Abbreviations: ADAM -a disintegrin and metalloproteinase. BSA -bovine serum albumin. CHO -Chinese hamster ovary cells. CTLD -C-type lectin-like domain. FBS -fetal bovine serum. MMPmatrix metalloproteinase. PBMC -peripheral blood mononuclear cells. PFA -paraformaldehyde. sCD93 -soluble CD93. ABSTRACTEfferocytosis -the phagocytic removal of apoptotic cells -is essential for the maintenance of homeostasis and prevention of the inflammatory and autoimmune diseases which can follow the lysis of uncleared apoptotic cells. CD93 is a transmembrane glycoprotein previously implicated in efferocytosis and angiogenesis, and upon mutation, results in the onset of efferocytosis-associated diseases such as atherosclerosis and rheumatoid arthritis. CD93 is produced as a cell surface protein which is shed as soluble CD93, but it is unknown how CD93 mediates efferocytosis or whether its efferocytic activity is mediated by the soluble or membrane-bound form. Herein, we demonstrate that the membrane bound form of CD93 has no phagocytic, efferocytic, or tethering activity, whereas soluble CD93 potently opsonizes apoptotic cells but not a broad range of Gram-Negative, Gram-Positive or fungal microorganisms. Using mass spectrometry, we identified the αxβ2 integrin as the receptor required for soluble CD93-mediated efferocytosis, and via deletion mutagenesis determined that soluble CD93 binds to apoptotic cells via its C-Type Lectin-Like domain, and to αxβ2 by its EGF-like repeats. This bridging of apoptotic cells to the αxβ2 integrin markedly enhanced efferocytosis by macrophages, and could be abrogated by knockdown of αxβ2 integrin. Combined, these data elucidate the mechanism by which CD93 regulates efferocytosis and identify a previously unreported opsonin-receptor system utilized by the immune system for the efferocytic clearance of apoptotic cells.Clearly, there is data supporting three distinct and mutually exclusive models of CD93 efferocytic function. In this study, we directly asses these three models of CD93 function, using in vitro models of binding, efferocytosis and opsonization. These experiments disfavor the efferocytic receptor and tethering receptor models of CD93 function, and instead demonstrate a strong opsonic effect of sCD93 against apoptotic cells but not against a panel of microorganisms. Using mass-spectrometry we then identified αxβ2 integrin as the efferocytic receptor which recognizes sCD93 opsonized targets, with apoptotic cells "bridged" by the CD93 CTLD domain to αxβ2 integrin on phagocytes via the CD93 EGFlike domain. This represents a previously undescribed opsonin-receptor system which aids in the efferocytosis of apoptotic cell by phagocytes such as macrophages. MATERIALS AND METHODS MaterialsDH5a Escherichia coli was a gift from Dr. John McCormick (University of Western Ontario), Rhodotorula minuta and Candida glabrate were gifts from Dr. Andre Lachance (University of Western Ontario). Escherichia coli K29 + and K29 -, and Burkholderia cenocepacia were gifts from Dr. Susan Koval (University of Wester...
Background: Post-translational modifications of tau modify its interaction with binding partners and cause tau mislocalisation and altered tau function in Alzheimer's disease (AD). The AD risk gene BIN1, is a binding partner for tau, however the mechanism by which BIN1 influences tau function is not fully understood. We hypothesised that BIN1 modulates AD risk by causing damaging tau mis-sorting to the synapse.Methods: Tau and BIN1 levels, distribution and interactions were assessed in post-mortem control and AD brain and in primary neurons. In primary neurons, tau was further examined using structured illumination microscopy and immunoblotting following BIN1 knockdown, BIN1-tau interactions were examined using proximity ligation assays and tau release from neurons was measured by sensitive sandwich ELISA.Results: Proline 216 in tau was identified as critical for tau interaction with the BIN1-SH3 domain, and tau phosphorylation at serine/threonine residues disrupted this interaction. Subcellular fractionation showed that BIN1 is lost from the cytoplasm of AD brain and this correlated with the mislocalisation of phosphorylated tau to synapses. Mimicking BIN1 loss in AD by knockdown of the protein in primary neurons altered the structure of dendritic spines, caused phosphorylated tau to mis-sort to synapses and reduced the physiological release of predominantly dephosphorylated tau.Conclusions: These data suggest that BIN1 loss in AD allows phosphorylated tau to be mis-sorted to synapses which likely alters the integrity of the post-synapse, alongside reducing the functionally important release of physiological forms of tau.
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