Mutational Analysis of the Active Site and Antibody Epitopes of the Complement-inhibitory Glycoprotein, CD59

The Ly-6 superfamily of cell surface molecules includes CD59, a potent regulator of the complement system that protects host cells from the cytolytic action of the membrane attack complex (MAC). Although its mechanism of action is not well understood, CD59 is thought to prevent assembly of the MAC by binding to the C8 and/or C9 proteins of the nascent complex. Here a systematic, structure-based mutational approach has been used to determine the region(s) of CD59 required for its protective activity. Analysis of 16 CD59 mutants with single, highly nonconservative substitutions suggests that CD59 has a single active site that includes Trp-40, Arg-53, and Glu-56 of the glycosylated, membrane-distal face of the disk-like extracellular domain and, possibly, Asp-24 positioned at the edge of the domain. The putative active site includes residues conserved across species, consistent with the lack of strict homologous restriction previously observed in studies of CD59 function. Competition and mutational analyses of the epitopes of eight CD59-blocking and non-blocking monoclonal antibodies confirmed the location of the active site. Additional experiments showed that the expression and function of CD59 are both glycosylation independent

Mapping the regions of the complement inhibitor CD59 responsible for its species selective activity

ABSTRACT: CD59 is a widely distributed membrane-bound glycoprotein that inhibits the formation of the cytolytic membrane attack complex (MAC) of complement on host cells. CD59 from different species varies in its capacity to inhibit heterologous complement, and this species selective function of CD59 contributes to the phenomenon of homologous restriction. Here, we demonstrate that human CD59 is not an effective inhibitor of rat complement, although rat CD59 inhibits rat and human complement equally well. By constructing human-rat CD59 chimeric proteins, we have mapped the residues important in conferring human CD59 species selectivity to two regions; 40-47 and 47-66 in the primary structure. Analysis of a model of the molecular surface of human CD59 revealed that residues 40-66 mapped to a region in the three-dimensional structure that surrounds residues previously identified as important for CD59 function. Activation of complement results in the formation of C3/ C5 convertase enzymes on the activating surface. The convertases serve to amplify the cascade and may lead to the assembly of the terminal complement proteins which form the cytolytic membrane attack complex (MAC). 1 Host cell membranes are protected from the proinflammatory and potentially cytolytic effects of the MAC by CD59, a widely distributed glycosylphosphatidylinositol (GPI)-linked membrane glycoprotein. CD59 functions by binding the terminal complement proteins C8 and C9 in the assembling MAC and interfering with its membrane insertion (1-4). An important feature of CD59 and other complement inhibitors is their species selectivity. This feature is responsible for the phenomenon of homologous restriction, whereby cells are largely resistant to lysis by homologous complement. The selective activity of CD59 is due to the species selective recognition of C8 and/or C9 in the assembling MAC. The species selective activity of CD59 appears to be well illustrated by the prolonged survival of transgenic pig organs expressing human CD59 following their transplantation into baboons (5, 6). Under normal circumstances, complement is involved in the rapid destruction of xenotransplanted tissue, a phenomenon known as hyperacute rejection. Nevertheless, species selective recognition is not absolute, and CD59's from different species vary in their effectiveness at inhibiting heterologous complement Mutational analysis of human CD59 has recently identified residues important for human CD59 function EXPERIMENTAL PROCEDURES Cells and DNA. Human and rat cDNA subcloned into the mammalian expression vector pCDNA3 (Invitrogen, Carlsbad, CA) was used for all DNA manipulations. Human CD59 cDNA was a gift from H. Okada (Nagoya City University, Nagoya, Japan), and rat CD59 cDNA was isolated as described previously (15). Chinese hamster ovary cells (CHO) were used for protein expression and were maintained in Dulbecco's modified essential medium (DMEM) supplemented with 10% FCS. pCDNA3 contains a G418 resistance marker, and stably transfected CHO cell clones and populations were selected following the cultivation of cells in the presence of G418 (Gibco, Gaithersburg, MD). Antibodies and Sera. Rabbit antiserum to CHO cell membranes that was used to sensitize CHO cells to complement was prepared by standard techniques (16). CHO cell membranes were prepared as described (17). Anti-rat CD59 monoclonal antibody 6D1 was described previously (18). mAb 2A10 (19) is directed against (NANP) n , a repeat domain of Plasmodium falciparum circumsporozoite protein, and was used to quantitate expression of epitope-tagged proteins. FITC-conjugated Abs used for flow cytometry were purchased from Sigma (St. Louis, MO). Normal human serum † This work was supported by NIH Grant AI 34451, a Grant in Aid from the American Heart Association (S.T.), and the Wellcome trust (B.P.M. and N.K.R

Soluble forms of Toll-like receptor (TLR)2 capable of modulating TLR2 signaling are present in human plasma and breast milk.

Dysregulation of the initial, innate immune response to bacterial infection may lead to septic shock and death. Toll-like receptors (TLRs) play a crucial role in this innate immune response, and yet the regulatory mechanisms controlling microbial-induced TLR triggering are still to be fully understood. We have therefore sought specific regulatory mechanisms that may modulate TLR signaling. In this study, we tested for the possible existence of a functionally active soluble form of TLR2. We demonstrated the existence of natural soluble forms of TLR2 (sTLR2), which we show to be capable of modulating cell activation. We found that blood monocytes released sTLR2 constitutively and that the kinetics of sTLR2 release increased upon cell activation. Analysis of cells expressing the human TLR2 cDNA or its c-myc-tagged version indicated that sTLR2 resulted from the posttranslational modification of the TLR2 protein in an intracellular compartment. Moreover, an intracellular pool of sTLR2 is maintained. sTLR2 was found naturally expressed in breast milk and plasma. Milk sTLR2 levels mirrored those of the TLR coreceptor soluble CD14. Depletion of sTLR2 from serum resulted in an increased cellular response to bacterial lipopeptide. Notably, serum sTLR2 was lower in tuberculosis patients. Coimmunoprecipitation experiments and computational molecular docking studies showed an interaction between sTLR2 and soluble CD14 in plasma and milk. These findings suggest the existence of a novel and specific innate immune mechanism regulating microbial-induced TLR triggering, and may lead to new therapeutics for the prevention and/or treatment of severe infectious diseases