markers, including glomerular filtration rate (GFR), but there was a weak inverse correlation between pathological scores and GFR (R2 = 0.292). Notably, this correlation gradually increased in patients with increasing CADM1 concentrations, and reached a maximum R2 (0.899) at a cutoff of 1,569 pg/mL. The results of this study suggest that urinary CADM1 is a useful marker indicating tubulointerstitial damage from elevated GFR levels in CKD.

Thrombin-cleaved syndecan-3/-4 ectodomain fragments mediate endothelial barrier dysfunction.

ObjectiveThe endothelial glycocalyx constitutes part of the endothelial barrier but its degradation leaves endothelial cells exposed to transmigrating cells and circulating mediators that can damage the barrier or promote intercellular gaps. Syndecan proteins are key components of the endothelial glycocalyx and are shed during disease states where expression and activity of proteases such as thrombin are elevated. We tested the ability of thrombin to cleave the ectodomains of syndecans and whether the products could act directly on endothelial cells to alter barrier function.Approach and resultsUsing transmission electron microscopy, we illustrated the presence of glycocalyx in human lung microvasculature. We confirmed expression of all syndecan subtypes on the endothelial surface of agarose-inflated human lungs. ELISA and western blot analysis suggested that thrombin can cleave syndecan-3/-4 ectodomains to produce fragments. In vivo, syndecan-3 ectodomain fragments increased extravasation of albumin-bound Evans blue in mouse lung, indicative of plasma protein leakage into the surrounding tissue. Syndecan-3/-4 ectodomain fragments decreased transendothelial electrical resistance, a measure of cell-cell adhesive barrier integrity, in a manner sensitive to a Rho kinase inhibitor. These effects were independent of glycosylation and thrombin receptor PAR1. Moreover, these cleavage products caused rapid VE-cadherin-based adherens junction disorganization and increased F-actin stress fibers, supporting their direct effect on endothelial paracellular permeability.ConclusionsWe suggest that thrombin can cleave syndecan-3/4 ectodomain into fragments which interact with endothelial cells causing paracellular hyperpermeability. This may have important implications in the pathogenesis of vascular dysfunction during sepsis or thrombotic disease states where thrombin is activated.

Decreased circulating ErbB4 ectodomain fragments as a read-out of impaired signaling function in amyotrophic lateral sclerosis

ErbB4 is a transmembrane receptor tyrosine kinase that binds to neuregulins to activate signaling. Proteolytic cleavage of ErbB4 results in release of soluble fragments of ErbB4 into the interstitial fluid. Disruption of the neuregulin-ErbB4 pathway has been suggested to be involved in the pathogenesis of amyotrophic lateral sclerosis (ALS). This study assesses whether soluble proteolytic fragments of the ErbB4 ectodomain (ecto-ErbB4) can be detected in cerebrospinal fluid (CSF) and plasma, and if the levels are altered in ALS. Immunoprecipitation combined with mass spectrometry or western blotting analyses confirmed the presence of ecto-ErbB4 in human CSF. Several anti-ErbB4-reactive bands, including a 55 kDa fragment, were detected in CSF. The bands were generated in the presence of neuregulin-1 (Nrg1) and were absent in plasma from ErbB4 knockout mice. Ecto-ErbB4 levels were decreased in CSF from ALS patients (n = 20) and ALS with concomitant frontotemporal dementia patients (n = 10), compared to age-matched controls (n = 13). A similar decrease was found for the short ecto-ErbB4 fragments in plasma of the same subjects. Likewise, the 55-kDa ecto-ErbB4 fragments were decreased in the plasma of the two transgenic mouse models of ALS (SOD1G93A and TDP-43A315T). Intracellular ErbB4 fragments were decreased in the frontal cortex from SOD1G93A mice, indicating a reduction in Nrg-dependent induction of ErbB4 proteolytic processing, and suggesting impaired signaling. Accordingly, overexpression of Nrg1 induced by an adeno-associated viral vector increased the levels of the ecto-ErbB4 fragment in the SOD1G93A mice. We conclude that the determination of circulating ecto-ErbB4 fragments could be a tool to evaluate the impairment of the ErbB4 pathway and may be a useful biomarker in ALS.

CSF-ApoER2 fragments as a read-out of reelin signaling: Distinct patterns in sporadic and autosomal-dominant Alzheimer disease

Reelin is a glycoprotein associated with synaptic plasticity and neurotransmission. The malfunctioning of reelin signaling in the brain is likely to contribute to the pathogenesis of Alzheimer's disease (AD). Reelin binding to Apolipoprotein E receptor 2 (ApoER2) activates downstream signaling and induces the proteolytic cleavage of ApoER2, resulting in the generation of soluble fragments. To evaluate the efficiency of reelin signaling in AD, we have quantified the levels of reelin and soluble ectodomain fragments of ApoER2 (ectoApoER2) in the cerebrospinal fluid (CSF). CSF from sporadic AD patients (sAD; n = 14, age 54–83 years) had lower levels of ecto-ApoER2 (~31% reduction; p = .005) compared to those in the age-matched controls (n = 10, age 61–80), and a higher reelin/ecto-ApoER2 ratio. In contrast, autosomal dominant AD patients, carriers of PSEN1 mutations (ADAD; n = 7, age 31–49 years) had higher ecto-ApoER2 levels (~109% increment; p = .001) and a lower reelin/ecto-ApoER2 ratio than the non-mutation carriers from the same families (n = 7, age 25–47 years). Our data suggest that the levels of ecto-ApoER2 in CSF could be a suitable read-out of an impaired reelin signaling in AD, but also indicate differences between sAD and ADAD.

Proteolytic cleavage of amyloid precursor protein by ADAM10 mediates proliferation and migration in breast cancer

BackgroundAmyloid precursor protein (APP), best known for its association with Alzheimer disease, has recently been implicated in breast cancer progression. However, the precise mechanism involved remains unclear. Here, we investigated the role of APP proteolytic cleavage in breast cancer functions.MethodsThe presence of APP proteolytic cleavage products was examined in breast cancer cell lines. The functional roles of APP in breast cancer were studied in vitro and tumor xenograft model using siRNA. The effects of full length APP and the α-secretase cleaved ectodomain fragment, soluble APPα (sAPPα) were further investigated for their overexpression in breast cancers. The α-secretase involved was identified. The α-secretase expression together with APP was examined in clinical breast cancers.ResultsWe showed that APP underwent proteolytic cleavage in breast cancer cells to generate sAPPα. The sAPPα and full length protein mediated breast cancer migration and proliferation, but in different functional extent. This proteolytic cleavage was mediated by ADAM10. Downregulation of APP and ADAM10 brought about similar functional effects. Overexpression of sAPPα reversed the effects of ADAM10 downregulation. Interestingly, in patients with non-luminal breast cancers, APP and ADAM10 expression correlated with each other and their co-expression was associated with the worst outcome.ConclusionsThese results demonstrated the contributory role of APP cleavage on its oncogenic roles in breast cancer. ADAM10 was the key α-secretase. APP and ADAM10 co-expression was associated with worse survival in non-luminal breast cancers. Targeting of APP or its processing by ADAM10 might be a promising treatment option in these cancers.

Justicidin A Reduces β-Amyloid via Inhibiting Endocytosis of β-Amyloid Precursor Protein.

β-amyloid precursor protein (APP) can be cleaved by α-, and γ-secretase at plasma membrane producing soluble ectodomain fragment (sAPPα). Alternatively, following endocytosis, APP is cleaved by β-, and γ-secretase at early endosomes generating β-amyloid (Aβ), the main culprit in Alzheimer’s disease (AD). Thus, APP endocytosis is critical for Aβ production. Recently, we reported that Monsonia angustifolia, the indigenous vegetables consumed in Tanzania, improved cognitive function and decreased Aβ production. In this study, we examined the underlying mechanism of justicidin A, the active compound of M. angustifolia, on Aβ production. We found that justicidin A reduced endocytosis of APP, increasing sAPPα level, while decreasing Aβ level in HeLa cells overexpressing human APP with the Swedish mutation. The effect of justicidin A on Aβ production was blocked by endocytosis inhibitors, indicating that the decreased APP endocytosis by justicidin A is the underlying mechanism. Thus, justicidin A, the active compound of M. angustifolia, may be a novel agent for AD treatment.