An ultra-specific avian antibody to phosphorylated tau protein reveals a unique mechanism for phosphoepitope recognition.

Highly specific antibodies to phosphoepitopes are valuable tools to study phosphorylation in disease states, but their discovery is largely empirical, and the molecular mechanisms mediating phosphospecific binding are poorly understood. Here, we report the generation and characterization of extremely specific recombinant chicken antibodies to three phosphoepitopes on the Alzheimer disease-associated protein tau. Each antibody shows full specificity for a single phosphopeptide. The chimeric IgG pT231/pS235_1 exhibits a K(D) of 0.35 nm in 1:1 binding to its cognate phosphopeptide. This IgG is murine ortholog-cross-reactive, specifically recognizing the pathological form of tau in brain samples from Alzheimer patients and a mouse model of tauopathy. To better understand the underlying binding mechanisms allowing such remarkable specificity, we determined the structure of pT231/pS235_1 Fab in complex with its cognate phosphopeptide at 1.9 Å resolution. The Fab fragment exhibits novel complementarity determining region (CDR) structures with a "bowl-like" conformation in CDR-H2 that tightly and specifically interacts with the phospho-Thr-231 phosphate group, as well as a long, disulfide-constrained CDR-H3 that mediates peptide recognition. This binding mechanism differs distinctly from either peptide- or hapten-specific antibodies described to date. Surface plasmon resonance analyses showed that pT231/pS235_1 binds a truly compound epitope, as neither phosphorylated Ser-235 nor free peptide shows any measurable binding affinity.

Characterization of the immunoglobulin repertoire of the spiny dogfish (Squalus acanthias).

The cartilaginous fish (chimeras, sharks, skates and rays) are the oldest group relative to mammals in which an adaptive immune system founded upon immunoglobulins has been found. In this manuscript we characterize the immunoglobulins of the spiny dogfish (Squalus acanthias) at both the molecular and expressed protein levels. Despite the presence of hundreds of IgM clusters in this species the serum levels of this isotype are comparatively low. However, analysis of cDNA sequences and serum protein suggests microheterogeneity in the IgM heavy chains and supports the proposal that different clusters are preferentially used in the two forms (monomer or pentamer) of this isotype. We also found that the IgNAR isotype in this species exists in a previously unknown multimeric format in serum. Finally, we identified a new form of the IgW isotype (the shark IgD orthologue), in which the leader is spliced directly to the first constant domain, resulting in a molecule lacking an antigen-binding domain.

ROCK2 associates with lectin-like oxidized LDL receptor-1 and mediates oxidized LDL-induced IL-8 production.

Oxidatively modified low-density lipoprotein (OxLDL) is a contributing factor of endothelial dysfunction, an early cellular event during atherogenesis. In endothelial cells, OxLDL has been shown to stimulate proinflammatory responses, increase lipid accumulation, and induce the expression of adhesion and extracellular matrix degrading molecules. The primary receptor for OxLDL on endothelial cells has been identified as a member of the scavenger receptor family called lectin-like OxLDL receptor-1 (LOX-1). A number of studies on LOX-1 have implicated its role in multiple cardiovascular diseases including atherosclerosis. To better understand the molecular mechanisms underlying the role of LOX-1 in endothelial cells, we identified interacting proteins in an affinity-purified LOX-1 receptor complex from human aortic endothelial HAECT cells by mass spectrometry. Two molecules involved in Rho signaling pathway, ARHGEF1 and ROCK2, were identified, and their associations with LOX-1 were confirmed in reciprocal immunoprecipitation studies. Particularly, ROCK2 was found to dynamically associate with LOX-1 in the presence of OxLDL. In addition, OxLDL treatment stimulated ROCK2 catalytic activity, and ROCK2 inhibition attenuated NF-kappaB activation and IL-8 production resulting from OxLDL activation of LOX-1. In summary, a functional proteomics approach has enabled us to identify novel LOX-1 interactors that potentially contribute to the cellular and signaling functions of LOX-1.

Oligomerization is required for the activity of recombinant soluble LOX-1.

LOX-1 is a scavenger receptor that functions as the primary receptor for oxidized low-density lipoprotein (OxLDL) in endothelial cells. The binding of OxLDL to LOX-1 is believed to lead to endothelial activation, dysfunction, and injury, which constitute early atherogenic events. Because of its potential pathological role in atherosclerosis, LOX-1 has been proposed as a therapeutic target for the treatment of this disease. In order to antagonize the ligand-binding function of cell surface LOX-1, we generated a series of recombinant human LOX-1-crystallizable fragment (Fc) fusion proteins and subsequently characterized their biochemical properties and ligand-binding activities in vitro. Consistent with the notion that oligomerization of cell surface LOX-1 is required for high-avidity binding of ligands, we found that LOX-1-Fc fusion protein containing four ligand-binding domains per Fc dimer, but not the one containing two ligand-binding domains, exhibited ligand-binding activity. Optimal ligand-binding activity could be achieved via crosslinking of LOX-1-Fc fusion proteins with a polyclonal antibody against Fc. The crosslinked LOX-1-Fc protein also effectively inhibited the binding and internalization of OxLDL by cell surface LOX-1. These findings demonstrate that functional oligomerization is required for recombinant LOX-1-Fc to function as an effective antagonist.

CRP is a novel ligand for the oxidized LDL receptor LOX-1.

C-reactive protein (CRP) is a risk factor for cardiovascular events and functions to amplify vascular inflammation through promoting endothelial dysfunction. Lectin-like oxidized low-density lipoprotein (oxLDL) receptor-1 (LOX-1) is the primary endothelial receptor for oxLDL, and both its expression and function are associated with vascular inflammation. As a scavenger receptor, LOX-1 is capable of binding to a variety of structurally unrelated ligands. Evidence is provided that demonstrates that CRP can act as a novel ligand for LOX-1. The direct interaction between these two proteins was demonstrated with purified protein in both ELISA and AlphaScreen assays. This interaction could be disrupted with known LOX-1 ligands, such as oxLDL and carrageenan. Moreover, the CRP interaction with cell surface-expressed LOX-1 was confirmed in cell-based immunofluorescent-binding studies. Mutagenesis studies demonstrated that the arginine residues forming the basic spine structure on the LOX-1 ligand-binding interface were dispensable for CRP binding, suggesting a novel ligand-binding mechanism for LOX-1, distinct from that used for oxLDL binding. The treatment of human endothelial cells with CRP led to the activation of proinflammatory genes including IL-8, ICAM-1, and VCAM-1. The inductions of these genes by CRP were LOX-1 dependent, as demonstrated by their attenuation in cells transfected with LOX-1 small-interfering RNA. Our study identifies and characterizes the direct interaction between LOX-1 and CRP and suggests that this interaction may mediate CRP-induced endothelial dysfunction.

LOX-1-dependent transcriptional regulation in response to oxidized LDL treatment of human aortic endothelial cells.

Oxidized low-density lipoprotein (OxLDL) has been implicated as a proatherogenic factor with a pathological role in the induction of endothelial dysfunction. Endothelial cells bind and uptake OxLDL primarily through the scavenger receptor lectin-like oxidized-low-density lipoprotein receptor-1 (LOX-1), which is believed to mediate critical effects of OxLDL in endothelial cells. To examine the biological events following LOX-1 activation by OxLDL, we used cDNA microarray analysis to globally analyze gene expression changes induced by OxLDL treatment of human aortic endothelial cell line (HAECT) cells overexpressing LOX-1. Consistent with reported functions of OxLDL, in control HAECT cells, OxLDL elicited gene changes in the oxidative stress pathway and other signaling pathways related to OxLDL. With OxLDL treatment, LOX-1-dependent gene expression changes associated with inflammation, cell adhesion, and signal transduction were observed. The transcripts of a number of cytokines and chemokines were induced, which included interleukin-8, CXCL2, CXCL3, and colony-stimulating factor-3. The secretion of these cytokines was confirmed by enzyme-linked immunosorbent assay analysis. In addition, our data revealed a novel link between LOX-1 and a number of genes, including Delta/notch-like epidermal growth factor repeat containing, stanniocalcin-1, cAMP response element modulator, and dual specificity phosphatase 1. Promoter analysis on the genes that changed as a result of LOX-1 activation by OxLDL allowed us to identify early growth response 1 and cAMP response element-binding protein as potential novel transcription factors that function downstream of LOX-1. Our study has enabled us to elucidate the gene expression changes following OxLDL activation of LOX-1 in endothelial cells and discover novel downstream targets for LOX-1.

Expression of the cysteine protease legumain in vascular lesions and functional implications in atherogenesis.

OBJECTIVE: The present study was conducted to characterize the expression of the cysteine protease legumain in murine and human atherosclerotic tissues, and to explore the molecular mechanisms by which legumain may contribute to the pathophysiology of atherosclerosis. METHODS AND RESULTS: Using microarray analysis, legumain mRNA expression was found to increase with development of atherosclerosis in the aorta of aging Apolipoprotein E deficient mice while expression remained at low level and unchanged in arteries of age-matched C57BL/6 control mice. In situ hybridization and immunohistochemical analysis determined that legumain was predominantly expressed by macrophages in the atherosclerotic aorta, in lesions at the aortic sinus and in injured carotid arteries of Apolipoprotein E deficient mice as well as in inflamed areas in advanced human coronary atherosclerotic plaques. In vitro, M-CSF differentiated human primary macrophages were shown to express legumain and the protein could also be detected in the culture media. When tested in migration assays, legumain induced chemotaxis of primary human monocytes and human umbilical vein endothelial cells. CONCLUSIONS: Legumain is expressed in both murine and human atherosclerotic lesions. The macrophage-specific expression of legumain in vivo and ability of legumain to induce chemotaxis of monocytes and endothelial cells in vitro suggest that legumain may play a functional role in atherogenesis.

SLIC-1/sorting nexin 20: a novel sorting nexin that directs subcellular distribution of PSGL-1.

P-Selectin glycoprotein ligand-1 (PSGL-1) is a mucin-like glycoprotein expressed on the surface of leukocytes that serves as the major ligand for the selectin family of adhesion molecules and functions in leukocyte tethering and rolling on activated endothelium and platelets. Previous studies have implicated the highly conserved cytoplasmic domain of PSGL-1 in regulating outside-in signaling of integrin activation. However, molecules that physically and functionally interact with this domain are not completely defined. Using a yeast two-hybrid screen with the cytoplasmic domain of PSGL-1 as bait, a novel protein designated selectin ligand interactor cytoplasmic-1 (SLIC-1) was isolated. Computer-based homology search revealed that SLIC-1 was the human orthologue for the previously identified mouse sorting nexin 20. Direct interaction between SLIC-1 and PSGL-1 was specific as indicated by co-immunoprecipitation and motif mapping. Colocalization experiments demonstrated that SLIC-1 contains a Phox homology domain that binds phosphoinositides and targets the PSGL-1/SLIC-1 complex to endosomes. Deficiency in the murine homologue of SLIC-1 did not modulate PSGL-1-dependent signaling nor alter neutrophil adhesion through PSGL-1. We conclude that SLIC-1 serves as a sorting molecule that cycles PSGL-1 into endosomes with no impact on leukocyte recruitment.

Transcriptional profiling of C2C12 myotubes in response to SHIP2 depletion and insulin stimulation.

Phosphoinositide lipids generated at the cell membrane are a key component of a variety of signaling pathways. Among several inositol phosphatases that regulate the availability of signaling phosphoinositide lipids, the type II SH2-domain-containing inositol 5-phosphatase (SHIP2; approved gene symbol Inppl1) is believed to have multiple functions, including the regulation of insulin signaling and cytoskeletal functions. To understand the function of SHIP2 in C2C12 muscle cells, we depleted SHIP2 through the use of RNA interference and analyzed the global effect of SHIP2 depletion on gene expression using Affymetrix microarrays containing approximately 45,000 mouse probe sets. Expression of SHIP2-targeting small-hairpin RNA in differentiated C2C12 muscle cells led to >80% decrease in SHIP2 mRNA and 60-80% decrease in SHIP2 protein, which resulted in significant gene expression changes linked to cytoskeletal functions, including altered expression of adducin-alpha, pallidin, stathmin-like-2, and synaptojanin-2 binding protein. Insulin treatment of C2C12 muscle cells caused transcriptional changes associated with known signaling pathways. However, SHIP2 depletion had no discernible effect on insulin-regulated gene expression. Taken together, our results suggest that SHIP2 is involved in the regulation of cytoskeletal functions, but a large reduction of SHIP2 in C2C12 muscle cells is not sufficient to affect insulin-mediated gene expression.

Inhibition of gluconeogenesis through transcriptional activation of EGR1 and DUSP4 by AMP-activated kinase.

Increased hepatic gluconeogenesis is an important contributor to the fasting hyperglycemia found in Type 2 diabetic patients. Low energy states activate the intracellular energy sensor AMP-activated kinase (AMPK). AMPK activation by the AMP mimetic AICAR (5-aminoimidazole-4-carboxamide riboside) has been shown to inhibit hepatic gluconeogenesis. We used transcriptional profiling to search for AICAR-regulated genes in hepatocyte cell lines. We report that a dual specificity phosphatase, Dusp4, is induced by AMPK in AML12, H4IIE, and Fao cells at both mRNA and protein levels. AMPK also induces the immediate early transcription factor Egr1 (early growth response 1), a known transcriptional activator of Dusp4, and it directly binds the Dusp4 promoter at its known binding site. Both reporter gene assays and real time PCR demonstrate that exogenous DUSP4 inhibits the promoter activity and expression of both glucose-6-phosphatase (Glc-6-P) and phosphoenolpyruvate carboxykinase (Pepck) to an extent similar to both AICAR and constitutively active AMPK. Conversely, depletion of EGR1 or DUSP4 using siRNA not only partially abrogates the inhibition of Pepck expression by AICAR, but also importantly affects glucose production by Fao cells. In Fao cells, small interfering RNA targeted EGR1 also depletes DUSP4 expression following treatment with AICAR, further supporting a direct link between EGR1 and DUSP4 activation. Expression of a constitutively active form of p38, a known effector of cAMP-mediated gluconeogenesis, rescues the DUSP4-mediated repression of PEPCK. These results suggest that the inhibition of hepatic gluconeogenesis by AMPK may, in part, be mediated by an immediate early gene response involving EGR1 and its target, DUSP4.

Expression, regulation, and triglyceride hydrolase activity of Adiponutrin family members.

Adiponutrin and a related protein, adipocyte triglyceride lipase (ATGL; also known as Desnutrin), were recently described as adipocyte-specific proteins with lipid hydrolase activity. Using bioinformatics, we identified three additional Adiponutrin family members (GS2, GS2-Like, and PNPLA1). Here, we report on the expression, regulation, and activity of GS2 and GS2-Like compared with Adiponutrin and Desnutrin/ATGL. GS2-Like is expressed and regulated in a manner similar to Adiponutrin; however, the absolute levels of mRNA are significantly lower than those of Adiponutrin or Desnutrin/ATGL. GS2 transcripts were identified only in humans and are highly expressed in adipose as well as other tissues. All four proteins show lipase activity in vitro, which is dependent on the presence of the active site serine for Adiponutrin, Desnutrin/ATGL, and GS2. Overexpression of Desnutrin/ATGL, GS2, and GS2-Like, but not Adiponutrin, decreases intracellular triglyceride levels. This is consistent with a function for Desnutrin/ATGL, GS2, and GS2-Like in lipolysis, but not for Adiponutrin. Consistent with previously reported data, Desnutrin/ATGL is upregulated by fasting in adipose tissue, whereas Adiponutrin is downregulated. Additionally, Adiponutrin and GS2-Like, but not Desnutrin/ATGL, are strongly induced in the liver of ob/ob mice. Our data support distinct functions for Adiponutrin and Desnutrin/ATGL and raise the possibility that GS2 may contribute significantly to lipolysis in human adipose tissue.