Search

Our findings support a model in which CSMD1 opposes actions of the complement cascade in neural tissues (top left). We investigated two models in which CSMD1/Csmd1 was genetically ablated: human cortical neurons derived from embryonic stem cells, and a back-crossed C57bl6-Tac mouse line (top right). CSMD1 is normally expressed by neurons and present at synapses where it can protect them from complement (bottom left); in the absence of CSMD1 (bottom right), we find more deposition of complement (on cultured human cortical neurons and in the mouse visual system), reduced numbers of synapses (in the mouse visual system), and synaptosomes that are more readily engulfed by microglia (in vitro). Created with BioRender.com. [Display omitted] • CSMD1, genetically associated with schizophrenia, localizes to synapses and interacts with complement in the brain. • CSMD1 KO mice have increased complement C3 at synapses and disrupted development of the retinogeniculate circuit. • Microglia show greater engulfment of CSMD1 KO synaptosomes in vitro, which is blocked by inhibition of complement receptor. • Human stem cell-derived neurons lacking CSMD1 were more vulnerable to complement deposition. • CSMD1 may function as a regulator of complement-mediated synapse elimination during brain development. Complement proteins facilitate synaptic elimination during neurodevelopmental pruning, but neural complement regulation is not well understood. CUB and Sushi Multiple Domains 1 (CSMD1) can regulate complement activity in vitro, is expressed in the brain, and is associated with increased schizophrenia risk. Beyond this, little is known about CSMD1 including whether it regulates complement activity in the brain or otherwise plays a role in neurodevelopment. We used biochemical, immunohistochemical, and proteomic techniques to examine the regional, cellular, and subcellular distribution as well as protein interactions of CSMD1 in the brain. To evaluate whether CSMD1 is involved in complement-mediated synapse elimination, we examined Csmd1 -knockout mice and CSMD1- knockout human stem cell-derived neurons. We interrogated synapse and circuit development of the mouse visual thalamus, a process that involves complement pathway activity. We also quantified complement deposition on synapses in mouse visual thalamus and on cultured human neurons. Finally, we assessed uptake of synaptosomes by cultured microglia. We found that CSMD1 is present at synapses and interacts with complement proteins in the brain. Mice lacking Csmd1 displayed increased levels of complement component C3, an increased colocalization of C3 with presynaptic terminals, fewer retinogeniculate synapses, and aberrant segregation of eye-specific retinal inputs to the visual thalamus during the critical period of complement-dependent refinement of this circuit. Loss of CSMD1 in vivo enhanced synaptosome engulfment by microglia in vitro, and this effect was dependent on activity of the microglial complement receptor, CR3. Finally, human stem cell-derived neurons lacking CSMD1 were more vulnerable to complement deposition. These data suggest that CSMD1 can function as a regulator of complement-mediated synapse elimination in the brain during development. [ABSTRACT FROM AUTHOR]

Abstract: To assess the influence of unknown factors in endotoxemia, a conditioned medium, achieved by the stimulation of THP1 monocytes with lipopolysaccharide (LPS) [4h], was used for the stimulation of human umbilical vein endothelial cells (HUVECs) [16h]. SVEP1, KIAA0247, and SRPX2 were selected after microarray analysis. To study their possible functions, siRNAs of SVEP1, KIAA0247, or SRPX2 were used for the transfection of HUVECs and cells were stimulated with conditioned medium [16h]. Inhibition of SVEP1 expression resulted in an increase of soluble intercellular adhesion molecule (sICAM) 1 (10%) and soluble E-selectin (sE-selectin) (19%). Inhibition of SRPX2 led to an increase of sICAM (11%) and sE-selectin (14%). KIAA0247 negative HUVECs showed a decrease in monocyte chemoattractant protein (MCP) 1 of 16%. SVEP1 and SRPX2 seemed to act as regulators of ICAM1 and E-selectin shedding and influence the expression of membrane bound adhesion molecules. [Copyright &y& Elsevier]

Abstract: C1r is a modular serine protease which is the autoactivating component of the C1 complex of the classical pathway of the complement system. We have determined the first crystal structure of the entire active catalytic region of human C1r. This fragment contains the C-terminal serine protease (SP) domain and the preceding two complement control protein (CCP) modules. The activated CCP1–CCP2–SP fragment makes up a dimer in a head-to-tail fashion similarly to the previously characterized zymogen. The present structure shows an increased number of stabilizing interactions. Moreover, in the crystal lattice there is an enzyme–product relationship between the C1r molecules of neighboring dimers. This enzyme–product complex exhibits the crucial S1–P1 salt bridge between Asp631 and Arg446 residues, and intermolecular interaction between the CCP2 module and the SP domain. Based on these novel structural information we propose a new split-and-reassembly model for the autoactivation of the C1r. This model is consistent with experimental results that have not been explained adequately by previous models. It allows autoactivation of C1r without large-scale, directed movement of C1q arms. The model is concordant with the stability of the C1 complex during activation of the next complement components. [Copyright &y& Elsevier]

Abstract: This study examines the effects of duplication and insertions-deletions (indels) by comparing human complement receptor 1 (CR1) and human CR1-like (CR1L) with syntenic genes from four other vertebrates (chimpanzee, baboon, rat, and mouse). By phylogenetic analysis, the domains of these genes can be classified into 10 distinct subfamilies (a, b, c, d, e, f, g-like, h, j, and k), which have been largely conserved throughout vertebrate and invertebrate evolution. In spite of many complex and diverse duplications and indels, the subfamily order of domains (a, j, e, f, b, k, d, g-like) has been maintained. The number of domain sets has increased progressively, thereby expanding the functional repertoire. [Copyright &y& Elsevier]

β2-Glycoprotein I (β2GPI) is a highly glycosylated phospholipid-binding plasma protein comprised of four complement control protein (CCP) domains and a distinct fifth domain. The structural organisation of human and bovine β2GPI in aqueous solution was studied by small-angle X-ray scattering (SAXS). Low-resolution models that match the SAXS experimental data best were independently constructed by three different ab initio 3D-reconstruction algorithms. Similar elongated S-shaped models with distinct side-arms, which were correlated to the position of the carbohydrate chains, were restored from all three algorithms. Due to an additional glycosylation site located on the CCP2 domain of bovine β2GPI a small change in the characteristic SAXS parameters was observed, which coincided with results obtained from SDS–PAGE. In comparison to the human analogue the corresponding restored low-resolution models displayed a similar S-shape with less bending in the middle part.As the experimental SAXS curves fit poorly to the simulated scattering curves calculated from the crystallographic coordinates of human β2GPI, the crystal structure was modified. First, additional carbohydrate residues missing from the crystal structure were modelled. Second, on the basis of the low-resolution models, the J-shaped crystal structure was rotated between CCP3 and CCP2 assuming the greatest interdomain flexibility between these domains. An S-shaped model with a tilt angle of ∼60° between CCP3 and CCP2 yielded the best fit to the experimental SAXS data. Since there is evidence that β2GPI can adopt different conformations, which reveal distinct differences in autoantibody recognition, our data clearly point to a reorientation of the flexible domains, which may be an essential feature for binding of autoantibodies. [Copyright &y& Elsevier]

The high affinity of human plasma Β2-glycoprotein I (Β2GPI), also known as apolipoprotein-H (Aport), for negatively charged phospholipids determines its implication in a variety of physiological pathways, including blood coagulation and the immune response. Β2GPI is considered to be a cofactor for the binding of serum autoantibodies from antiphospholipid syndrome (APS) and correlated with thrombosis, lupus erythematosus and recurrent fetal loss. We solved the Β2GPI structure from a crystal form with 84% solvent and present a model containing all 326 amino acid residues and four glycans. The structure reveals four complement control protein modules and a distinctly folding fifth C-terminal domain arranged like beads on a string to form an elongated J-shaped molecule. Domain V folds into a central Β-spiral of four antiparallel Β-sheets with two small helices and an extended C-terminal loop region. It carries a distinct positive charge and the sequence motif CKNKEKKC close to the hydrophobic loop composed of residues LAFW (313–316), resulting in an excellent counterpart for interactions with negatively charged amphiphilic substances. The Β2GPI structure reveals potential autoantibody-binding sites and supports mutagenesis studies where Trp316 and CKNKEKKC have been found to be essential for the phospholipid-binding capacity of Β2GPI. [ABSTRACT FROM AUTHOR]