Biochemical analysis of molecular interactions in specific genomic regions requires their isolation while retaining molecular interactions in vivo. Here, we report isolation of telomeres by engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) using a transcription activator-like (TAL) protein recognizing telomere repeats. Telomeres recognized by the tagged TAL protein were immunoprecipitated with an antibody against the tag and subjected to identification of telomere-binding molecules. enChIP-mass spectrometry (enChIP-MS) targeting telomeres identified known and novel telomere-binding proteins. The data have been deposited to the ProteomeXchange with identifier PXD000461. In addition, we showed that RNA associated with telomeres could be isolated by enChIP. Identified telomere-binding molecules may play important roles in telomere biology. enChIP using TAL proteins would be a useful tool for biochemical analysis of specific genomic regions of interest.

Secretory phospholipase A2 group IIA (sPLA2-IIA) plays an important role in the pathogenesis of inflammatory diseases. Catalytic activity of this enzyme that generates arachidonic acid is a major target for development of anti-inflammatory agents. Independent of its catalytic activity, sPLA2-IIA induces pro-inflammatory signals in a receptor-mediated mechanism (e.g. through the M-type receptor). However, the M-type receptor is species-specific: sPLA2-IIA binds to the M-type receptor in rodents and rabbits, but not in human. Thus sPLA2-IIA receptors in human have not been established. Here we demonstrated that sPLA2-IIA bound to integrin ␣v␤3 at a high affinity (K D ‫؍‬ 2 ؋ 10 ؊7 M). We identified amino acid residues in sPLA2-IIA (Arg-74 and Arg-100) that are critical for integrin binding using docking simulation and mutagenesis. The integrin-binding site did not include the catalytic center or the M-type receptor-binding site. sPLA2-IIA also bound to ␣4␤1. We showed that sPLA2-IIA competed with VCAM-1 for binding to ␣4␤1, and bound to a site close to those for VCAM-1 and CS-1 in the ␣4 subunit. Wild type and the catalytically inactive H47Q mutant of sPLA2-IIA induced cell proliferation and ERK1/2 activation in monocytic cells, but the integrin binding-defective R74E/R100E mutant did not. This indicates that integrin binding is required, but catalytic activity is not required, for sPLA2-IIA-induced proliferative signaling. These results suggest that integrins ␣v␤3 and ␣4␤1 may serve as receptors for sPLA2-IIA and mediate pro-inflammatory action of sPLA2-IIA, and that integrinsPLA2-IIA interaction is a novel therapeutic target. The phospholipase A2 (PLA2)2 family is a group of intracellular and secreted enzymes that hydrolyzes the sn-2 ester bond in the glyceroacyl phospholipids present in lipoproteins and cell membranes to form nonesterified fatty acids and lysophospholipids. These products act as intracellular second messengers or are further metabolized into potent mediators of a broad range of cellular processes, including inflammation, apoptosis, and atherogenesis (1). The mammalian secretory PLA2 isoforms are comprised of the groups named IB, IIA, IIC, IID, IIE, IIF, V, X, and XII (2, 3). All secretory PLA2 isoforms have in common a Ca 2ϩ -dependent catalytic mechanism, a low molecular mass (13-16 kDa), several disulfide bridges, and a wellconserved overall three-dimensional structure (2, 4, 5). Secretory PLA2 type IIA (sPLA2-IIA) was first isolated and purified from rheumatoid synovial fluid. sPLA2-IIA is an acute phase reactant and is found in markedly increased plasma concentrations in diseases that involve systemic inflammation such as sepsis, rheumatoid arthritis, and cardiovascular disease (up to 1000-fold and Ͼ1 g/ml). Inflammatory cytokines such as IL-6, TNF-␣, and IL-1␤ induce synthesis and release of sPLA2-IIA in arterial smooth muscle cells and hepatocytes, which are the major sources of the plasma sPLA2-IIA in these systemic inflammatory conditions (6, 7). In addition to being a pro-inflammatory ...

Hepatic stellate cells (HSC), the key fibrogenic cells of the liver, transdifferentiate into myofibroblasts upon phagocytosis of apoptotic hepatocytes. Galectin-3, a β-galactoside-binding lectin, is a regulator of the phagocytic process. In this study, our aim was to study the mechanism by which extracellular galectin-3 modulates HSC phagocytosis and activation. The role of galectin-3 in engulfment was evaluated by phagocytosis and integrin binding assays in primary HSC. Galectin-3 expression was studied by real-time PCR and enzyme-linked immunosorbent assay, and in vivo studies were done in wild-type and galectin-3(-/-) mice. We found that HSC from galectin-3(-/-) mice displayed decreased phagocytic activity, expression of transforming growth factor-β1, and procollagen α1(I). Recombinant galectin-3 reversed this defect, suggesting that extracellular galectin-3 is required for HSC activation. Galectin-3 facilitated the α(v)β(3) heterodimer-dependent binding, indicating that galectin-3 modulates HSC phagocytosis via cross-linking this integrin and enhancing the tethering of apoptotic cells. Blocking integrin α(v)β(3) resulted in decreased phagocytosis. Galectin-3 expression and release were induced in active HSC engulfing apoptotic cells, and this was mediated by the nuclear factor-κB signaling. The upregulation of galectin-3 in active HSC was further confirmed in vivo in bile duct-ligated (BDL) rats. Galectin-3(-/-) mice displayed significantly decreased fibrosis, with reduced expression of α-smooth muscle actin and procollagen α1(I) following BDL. In summary, extracellular galectin-3 plays a key role in liver fibrosis by mediating HSC phagocytosis, activation, and subsequent autocrine and paracrine signaling by a feedforward mechanism.

Many eukaryotic proteins are anchored to the cell surface via the glycolipid glycosylphosphatidylinositol (GPI). Mammalian GPIs have a conserved core but exhibit diverse N-acetylgalactosamine (GalNAc) modifications, which are added via a yet unresolved process. Here we identify the Golgi-resident GPI-GalNAc transferase PGAP4 and show by mass spectrometry that PGAP4 knockout cells lose GPI-GalNAc structures. Furthermore, we demonstrate that PGAP4, in contrast to known Golgi glycosyltransferases, is not a single-pass membrane protein but contains three transmembrane domains, including a tandem transmembrane domain insertion into its glycosyltransferase-A fold as indicated by comparative modeling. Mutational analysis reveals a catalytic site, a DXD-like motif for UDP-GalNAc donor binding, and several residues potentially involved in acceptor binding. We suggest that a juxtamembrane region of PGAP4 accommodates various GPI-anchored proteins, presenting their acceptor residue toward the catalytic center. In summary, we present insights into the structure of PGAP4 and elucidate the initial step of GPI-GalNAc biosynthesis.

We recently found that integrin αvβ3 binds to fibroblast growth factor (FGF)-αvβ31 (FGF1), and that the integrin-binding defective FGF1 mutant (Arg-50 to glutamic acid, R50E) is defective in signalling and antagonistic to FGF1 signalling. R50E suppressed angiogenesis and tumour growth, suggesting that R50E has potential as a therapeutic. However, FGF1 is unstable, and we had to express R50E in cancer cells for xenograft study, since injected R50E may rapidly disappear from circulation. We studied if we can develop antagonist of more stable FGF2. FGF2 is widely involved in important biological processes such as stem cell proliferation and angiogenesis. Previous studies found that FGF2 bound to αvβ3 and antagonists to αvβ3 suppressed FGF2-induced angiogenesis. However, it is unclear how FGF2 interacts with integrins. Here, we describe that substituting Lys-119/Arg-120 and Lys-125 residues in the predicted integrin-binding interface of FGF2 to glutamic acid (the K119E/R120E and K125E mutations) effectively reduced integrin binding to FGF2. These FGF2 mutants were defective in signalling functions (ERK1/2 activation and DNA synthesis) in NIH3T3 cells. Notably they suppressed, FGF2 signalling induced by WT FGF2 in endothelial cells, suggesting that the FGF2 mutants are antagonists. The FGF2 mutants effectively suppressed tube formation in vitro, sprouting in aorta ring assays ex vivo and angiogenesis in vivo. The positions of amino acids critical for integrin binding are different between FGF1 and FGF2, suggesting that they do not interact with integrins in the same manner. The newly developed FGF2 mutants have potential as anti-angiogenic agents and useful tools for studying the role of integrins in FGF2 signalling.

CD40L plays a major role in immune response and is a major therapeutic target for inflammation. Integrin a5b1 and CD40 simultaneously bind to CD40L. It is unclear if a5b1 and CD40 work together in CD40/CD40L signaling or how a5b1 binds to CD40L. In this article, we describe that the integrin-binding site of human CD40L is predicted to be located in the trimeric interface by docking simulation. Mutations in the predicted integrin-binding site markedly reduced the binding of a5b1 to CD40L. Several CD40L mutants defective in integrin binding were defective in NF-kB activation and B cell activation and suppressed CD40L signaling induced by wild-type CD40L; however, they still bound to CD40. These findings suggest that integrin a5b1 binds to monomeric CD40L through the binding site in the trimeric interface of CD40L, and this plays a critical role in CD40/CD40L signaling. Integrin avb3, a widely distributed vascular integrin, bound to CD40L in a KGD-independent manner, suggesting that avb3 is a new CD40L receptor. Several missense mutations in CD40L that induce immunodeficiency with hyper-IgM syndrome type 1 (HIGM1) are clustered in the integrin-binding site of the trimeric interface. These HIGM1 CD40L mutants were defective in binding to a5b1 and avb3 (but not to CD40), suggesting that the defect in integrin binding may be a causal factor of HIGM1. These findings suggest that a5b1 and avb3 bind to the overlapping binding site in the trimeric interface of monomeric CD40L and generate integrin-CD40L-CD40 ternary complex. CD40L mutants defective in integrins have potential as antagonists of CD40/CD40L signaling.

Leukocyte arrest on the endothelial cell surface during leukocyte extravasation is induced by rapid integrin activation by chemokines. We recently reported that fractalkine induces integrin activation without its receptor CX3CR1 through binding to the allosteric site (site 2) of integrins. Peptides from site 2 bound to fractalkine and suppressed integrin activation by fractalkine. We hypothesized that this is not limited to membrane-bound fractalkine. We studied whether stromal cell-derived factor-1 (SDF1), another chemokine that plays a critical role in leukocyte arrest, activates integrins through binding to site 2. We describe here that (1) SDF1 activated soluble integrin αvβ3 in cell-free conditions, suggesting that SDF1 can activate αvβ3 without CXCR4; (2) site 2 peptide bound to SDF1, suggesting that SDF1 binds to site 2; (3) SDF1 activated integrins αvβ3, α4β1, and α5β1 on CHO cells (CXCR4-negative) and site 2 peptide suppressed the activation; (4) A CXCR4 antagonist AMD3100 did not affect the site 2-mediated integrin activation by SDF1; (5) Cell-surface integrins were fully activated in 1 min (much faster than activation of soluble αvβ3) and the activation lasted at least for 1 h. We propose that the binding of SDF1 to cell-surface proteoglycan facilitates the allosteric activation process; (6) Mutations in the predicted site 2-binding site in SDF1 suppressed integrin activation. These results suggest that SDF1 (e.g. presented on proteoglycans) can rapidly activate integrins in an allosteric manner by binding to site 2 in the absence of CXCR4. The allosteric integrin activation by SDF1 is a novel target for drug discovery.

We have characterized the microstructures of as-sintered and optimally post-sinter annealed Nd-rich Ga-doped Nd-Fe-B magnets by scanning electron microscopy (SEM) and aberration-corrected scanning transmission electron microscopy (STEM). While the Nd 2 Fe 14 B grains in the as-sintered sample with a coercivity of 0.99 T are in direct contact with each other, those in the optimally annealed sample with a coercivity of 1.8 T are completely enveloped by typically 10-nm-thick Nd-rich phase that contains little Fe. This strongly suggests that the Nd 2 Fe 14 B grains in the optimally annealed Nd-rich Ga-doped Nd-Fe-B magnets are exchange decoupled in contrast to those in the commercial sintered magnets.