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Department of Biochemistry, The Skirball Institute, New York 10016
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Abstract |
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Activation of complement results in the formation of the proteolytic C3/C5 convertases, which can initiate formation of the cytolytic membrane attack complex (MAC)1. The MAC is formed by the sequential assembly of the terminal complement proteins C5b, C6, C7, C8, and multiple C9s. Under normal circumstances, complement is activated by, and directed against invading microorganisms. However, under certain circumstances, most notably in some autoimmune and inflammatory conditions, complement can also become deposited on host cells. In addition to causing cell lysis, sublethal concentrations of MAC assembled on host cells mediate various inflammatory processes that can elicit severe pathophysiological effects. Host cell membranes are protected from homologous MAC by CD59, a small glycoprotein attached to the plasma membrane by a glycosylphosphatidylinositol (GPI) anchor. The mature protein consists of 77 amino acids arranged in a single cysteine-rich domain composed of two antiparallel β-sheets, 5 protruding surface loops and a short helix (1, 2). CD59 functions by binding to C8
Several studies have demonstrated the potential usefulness of recombinant soluble complement regulatory proteins for therapy of autoimmune and inflammatory disease (7–11). In addition, MAC-mediated tissue destruction is primarily responsible for hyperacute rejection of porcine organs transplanted into primates, a result of complement activation by natural antibodies (12). CD59 and other complement inhibitors display varying degrees of species selectivity. The expression of human CD59 on transgenic animal organs protects them from human complement-mediated damage and prolongs their survival after transplantation into baboons (13–16). There is thus much interest in the potential use of CD59 and other complement inhibitors, either soluble or expressed on the surface of transgenic pig organs, in human transplantation.
CD59 belongs to the Ly6 superfamily of proteins which includes functional CD59 analogues from other species, snake venom neurotoxins, urokinase-type plasminogen activator receptor and murine Ly6 differentiation antigens (17). Mouse Ly6E antigen (18) is a structural but not functional analogue of CD59 (see Fig. 1). In the approach reported here, functionally important regions of CD59 were determined by replacing regions of Ly6E with corresponding regions from CD59 and assaying expressed chimeric proteins for activity. The active site was then further defined by a series of site-specific mutations, selected as a result of comparing evolutionary conserved residues and modeling of the molecular surface of CD59. 
and/or C9 in the assembling MAC and interfering with C9 membrane insertion and polymerization (3–6).
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Materials and Methods |
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Antibodies and Serum.
Rabbit antisera to CHO cell membranes, CD59 and to CD59-specific peptide were prepared by standard techniques (19). CHO cell membranes were prepared as described (20). CD59 antigen was prepared from human erythrocytes as described (21). The peptide antigen corresponded to the COOH-terminal sequence of mature CD59 (CKKDLCNFNEQLE) and was conjugated to KLH for immunization. Peptide synthesis and KLH conjugation was performed by Genemed (South San Francisco, CA). Anti-CD59 peptide Ab was affinity purified by means of peptide immobilized onto CNBr-activated sepharose (Pharmacia, Piscataway, NJ). Anti-CD59 mAbs YTH53.1, MEM43/5, and HC1 were kindly provided by Dr. B.P. Morgan (University of Wales, Cardiff, UK). Each recognize nonoverlapping conformational epitopes on CD59 (22). mAb 2A10 (23) is directed against (NANP)n, a repeat domain of Plasmodium falciparum circumsporozoite protein. FITC-conjugated Abs used for flow cytometry were purchased from Sigma Chem. Co. (St. Louis, MO). Normal human serum (NHS) was obtained from the blood of healthy volunteers in the laboratory and stored at –70°C.
Mutant Construction.
Based on the alignment of CD59 and Ly6E amino acid sequences (Fig. 1), cDNA encoding the chimeric constructs depicted in Fig. 2 were prepared. Segments of either CD59 or Ly6E cDNA were generated and joined using PCR. The general procedure used for the generation of chimeric constructs was as described (24). The 5' and 3' end primers, which matched an untranslated sequence of either CD59 or Ly6E, included a HindIII and ApaI site, respectively. Using either CD59 or Ly6E cloned into pCDNA3 as template, these primers were paired in a PCR with CD59-Ly6E chimeric primers that spanned the desired crossover point. The PCR products were purified from agarose gel after electrophoresis and used in a second amplification with the primers corresponding to 5' or 3' untranslated regions. The resulting full-length CD59-Ly6E chimeric cDNAs were cloned into the HindIII/ApaI sites of pCDNA3 for sequencing and expression. Five site-specific point mutations in CD59 (N18Q, F23D, Y36F, W40Y, and L54N) were prepared by PCR using similar techniques. In the first PCR amplification, 5' and 3' primers to CD59 untranslated region were paired with primers spanning the target site, and which contained a substituted codon at the target site. An epitope tag was engineered into CD59-Ly6E chimeric proteins between the first and second amino acids of mature CD59. The NH2-terminal Leu was duplicated at the COOH-terminus of the tag. A 36mer oligonucleotide encoding NANPNANPNA and with appropriate overhangs was inserted at the PstI restriction site of each point-specific mutant, and of CD59 and CD-Ly constructs (chimeras with CD59 at the NH2 terminus).
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Flow Cytometry.
Analysis of cell surface protein expression was performed by flow cytometry using a Beckton Dickinson FACScan®. Detached CHO cells were incubated with appropriate Ab for 30 min at 4°C, washed by centrifugation, incubated with appropriate FITC-conjugated second Ab (30 min/4°C), and washed again before fixation with 2% paraformaldehyde in PBS. All incubations and washing were performed in DMEM/10% FCS. The following Abs and concentrations were used: rabbit antiCOOH-terminal CD59 peptide Ab (30 µg/ml); rabbit anti-CD59 antiserum (1:100); anti-CD59 mAb YTH53.1 IgG (10 µg/ml); antitag mAb 2A10 and anti-CD59 mAb MEM 43/5 ascites (1:250); anti-CD59 mAb HC1 culture supernatant (1:25). FITC-conjugated secondary Abs were used between 1:100 and 1:400 final concentration. Cells for sorting were fluorescently labeled following the procedure above but without fixation. Sorting was done in a Coulter Epics Elite with EPS sort module.
Cell Lysis Assays.
Subconfluent CHO cells were detached with versene (GIBCO BRL), washed once and resuspended to 1 x 106/ml in DMEM/10% FCS. An equal volume of calcein-AM (5 µg/ml) (Molecular Probes, Eugene, OR) in DMEM was added and the cells incubated for 15 min at 37°C. The cells were then washed twice, resuspended to their original concentration in DMEM/10% FCS and incubated with rabbit anti-CHO cell membrane antiserum (20% final concentration) for 30 min on ice. The sensitized cells were then centrifuged, resuspended to their original concentration and an equal volume of NHS diluted in DMEM added. After 1 h at 37°C, the cells were centrifuged and cell lysis assessed by measuring released fluorescence in the supernatant. Measurements were performed in microtiter plates using a Labsystems fluoroscan II set to read at excitation 485 nm and emission 538 nm. Cells were lysed with 0.01% saponin for 100% lysis controls. Cell lysis assays were typically performed in 1.5-ml microfuge tubes in a final volume of 200 µl (1 x 105 cells). Cell lysis assays were also performed using propidium iodide, which fluorescently stains dead cells. CHO cells were sensitized to complement as described and 100 µl cells (1 x 106/ml) mixed with NHS dilutions. After 60 min at 37°C, propidium iodide was added (10 µg/ml final) and cell lysis determined by flow cytometry.
Molecular Modeling.
A CD59 analytical molecular surface, which is defined as the smooth envelope touching the van der Waals surface of atoms as the solvent probe rolls over the protein molecule, was built with the contour-buildup algorithm (25) as implemented in the ICM program (26). A probe sphere of 1.4 A was used. Molecular models of the point mutants were built and analyzed using the following procedures: (a) the structure of CD59 (pdb-code 1cds) solved by NMR (1) was carefully energy minimized; (b) appropriate residue modifications introduced; (c) the mutated side-chains were energy minimized using the biased probability Monte Carlo sample of the side-chain torsions (27), (d) interactions were analyzed by visual inspection using the ICM program.
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Results |
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CHO cell populations expressing similar levels of recombinant CD59 or chimeric proteins were assayed for their susceptibility to human complement. The functional data obtained with the CD-Ly chimeras (CD59 at NH2 terminus) revealed that the region of CD59 which lies COOHterminal to residue 57 is not required for function; both CD-Ly 57 and CD-Ly 64 possessed the same specific activity as wild type CD59 (Fig. 4). The other CD-Ly chimeras which contained shorter CD59-specific sequences had no complement inhibitory activity. Of the Ly-CD chimeras (Ly6E at NH2 terminus), only Ly-CD 16 possessed any functional activity (Fig. 4). The specific activity of Ly-CD 16 was 
85% that of wild-type CD59. Taken together, these data indicate that the functionally important region of CD59 is located between amino acids 16 and 57 in the primary structure.
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Effect of Point Mutations within the Predicted Functional Site of CD59.
The three conserved residues (shown in green, Fig. 7 b) and the conserved hydrophobic residue at position F23 within the putatively identified binding groove of CD59 were individually targeted for site-specific mutagenesis. The four mutant CD59s prepared were F23D (Phe at position 23 changed to Asp), Y36F, W40Y and L54N. The amino acids used for the substitutions were chosen to have similar shape but different physico-chemical properties. The W40Y mutant was not stably expressed on the surface of transfected CHO cells. Of the three expressed mutant proteins, L54N had almost no function, F23D retained some but significantly reduced function, and Y36F had normal function (Fig. 8). For unknown reasons, only relatively moderate levels of the L54N mutant protein could be expressed on CHO cells, but activity was compared with CHO cells expressing similar levels of wild-type CD59 (see Table 1). In an attempt to assess whether the mutant proteins were correctly folded, we studied their reactivity with three different mAbs that each bind to nonoverlapping conformational epitopes on CD59. All mutant proteins were recognized by these antibodies (Table 1).
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Discussion |
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Taken together, these data indicate that the active site of CD59 is on the front (facing away from membrane) face of the molecule and involves residues located along a hydrophobic groove defined by evolutionary conserved residues. In further support of this conclusion, it was recently shown that a correctly folded W40E mutant (residue located within hydrophobic groove, Fig. 7 b) expressed on the surface of CHO cells was inactive (Rushmere, N.K., D.L. Bodian, S.J. Davis, S. Tomlinson, and B.P. Morgan. 1996. XVIth International Complement Workshop. Boston, MA). In a previous report, Kieffer et al. (2) suggested that a relatively hydrophobic strip composed of exposed Tyr and Phe residues may constitute the functional site of CD59. Of note however, these residues are located on the opposite face and perpendicular to the hydrophobic groove identified in this report.
All but one of the Ly-CD and CD-Ly chimeric proteins expressed on CHO cells possessed either full or zero activity, making interpretation of data simple. The Ly-CD 16 chimera displayed slightly less (15%) activity than wild type CD59. The simplest explanation for this, taking into account the modeling and point mutation data, is that the NH2-terminal sequence of Ly6E causes a change in the tertiary conformation of the active site of CD59. We cannot exclude however, that the NH2-terminal sequence of CD59 may contribute to its activity.
The rationale in choosing the substituted residues in the CD59 point mutants was as follows: (a) F23D. Aspartate is smaller than phenylalanine, but has a consistent shape with branching at C
carbon; (b) Y36F. Phenylalanine is lacking the hydroxyl group of tyrosine, and based on its conservation, this group may be important; (c) W40Y. Tyrosine is smaller than tryptophane and the aromatic ring superimposes well onto the first ring of tryptophane; (d) L54N. Asparagine is very close to leucine in shape and both sidechains branch at C, although the arrangement at C is tetrahedral for leucine and flat for asparagine. Of the four point mutants prepared, one (Y36F) retained function, two (F23D and L54N) disrupted function but preserved protein topology, and one (W40Y) could not be stably expressed. By building an explicit model of this mutant we found that mutation of Trp40 to Tyr40 leads to a severe clash of the hydroxyl group with the backbone H alpha atom of Val50. This clash cannot easily be resolved and likely leads to the loss of tertiary structure. The near full functional activity of Y36F suggests either that this residue is not involved in ligand binding, or that ligand interaction is primarily with the phenol ring, rather than hydrogen bonding via the hydroxyl group. A further series of multiple residue mutations within and around the hydrophobic groove will better define the active site.
It is not clear what function the N-linked carbohydrate of CD59 may serve. It may be necessary for costimulation of T cell activation (31, 32); it has been reported that the ability of CD59 to enhance CD58-dependent T cell responses is dependent on the N-linked carbohydrate (33). Earlier reports are in conflict over the requirement of N-linked glycosylation for CD59 complement inhibitory function (28– 30). Here we confirm that glycosylation at Asn18 is not required for the inhibition of C5b-9 assembly on the cell surface. In fact, the absence of the carbohydrate enhances slightly, but significantly, the complement inhibitory activity of the protein. The mechanism for this increase in inhibitory activity is not clear, but it is intriguing that membrane sialic acid has recently been described as an acceptor for C5b6, thus enhancing C5b-9–mediated lysis (34). The absence of the sialylated carbohydrate of CD59 may therefore decrease, in a non-specific manner, C5b6 binding to the cell surface. Whatever the mechanisms involved, our data indicate that CD59 lacking N-linked carbohydrate yields an improved inhibitor, an important consideration in designing complement inhibitors for therapeutic applications and in engineering animal organs for human transplantation.
CD59 interferes with the final stages of MAC assembly either by binding to the -chain of C8 in C5b-8 and blocking the binding of C9, by binding to C9 in the nascent C5b-9 complex and preventing proper membrane insertion and C9 polymerization, or by both mechanisms (3, 5, 6). If both mechanisms are operative, then the identification of a single functionally important site in CD59 would suggest that both C8 and C9 bind at this site. The putative CD59 binding site lies in the vicinity of a deep extended linear and hydrophobic groove. These characteristics are consistent with the ligand being an unfolded polypeptide chain with exposed hydrophobic residues, as is postulated to occur when C8 and C9 interact to form the MAC. Of possible relevance, the molecular surface contains a long tunnel under loop Asn8 to Cys13. There are two side chains facing the inner surface of the tunnel, the conserved Val35 as well as residues Asn, Ser, or Gln at position 72. This channel may represent a secondary binding site, but one that may not be required for its complement inhibitory function.
The intense current interest in inhibitors of complement is due to their potential use as anti-inflammatory agents and for preventing unwanted complement-mediated cytolysis. In addition to its role in hyperacute rejection of xenografts, there is evidence indicating that complement-mediated cytolysis is important in the pathogenesis of many autoimmune and inflammatory diseases (35). Understanding precisely how CD59 and other complement inhibitors function will provide a foundation for designing functional inhibitors, possibly multivalent and with enhanced inhibitory activity. The identification of the active site for CD59 may also enable the design of CD59 inhibitors, quite possibly with affinities higher than those of its natural ligands (36); CD59 and other membrane-bound inhibitors of complement are functionally expressed on tumor cells (37, 38), and inhibiting CD59 on tumor cells may enhance their lysis when targeted by complement activating tumor-specific antibodies.
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Acknowledgments |
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This work was supported by National Institutes of Health grants AI34451 and AI08499, and by a Grant in Aid from the American Heart Association.
Submitted: 8 July 1996
Revised: 27 November 1996
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References |
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