Structural composition and functional characterization of soluble CD59: heterogeneity of the oligosaccharide and glycophosphoinositol (GPI) anchor revealed by laser-desorption mass spectrometric analysis (original) (raw)
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Membrane defence against complement lysis: The structure and biological properties of CD59
Immunologic Research, 1993
The complement system is an important branch of the innate immune response, constituting a first line of defence against invading microorganisms which activate complement via both antibody-dependent and -independent mechanisms. Activation of complement leads to (a) a direct attack upon the activating cell surface by assembly of the pore-forming membrane attack complex (MAC), and (b) the generation of inflammatory mediators which target and recruit other branches of the immune system. However, uncontrolled coinplement activation can lead to widespread tissue damage in the host, since certain of the activation products, notably the fragment C3b and the C5b-7 complex, can bind nonspecifically to any nearby cell membranes. Therefore it is important that complement activation is tightly regulated. Our own cells express a number of membrane-bound control proteins which limit complement activation at the cell surface and prevent accidental complement-mediated damage. These include decay-accelerating factor, complement receptor 1 and membrane cofactor protein, all of which are active at the level of C3/C5 convertase formation. Until recently, cell surface control of MAC assembly had been attributed to a single 65-kD membrane protein called homologous restriction factor (alternatively named CS-binding protein and MAC-inhibiting protein). However a second MAC-inhibiting protein has since been discovered and it is now clear that this protein plays a major role in the control of membrane attack. This review charts the rapid progress made in elucidating the protein and gene structure, and the mechanism of action of this most recently discovered complement inhibitor, CD59. Dr. Alexandra Davies 9 1993 MRC Centre S. Karger AG, Basel Hills Road 0257-277X/93/ Cambridge CB2 2QH (UK) 0123-025852.75/0
Insights into the Human CD59 Complement Binding Interface Toward Engineering New Therapeutics
Journal of Biological Chemistry, 2005
CD59 is a 77-amino acid membrane glycoprotein that plays an important role in regulating the terminal pathway of complement by inhibiting formation of the cytolytic membrane attack complex (MAC or C5b-9). The MAC is formed by the self assembly of C5b, C6, C7, C8, and multiple C9 molecules, with CD59 functioning by binding C5b-8 and C5b-9 in the assembling complex. We performed a scanning alanine mutagenesis screen of residues 16-57, a region previously identified to contain the C8/C9 binding interface. We have also created an improved NMR model from previously published data for structural understanding of CD59. Based on the scanning mutagenesis data, refined models, and additional site-specific mutations, we identified a binding interface that is much broader than previously thought. In addition to identifying substitutions that decreased CD59 activity, a surprising number of substitutions significantly enhanced CD59 activity. Because CD59 has significant therapeutic potential for the treatment of various inflammatory conditions, we investigated further the ability to enhance CD59 activity by additional mutagenesis studies. Based on the enhanced activity of membrane-bound mutant CD59 molecules, clinically relevant soluble mutant CD59-based proteins were prepared and shown to have up to a 3-fold increase in complement inhibitory activity. Complement is an important component of host defense and is an effector mechanism for both innate and adaptive immune responses. Complement also plays important roles in enhancing the induction of both humoral and cellular immunity, regulating tolerance to self-antigens, and in the clearance of immune complexes and apoptotic cells. These effects of complement are mediated either directly or indirectly by bioactive cleaved protein fragments or by a terminal cytolytic protein assembly, termed the membrane attack complex (MAC 3 or C5b-9). Generation of the MAC during the complement cascade is initiated by cleavage of C5, which yields C5b and results in the sequential binding of C6, C7, C8, and multiple C9 molecules. Necessarily, complement effector mechanisms are under tight control to prevent damage to host cells, and MAC formation is under the control of CD59, a widely distributed 18-21-kDa (77 amino acids) glycoprotein attached to the plasma mem
Mapping the Active Site of CD59
Journal of Experimental Medicine, 1997
CD59 is a widely distributed membrane-bound inhibitor of the cytolytic membrane attack complex (MAC) of complement. This small (77 amino acid) glycoprotein is a member of the Ly6 superfamily of proteins and is important in protecting host cells from the lytic and proinflammatory activity of the MAC. CD59 functions by binding to C8 and/or C9 in the nascent MAC and interfering with C9 membrane insertion and polymerization. We present data obtained from a combination of molecular modeling and mutagenesis techniques, which together indicate that the active site of CD59 is located in the vicinity of a hydrophobic groove on the face of the molecule opposite to a “hydrophobic strip” suggested earlier. In addition, removal of the single N-linked glycosylation site at Asn18 of CD59 resulted in an enhancement of complement inhibitory activity.
Journal of Biological Chemistry, 2011
CD59 is a glycosylphosphatidylinositol-anchored protein that inhibits the assembly of the terminal complement membrane attack complex (MAC) pore, whereas Streptococcus intermedius intermedilysin (ILY), a pore forming cholesterol-dependent cytolysin (CDC), specifically binds to human CD59 (hCD59) to initiate the formation of its pore. The identification of the residues of ILY and hCD59 that form their binding interface revealed a remarkably deep correspondence between the hCD59 binding site for ILY and that for the MAC proteins C8α and C9. ILY disengages from hCD59 during the prepore to pore transition, suggesting that loss of this interaction is necessary to accommodate specific structural changes associated with this transition. Consistent with this scenario, mutants of hCD59 or ILY that increased the affinity of this interaction decreased the cytolytic activity by slowing the transition of the prepore to pore but not the assembly of the prepore oligomer. A signature motif was also identified in the hCD59 binding CDCs that revealed a new hCD59-binding member of the CDC family. Although the binding site on hCD59 for ILY, C8α, and C9 exhibits significant homology, no similarity exists in their binding sites for hCD59. Hence, ILY and the MAC proteins interact with common amino acids of hCD59 but lack detectable conservation in their binding sites for hCD59.
Production and functional characterization of a soluble recombinant form ofmouse CD59
Immunology, 2000
This report describes the engineering, expression, puri®cation and functional characterization of a soluble recombinant form of murine CD59 (srMoCD59). We report the expression in Chinese hamster ovary (CHO) cells of a modi®ed mouse CD59 cDNA that had been truncated at D-74, resulting in the loss of the glycosylphosphatidyl inositol (GPI) anchor, and containing six additional C-terminal histidines. The expressed srMoCD59 was puri®ed from tissue culture supernatant by means of its poly-histidine tag using immobilized metal af®nity chromatography. In comparison with CD59 on mouse erythrocytes, the srMoCD59 had a reduced molecular weight (18±20 000 as compared with 20±28 000 for GPI-anchored srMoCD59). The terminal complement inhibitory capacity of this soluble recombinant protein was assessed using two methods: a cobra venom factor (CVF)-triggered`reactive-lysis' system and a C5b-7 site assay. In both assays, srMoCD59 inhibited lysis by the sera from all three species tested in the rank order mouse>rat>>human. The amount of srMoCD59 required to produce 50% inhibition of lysis in the C5b-7 site assay, using puri®ed terminal components to develop lysis, was 10-fold less than that required in the same assay when EDTA serum was used as a source of C8 and C9, or in the CVF reactive lysis system. These data indicate that the presence of serum markedly interfered with the activity of srMoCD59 and have important implications for the use of recombinant soluble CD59 analogues as therapeutic agents in complement-mediated diseases.
Diabetes, 2004
Micro-and macrovascular diseases are major causes of morbidity and mortality in the diabetic population, but the cellular and molecular mechanisms that link hyperglycemia to these complications remain incompletely understood. We proposed that in human diabetes, inhibition by glycation of the complement regulatory protein CD59 increases deposition of the membrane attack complex (MAC) of complement, contributing to the higher vascular risk. We report here 1) the generation and characterization of an anti-glycated human CD59 (hCD59) specific antibody, 2) the detection with this antibody of glycated hCD59 colocalized with MAC in kidneys and nerves from diabetic but not from nondiabetic subjects, and 3) a significantly reduced activity of hCD59 in erythrocytes from diabetic subjects, a finding consistent with glycation inactivation of hCD59 in vivo. Because hCD59 acts as a specific inhibitor of MAC formation, these findings provide a molecular explanation for the increased MAC deposition reportedly found in the target organs of diabetic complications. We conclude that glycation inactivation of hCD59 that leads to increased MAC deposition may contribute to the extensive vascular pathology that complicates human diabetes.
Soluble CD59-ANTIGEN Levels Are Increased in Renal Disease Plasma
American Journal of Biochemistry and Biotechnology, 2013
Cluster of Differentiation antigen 59 (CD59) is a glycosyl phosphatidylinositol-linked protein that protects cells from damage by lytic terminal complexes of complement. Soluble forms of CD59 in serum have been little investigated in disease states. In the present study, by using a sandwich ELISA, reactivities with antibodies to CD59 were compared for sera and plasma from normal individuals and those with renal, leukemia, heart, liver and lung diseases. In all of 44 renal patients, reactivities were up to threefold higher than those of normals and patients with other conditions. The soluble CD59-related antigen displayed epitopes recognized by an anti-CD59 rabbit antiserum and CD59 monoclonal antibodies MEM-43 and YTH 53.1. Failure to bind the hydrophobic matrix phenyl Sepharose suggested that the CD59-related antigen lacked a membrane-anchoring structure. Although the cellular origin of soluble CD59-related antigen is unknown, some effect common to different renal diseases can promote increased plasma levels of this antigen. In two individuals, monitored before and after renal transplantation, plasma levels of CD59-related antigen decreased towards normal after receipt of a kidney.
European Journal of Immunology, 1993
Many membrane proteins are attached via a glycosyl phosphatidylinositol (GPI) anchor. Proteins anchored in this way make no direct contact with the interior of the cell, therefore a role in signaling or activation would seem unlikely. Nevertheless, cross-linking of GPI-anchored proteins on human and murine T lymphocytes has been shown to cause calcium transients and cell activation. Our studies address the non-lethal events caused by the membrane attack complex of complement, which include release of Ca2+ from intracellular stores, and have suggested that the GPI-anchored complement inhibitor CD59 may be involved in signaling these events. We here report that cross-linking of CD59 on human neutrophils using specific monoclonal antibody and second antibody caused a rapid increase in intracellular free Ca2+ concentration (Ca2+ transient) due to release of Ca2+ from stores and also caused neutrophil oxidase activation. All antibodies against CD59 tested were effective and cross-linking of any other GPI-anchored protein expressed on neutrophils also initiated an increase in intracellular free Ca2+ concentration, whereas cross-linking of transmembrane proteins caused little or no response. A tyrosine kinase-dependent activation pathway was indicated by the demonstration of tyrosine phosphorylation on cross-linking and by blocking of the Ca2+ transient with the tyrosine kinase inhibitor herbimycin.