©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Identity of the Segment of Human Complement C8 Recognized by Complement Regulatory Protein CD59 (*)

(Received for publication, May 10, 1995)

Dara H. Lockert (1) Kenneth M. Kaufman (2) Chi-Pei Chang (1) Thomas Hüsler (1) James M. Sodetz (2) Peter J. Sims (1)(§)

From the  (1)Blood Research Institute, Blood Center of Southeastern Wisconsin, Milwaukee, Wisconsin 53233 and the (2)Department of Chemistry and Biochemistry, School of Medicine, University of South Carolina, Columbia, South Carolina 29208

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

CD59 antigen is a membrane glycoprotein that inhibits the activity of the C5b-9 membrane attack complex (MAC), thereby protecting human cells from lysis by human complement. The inhibitory function of CD59 derives from its capacity to interact with both the C8 and C9 components of MAC, preventing assembly of membrane-inserted C9 polymer. MAC-inhibitory activity of CD59 is species-selective and is most effective when both C8 and C9 derive from human or other primate plasma. Rabbit C8 and C9, which can substitute for human C8 and C9 in MAC, mediate virtually unrestricted lysis of human cells expressing CD59. In order to identify the segment of human C8 that is recognized by CD59, recombinant peptides containing human or rabbit C8 sequence were expressed in Escherichia coli and purified. CD59 was found to specifically bind to a peptide corresponding to residues 334-385 of the human C8 alpha-subunit, and to require a disulfide bond between Cys and Cys. No specific binding was observed to the corresponding sequence from rabbit C8alpha (residues 334-386). To obtain functional evidence that this segment of human C8alpha is selectively recognized by CD59, recombinant C8 proteins were prepared by co-transfecting COS-7 cells with human/rabbit chimeras of the C8alpha cDNA, and cDNAs encoding the C8beta and C8 chains. Hemolytic activity of MAC formed with chimeric C8 was analyzed using target cells reconstituted with CD59. These experiments confirmed that CD59 recognizes a conformationally sensitive epitope that is within a segment of human C8alpha internal to residues 320-415. Our data also suggest that optimal interaction of CD59 with this segment of human C8alpha is influenced by N-terminal flanking sequence in C8alpha and by human C8beta, but is unaffected by C8.


INTRODUCTION

Human CD59 antigen is a 18-21-kDa plasma membrane protein that functions as an inhibitor of the C5b-9 membrane attack complex (MAC) (^1)of human (hu) complement(1) . CD59 interacts with both the C8 and C9 components of MAC during its assembly at the cell surface, thereby inhibiting formation of the membrane-inserted C9 homopolymer responsible for MAC cytolytic activity (2, 3) . This serves to protect hu blood and vascular cells from injury arising through activation of complement in plasma. CD59's inhibitory activity is dependent upon the species of origin of C8 and C9, with greatest inhibitory activity observed when C8 and C9 are from hu or other primates. By contrast, CD59 exerts little or no inhibitory activity toward C8 or C9 of most other species, including rabbit (rb) (4, 5, 6) .

Human C9 is a single-chain 72-kDa polypeptide, whereas C8 consists of a heterotrimer of polypeptides, arranged as a disulfide-linked alpha- subunit that is non-covalently associated with a beta-chain(7, 8) . C8alpha and C8beta chains exhibit extensive sequence homology with C9. Analysis of the physical association of CD59 with components of MAC suggested that separate binding sites for CD59 are contained within the alpha-chain of hu C8 and within hu C9.(9) Consistent with the evidence for a CD59 binding site in hu C8alpha, C8 hybrids formed by combining rb C8alpha- with hu C8beta displayed unrestricted hemolytic activity toward hu erythrocytes (huE)(10) . Whereas the CD59 recognition site in hu C9 has been localized to a segment of the polypeptide spanning residues 359-415, the corresponding site within hu C8alpha remains to be identified(11, 12) . Comparison of the aligned sequences of hu and rb C8 alpha-chains revealed maximal divergence of sequence between residues 349-385, suggesting that this segment of the polypeptide might contain the site within hu C8 that is selectively recognized by CD59(10) . In this study, we use recombinant C8alpha peptides and hemolytically active C8 hybrids and chimeras containing hu and rb polypeptide sequence to determine the contribution of this sequence to the species-selective interaction of CD59 with the C8 component of hu MAC.


EXPERIMENTAL PROCEDURES

Materials

Human complement proteins C5b6, C7, C8, and C9 and huE glycoprotein CD59 were purified and assayed as described previously(3, 13, 14) . Rb C8 and C9 were purified as described previously(9, 10) . Chicken erythrocytes (chE) were from Cocalico Biologics, Inc. (Reamstown, PA). COS-7 cells were from American Tissue Culture Collection (Rockville, MD). Escherichia coli strain JS5 was from Bio-Rad. Dulbecco's modified Eagle's medium was from Mediatech Inc. (Herndon, VA). Opti-MEM I was from Life Technologies, Inc. Heat-inactivated fetal calf serum was from Biocell (Rancho Dominguez, CA). Oligonucleotides were synthesized by the Molecular Biology Core Laboratories, Blood Research Institute, Milwaukee, WI.

Solutions

MBS consisted of 150 mM NaCl, 10 mM MOPS, pH 7.4. Washing buffer consisted of 0.1% (w/v) BSA, 0.1% (v/v) Nonidet P-40 in MBS. GVBS consisted of 150 mM NaCl, 3.3 mM sodium barbital,0.1%(w/v) gelatin, pH 7.4. GVBE consisted of 150 mM NaCl, 3.3 mM sodium barbital, 10 mM EDTA, 0.1%(w/v) gelatin, pH 7.4.

Labeling of CD59

CD59 was biotinylated by incubation (1 h, 23 °C) with a 20-fold molar excess of NHS-LC-biotin in MBS adjusted to pH 8.5 with 100 mM sodium carbonate. Unincorporated label was removed by gel filtration on Sephadex G-25 (PD-10; Pharmacia Biotech Inc.), followed by exhaustive dialysis.

Expression and Purification of Recombinant C8alpha Peptides

Fusion proteins containing the maltose-binding protein (MBP) and a defined segment of hu or rb C8alpha were expressed in E. coli, purified, and cleaved with factor Xa to release recombinant C8alpha peptides. cDNAs encoding subunits of hu C8 and rb C8 are described elsewhere(10, 15, 16, 17) . cDNA fragments encoding hu C8alpha residues 334-385 and rb C8alpha 334-386 were prepared by PCR amplification of the respective cDNAs. Primers were designed to produce blunt ends with an in-frame stop codon at the 3` end of the coding strand. PCR products were cloned into the XmnI site of the pMAL-p2 fusion protein expression vector (New England Biolabs). Fidelity of PCR and cloning was confirmed by dideoxy sequencing using Sequenase (United States Biochemical Corp.). E. coli KS1000 cells were used for propagation of the pMAL-p2 constructs and expression of MBP-C8alpha fusion proteins. Control expressions used pMAL-p2 plasmid with no insert, which yields MBP-lacZalpha as the fusion product.

Fusion proteins were purified from periplasmic extracts by amylose affinity chromatography, according to the manufacturer's recommendation. Digestions with factor Xa were performed in 20 mM Tris, 20 mM NaCl, pH 7.4 (pH 7.0 for rb C8alpha), after which the sample was applied directly to a Q-Sepharose (Sigma) column equilibrated in the same buffer. Released peptide was collected in the wash and determined to be >95% pure when analyzed on 15% acrylamide/Tricine SDS-polyacrylamide gel electrophoresis gels. Electrophoretic mobility differences observed upon reduction of the isolated peptide suggest that the single cysteine pair (corresponding to Cys and Cys in hu C8alpha) spontaneously forms an intrachain disulfide bond. Identity of purified peptides was confirmed by amino acid analysis.

To prepare Cys Gly mutants of hu C8alpha-derived peptide 334-385, PCR-mediated site-directed mutagenesis was performed using overlapping primers to change the two Cys codons(18) . The product was cloned into pMAL-p2 and expressed as a fusion protein. The peptide was released with factor Xa and purified as described above. For certain experiments, cysteine residues of the purified peptides were reduced and alkylated by incubation (37 °C, 60 min) with 10 mM dithiothreitol, followed by 30 mM iodoacetamide.

CD59 Binding to C8alpha-derived Peptides

The binding of CD59 to C8, recombinant C8alpha-derived peptides, and to peptides expressed as MBP fusion proteins was performed by modification of CD59 plate-binding assay reported previously(9, 11) . Plasma-derived C8, MBP-C8alpha peptide fusion proteins, or the purified peptides in 0.1 M sodium bicarbonate, pH 8.5, were adsorbed to 96-well polyvinyl microtiter plates by overnight incubation at 4 °C. After blocking (2 h, 23 °C) with 1% (w/v) BSA, the wells were washed and 0.4 µg/ml biotin-CD59 in washing buffer added. To determine nonspecific binding, biotin-CD59 was first mixed with 20 µg/ml unlabeled CD59. After a 2-h incubation at 23 °C, the wells were rinsed with washing buffer and incubated (1 h) with 1 µg/ml streptavidin-alkaline phosphatase. After rinsing, color was developed by addition of 1 mg/ml p-nitrophenyl phosphate and optical density at 405 nm recorded (Vmax Microplate Reader, Molecular Devices, Inc). CD59 binding to MBP-C8alpha fusion proteins was corrected for nonspecific binding to wells coated with MBP-lacZalpha, generated by expression of pMAL-p2.

Construction of C8 and Chimeric C8alpha Expression Vectors

To express hu or rb C8 in COS-7 cells, cDNAs encoding full-length C8alpha, C8beta, and C8 subunits were cloned into the expression vector pcDNA3 (Invitrogen) as described elsewhere. (^2)The hu/rb chimeras of C8alpha were prepared either in pBluescript II (Stratagene) or by modifying full-length pcDNA3 constructs. Chimeras H1-319/R320-555 and R1-319/H320-554 (see Fig. 5) were prepared in the former by exchanging cDNA fragments generated from cleavage at the polylinker and at a conserved BamHI site located 5` of the candidate CD59 recognition site in C8alpha. Full-length chimeric inserts were released and subsequently cloned into pcDNA3. Chimeras H1-319/R320-416/H416-554 and R1-319/H320-415/R417-555 were generated directly in pcDNA3 by utilizing the BamHI site and a conserved 3`-oriented ClaI site to exchange internal cDNA segments. All constructs were sequenced across junction sites to verify cloning fidelity.


Figure 5: Inhibitory function of CD59 requires hu C8alpha residues 320-415. Bar graph (right panel) summarizes combined results of all experiments performed under conditions of Fig. 1with recombinant hu C8 containing chimeric alpha-subunits. From each C8 titration, the inhibitory activity of CD59 (expressed as the percentage of inhibition of hemolysis due to CD59, ordinate) was calculated at the concentration of C8 resulting in 50% hemolysis in the absence of CD59. Error bars denote mean ± S.D. (n = 5); asterisks indicate significance (p < 0.01) compared to recombinant rb C8. To the left of each data bar, the protein tested is depicted so as to designate those portions containing hu (open) or rb (shaded) C8alpha sequence. Human C8 and rabbit C8 denote C8 purified from hu and rb plasma, respectively. Recombinant C8 proteins contain hu (H) or rb (R) alpha-chain sequence numbered according to the deduced primary structure of the hu or rb mature polypeptide, respectively. In some chimeric constructs, numbering appears discontinuous because of a gap in the alignment of the rb and hu C8alpha sequences. In all cases, chimeric alpha-chains were expressed with hu C8 and hu C8beta chains. Domains depicted in the proposed structure of C8 include thrombospondin type 1 (TS), low density lipoprotein receptor (LDLR), hinge (Hinge), membrane binding (MB), and epidermal growth factor precursor (EGFP). Figure is adapted from (8) .




Figure 1: Effect of CD59 on hemolytic activity of recombinant C8. Functional assay of hu, rb, and hu/rb hybrids of complement C8 measured in the absence () or presence (bullet) of cell-surface CD59. Hemolytic activity of each protein (ordinate) was determined by titration of each C8 construct in the presence of 50 ng/ml rb C9, using hu C5b-7, chE target cells reconstituted with hu CD59 (see ``Experimental Procedures''). Results shown are for plasma-derived hu C8 (panel A), plasma-derived rb C8 (panel B), recombinant hu C8 (panelC), and recombinant hybrid C8 containing rb C8alpha, hu C8, and hu C8beta (panel D).



Transfection of COS-7 Cells

Plasmids were propagated in E. coli JS5. Plasmid DNA was isolated using Qiagen-tips (Qiagen Inc., Chatsworth, CA) and was used with or without further purification through a CsCl gradient. COS-7 cells were transfected using DEAE-dextran. After transfection, cells were grown for 24 h in Dulbecco's modified Eagle's medium (Mediatech Inc.) supplemented with 10% fetal bovine calf serum plus 2 mM L-glutamine, after which this medium was replaced by Opti-MEM I (Life Technologies, Inc.). Cell supernatants were harvested after 48-65 h, phenylmethylsulfonyl fluoride (1 mM), benzamidine (1 mM), and EDTA (10 mM) were added, and the supernatants concentrated at 4 °C (Centricon 30, Amicon)(12) .

Analysis of the Inhibitory Function of CD59 toward Recombinant C8 Hybrids and C8 Chimeras

Hemolytic activity of each recombinant C8 was assayed using as target cells chE that were reconstituted with purified hu CD59(12) . chE were washed extensively and suspended in GVBS, and the membrane C5b67 complex assembled by mixing cells (1.4 10^9/ml final concentration) with hu C5b6 (13 µg/ml final concentration) followed by addition of hu C7 (1 µg/ml). After 10 min, the C5b67 chE were diluted to 1.4 10^8/ml in GVBE and incubated (10 min, 37 °C) with 0 or 750 ng/ml CD59. After washing in ice-cold GVBE, 25 µl containing 2.8 10^6 of these cells were mixed with 50 µl of Opti-MEM I containing 0-3 ng of C8 (hu, rb, or recombinant). After 15 min (37 °C), rb C9 (5 ng in 25 µl of GVBE) was added (final volume, 100 µl). Hemolysis was determined after 30 min at 37 °C, with correction for nonspecific lysis (always <5%) measured in the absence of C9.


RESULTS

Contribution of hu C8 alpha, beta, and Chains to Recognition by CD59

Previous studies suggested that CD59's selective inhibition of hu MAC reflected its affinity for a binding site unique to hu C9 as well as a binding site contained within the alpha-subunit of hu C8(9, 10) . In order to confirm the importance of the hu C8alpha-chain to CD59's selective regulation of the lytic activity of hu MAC, recombinant C8 and hybrids of C8 containing hu and rb subunits were expressed in COS-7 by co-transfection with cDNAs for each of the polypeptide chains of the hu or rb protein. Analysis by Western blotting revealed that all three C8 chains are secreted and that the C8alpha and C8 chains form the expected disulfide-linked C8alpha- dimer.^2 Expressed proteins were then tested for hemolytic activity against C5b67-coated erythrocytes, measured in the presence or absence of hu CD59. In these assays, rb C9 was used to complete MAC assembly, so as to circumvent CD59's interaction with hu C9(9, 11, 12) . As illustrated in Fig. 1, CD59 was observed to markedly inhibit the lytic activity of MAC assembled with hu C8 (panel A), but showed negligible activity when rb C8 was used to assemble MAC (panel B). When recombinant hu C8, expressed from hu C8 alpha, beta, and cDNAs, was used to assemble MAC, sensitivity to inhibition by CD59 was similar to that of plasma-derived hu C8 (cf. panels A and C). By contrast, a recombinant hybrid C8, prepared by co-transfection with rb C8alpha, hu C8beta, and hu C8 cDNAs (panel D), resembled rb C8, and was only slightly inhibited by CD59. We next used this assay to analyze all possible hybrids formed by interchanging the alpha, beta, and subunits of hu and rb C8. These hybrids were analyzed in multiple hemolytic titrations performed under the conditions of Fig. 1, and the cumulative data for each are summarized in Fig. 2. Inspection of these data revealed the following. 1) CD59 exerted maximal inhibition toward MAC assembled from hu C8, recombinant or plasma-derived (cf. human C8, rabbit C8, and constructs 1 and 2). 2) All C8 hybrids produced with the rb alpha-chain (constructs 3-5) were indistinguishable from native rb C8 and only weakly inhibited by CD59. This included a hybrid containing both hu C8 and hu C8beta (construct 5). 3) In C8 hybrids that contained hu alpha-chain, replacement of hu C8 by rb C8 resulted in negligible loss of recognition by CD59 (construct 6), whereas replacement of hu C8beta by rb C8beta (constructs 7 and 8) resulted in a reduction in inhibition by CD59 relative to that observed for hu C8. Taken together, these data suggest that CD59's inhibitory activity is dependent on a motif that is contained within the hu C8 alpha-chain and is not influenced by the species of origin of C8. Moreover, interaction of CD59 with this site in C8alpha is optimal only when C8 contains the hu C8beta-chain. By contrast, neither hu C8 nor hu C8beta alone or in combination conferred recognition by CD59, when C8 was assembled using rb C8alpha (constructs 3-5).


Figure 2: The rb/hu C8 hybrids reveal role of the hu C8alpha chain in recognition by CD59. C8 hybrids composed of either rb or hu alpha, beta, and subunits were expressed in COS-7 and assayed for functional inhibition by CD59 under conditions described for Fig. 1. From each C8 titration performed as in Fig. 1, the inhibitory activity of CD59 (expressed as the percentage of inhibition of hemolysis due to CD59, ordinate) was calculated at the concentration of C8 that resulted in 50% hemolysis in the absence of CD59. HumanC8 and rabbitC8 represent the plasma-derived proteins; constructs 1-8 represent recombinant C8 containing the indicated subunits. Bargraph summarizes combined results of all experiments, normalized to results obtained in each experiment for recombinant hu C8 (100%). Errorbars denote mean ± S.D.; parentheses indicate number of independent experiments; asterisks indicate significance (p < 0.01) compared to recombinant rb C8. , significance (p < 0.06)



Mapping of the CD59 Binding Site within the alpha-Subunit of hu C8

We have shown previously that CD59 selectively binds to the hu C8alpha-chain, but not to rb C8alpha, and that this interaction is lost upon reduction of intrachain disulfide bonds(9) . Alignment of the hu and rb C8 alpha-chains revealed a single segment of highly divergent sequence between hu residues 349-385 (corresponding to 349-386 in rb C8alpha), suggesting that this particular segment of C8alpha might confer species-selective recognition of hu C8 by CD59(10) . In order to explore this hypothesis, we expressed peptides containing this segment of hu and rb C8alpha and tested their capacity to bind CD59. CD59 was observed to specifically bind to the peptide corresponding to hu C8alpha 334-385, whereas no specific binding was detected for the corresponding segment of rb C8alpha (Fig. 3). This segment of C8alpha contains two conserved Cys residues (Cys and Cys in hu C8alpha), which are predicted to form an intrachain disulfide bond(19, 20) . Because disulfide reduction of hu C8alpha resulted in the loss of detectable affinity for CD59(9) , we considered the possibility that the putative Cys-Cys disulfide is required for expression of the CD59 binding site. As shown in Fig. 3, whereas CD59 bound specifically to the peptide corresponding to hu C8alpha 334-385, such binding was eliminated when cysteine residues 345 and 369 were replaced by glycine. Similar results were obtained when CD59 binding to native versus disulfide-reduced hu C8alpha 334-385 peptide was compared (Fig. 4).


Figure 3: CD59 binding to C8alpha-chain peptide. Microtiter plate wells were coated with 10 µg/ml of hu C8 or the C8alpha peptide indicated (abscissa) and CD59 binding performed as described under ``Experimental Procedures.'' Solid bars represent total binding; open bars represent nonspecific binding obtained in presence of 50-fold excess unlabeled CD59. All absorbances (405 nm) are normalized to that obtained in each separate assay for wells coated with 10 µg/ml hu C8 (OD = 1.0), after correction for nonspecific binding to wells coated with BSA. Error bars denote mean ± S.D. of all results obtained from six separate experiments so performed. Results shown are for plasma-derived hu C8, hu C8alpha peptide 334-385, rb C8alpha peptide 334-386, and hu C8alpha peptide 334-385 containing Cys Gly mutations at residues 345 and 369.




Figure 4: Role of hu C8alpha Cys/Cys disulfide in expression of CD59 binding site. Microtiter plate binding of biotin-CD59 was performed for wells coated with 10 µg/ml MBP-C8alpha 334-385 or MBP-C8alpha 334-385 containing Cys Gly mutations at Cys and Cys. hu C8 refers to wells coated with plasma-derived hu C8. Solid bars denote specific binding to each protein that was plate-coated without prior disulfide reduction; hatched bars denote proteins that were reduced and alkylated prior to plate coating (see ``Experimental Procedures''). MBP itself contains no Cys residues, and therefore MBP fusion proteins were employed for these experiments to circumvent potential changes in plate-coating efficiency of free peptides after reduction and alkylation. Absorbances (OD; ordinate) are normalized to data obtained in each assay for hu C8 (OD = 1), after correction for nonspecific binding to MBP-lacZalpha. Error bars denote mean ± S.D. of all results obtained from six separate experiments so performed.



Analysis of Recombinant C8 Containing Chimeric alphaChain

To further analyze the contribution of this segment of hu C8alpha to the recognition of hu MAC by CD59, we expressed recombinant hu C8 containing chimeric C8alpha in which this segment was exchanged between the hu and rb polypeptides. In order to produce hu/rb chimeras of the C8 alpha-chain, we took advantage of conserved restriction sites flanking the region of interest (see ``Experimental Procedures``). Chimeric C8alpha chains were co-expressed with hu C8 and hu C8beta and tested for hemolytic activity and sensitivity to inhibition by CD59 (Fig. 5). These experiments revealed the following. 1) Substitution of hu C8alpha residues 320-554 (construct 3) or 320-415 (construct 4) with corresponding rb sequence resulted in proteins that were indistinguishable from rb C8, even though both contained hu C8 and hu C8beta. 2) Substitution of rb C8alpha residues 320-555 (construct 5) or 320-416 (construct 6) with corresponding hu sequence partially restored the capacity of CD59 to inhibit the lytic activity of C8, implying selective recognition of residues 320-415 of the hu sequence within the chimeric alpha-chains. 3) Although all constructs containing hu C8alpha 320-415 showed increased sensitivity to inhibition by CD59, maximal inhibition was observed only when C8alpha consisted entirely of hu sequence (cf. constructs 5 and 6 with construct 1). Taken together, these data suggest that the species-selective interaction of CD59 with hu MAC is conferred primarily by residues contained within the segment 320-415 of hu C8 alpha-chain, but that optimal interaction with this site requires the presence of additional hu sequence in the N-terminal flanking portion of the polypeptide.


DISCUSSION

The data of the present study identify the sequence between residues 320-415 of the hu C8 alpha-chain as critical to the selective recognition of hu C8 by cell-surface CD59. Our experiments also suggest that a disulfide-bonded loop contained within residues 334-385 (Cys-Cys) is required for CD59 binding, providing an initial clue to the structural motif through which this inhibitor selectively regulates the lytic activity of hu MAC. These data further suggest that the capacity of CD59 to interact with this segment of C8alpha is influenced by other portions of C8. In particular, we note that replacing hu C8beta with rb C8beta decreased the inhibitory activity of CD59 toward hu C8 (see constructs 7 and 8 in Fig. 2). Similarly, retention of N-terminal flanking rb sequence in chimeric C8alpha constructs containing hu residues 320-415 partially decreases the inhibitory activity of CD59 toward an otherwise hu C8 (see constructs 5 and 6 in Fig. 5). By contrast to the partial loss of CD59 inhibitory function observed for the above C8 constructs, mere substitution of C8alpha residues 320-415 in hu C8 with corresponding rb sequence (construct 4 in Fig. 5) is sufficient to completely abrogate CD59's selective inhibition of the lytic activity of hu C8, resulting in a protein that is functionally indistinguishable from rb C8. Taken together, these data support the interpretation that C8alpha residues 320-415 contain the CD59 binding site in hu C8, but that the conformation of this site is allosterically influenced by the hu C8beta subunit as well as by C8alpha sequence flanking this site. Our interpretation that CD59's selective inhibitory activity toward hu C8 reflects its capacity to bind hu C8alpha residues 320-415 is consistent with the observed species-selective binding of CD59 to both hu C8alpha and to the hu C8alpha-derived peptide 334-385 (Fig. 3), and by the lack of detectable CD59 binding to either hu C8beta or C8 subunits (9) . Nevertheless, we cannot exclude the possibility that CD59 also interacts with these other regions of C8 that are potentially exposed during MAC assembly.

Our conclusion that CD59 recognizes sequence unique to hu C8alpha as derived from the present analysis of the interaction of recombinant C8 hybrids with purified and membrane-reconstituted CD59 is consistent with results of earlier experiments in which the lytic activity of plasma-derived forms of C8 toward huE were evaluated. In particular, it was observed that hu C8 and a derivative of hu C8 that lacked C8 were equally restricted in their capacity to lyse huE, suggesting that the C8 subunit does not contribute to the mechanism by which CD59 or other huE complement regulatory factors restrict MAC assembly(21) . Furthermore, a C8 hybrid formed by combining the isolated rb C8alpha- and hu C8beta subunits exhibited the same level of unrestricted lytic activity toward huE as was observed for rb C8, implicating C8alpha as the determinant of species-selective restriction of C8 lytic activity(10) . As discussed above, measurement of the physical association of CD59 with isolated hu C8 subunits also revealed that whereas CD59 binds specifically to C8alpha- or C8alpha, such binding is not detected for either C8 or C8beta(9) . Taken together with the results of the present study, these data provide compelling evidence that the primary CD59 binding site in hu C8 is within the C8alpha subunit, centered on residues 334-385.

In addition to interacting with the C8 component of hu MAC, CD59 is also known to interact with hu C9, a polypeptide that exhibits sequence similarity to hu C8alpha and to several other MAC components(9, 11, 12) . The CD59 recognition domain in hu C9, as deduced from analysis of the lytic activity of hu/rb C9 chimeras, was localized to residues 334-415, whereas analysis of CD59 binding to hu C9-derived peptides suggested that C9 residues 359-411 were required for specific binding (11, 12) . It is of interest to note that from the aligned sequences of hu C8alpha and hu C9, the site in C8alpha that we now identify as conferring recognition by CD59 (C8alpha 320-415) overlaps the same region of C9 that was shown to contain the CD59 recognition domain (Fig. 6). Surprisingly, inspection of the aligned amino acid sequences of hu C8alpha and C9 that span the respective CD59 recognition sites of these two proteins reveals very limited sequence identity and unremarkable sequence homology. This suggests that the CD59 binding sites expressed by these two MAC components are not structurally identical, but comprise distinct motifs that are individually recognized by the inhibitor. In this context, it is of interest to note that by contrast to the apparent conformational sensitivity of the CD59 recognition site in hu C8alpha (see above), the segment of hu C9 recognized by CD59 shows virtually no dependence on flanking C9 sequence, nor does it require formation of the intrachain disulfide (between C9 Cys-Cys, corresponding to C8alpha Cys-Cys) that is normally present in hu C9(11, 19, 20, 22, 23) .


Figure 6: Alignment of sequence within the CD59 recognition sites in hu C8alpha and C9. The segment of hu C8alpha identified to contain the site recognized by CD59 is aligned to the corresponding segment of hu C9. Identity of this segment in C8alpha (residues 320-415) is from data of Fig. 5. Alignment is based upon the entire amino acid sequence of the mature hu C8alpha and C9 polypeptides (overall identity = 32%). Arrows depict the peptide sequence from C8alpha (residues 334-385; see Fig. 3and Fig. 4) and from C9 (residues 359-411; from (11) ) shown to specifically bind CD59. Asterisks denote sequence identity; dashed lines denote the putative intrachain disulfide bond contained in this segment of each polypeptide (see ``Discussion''). Domain structure of the polypeptides is depicted as described in Fig. 5, except that C9 lacks the C-terminal thrombospondin domain contained in C8alpha.



It remains unresolved how the interaction of CD59 with its binding site in C8alpha serves to inhibit MAC assembly. In membranes expressing CD59, there is reduced incorporation of C9 and reduced lytic activity of MAC, suggesting that CD59 inhibits C9 binding to C5b-8 or subsequent conformational changes required for membrane insertion and C9 polymer formation(2, 3, 5) . The capacity of CD59 to restrict C9 binding to C5b-8 as well as to inhibit MAC lytic activity after C9 is initially bound implies potential inhibition at both steps of MAC assembly(3, 5, 24) . In addition to providing a binding site for CD59, the alpha-chain of C8 also has the capacity to bind C9, and therefore this subunit of the C5b-8 complex is thought to mediate binding and incorporation of C9 into MAC(7, 9, 25) . This raises the possibility that the segment of C8alpha to which CD59 binds either overlaps or is closely apposed to a segment of C8alpha that interacts with C9, accounting for CD59's inhibitory effect on MAC assembly. Of note, whereas CD59 binding to C8 is highly species-selective, such species selectivity is not observed for the interaction of C9 with C5b-8, in the absence of CD59(5, 12) . This suggests that the C9 and CD59 binding sites in C8alpha that are both exposed when C8 incorporates into MAC are not identical.


FOOTNOTES

*
This work was supported by Grants HL36061 (to P. J. S.) and GM42898 (to J. M. S.) from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Blood Center of Southeastern Wisconsin, P. O. Box 2178, Milwaukee, WI 53233. Tel.: 414-937-3850; Fax: 414-937-6284.

(^1)
The abbreviations used are: MAC, C5b-9 membrane attack complex of complement; hu, human; rb, rabbit; huE, human erythrocyte; chE, chicken erythrocyte; BSA, fatty-acid and globulin-free bovine serum albumin; MBP, maltose-binding protein; PCR, polymerase chain reaction; Tricine, N-[2-hydroxy-1,1,bis(hydroxymethyl)ethyl]glycine; MOPS, 4-morpholinepropanesulfonic acid.

(^2)
K. M. Kaufman, C. S. Letson, P. L. Platteborze, G. A. Michelotti, and J. M. Sodetz, manuscript in preparation.


ACKNOWLEDGEMENTS

We gratefully acknowledge technical assistance from Diana Schick, Lilin Li, and Randal Orchekowski.


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  14. Wiedmer, T., and Sims, P. J. (1985) J. Biol. Chem. 260,8014-8019 [Abstract/Free Full Text]
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  16. Rao, A. G., Howard, O. M., Ng, S. C., Whitehead, A. S., Colten, H. R., and Sodetz, J. M. (1987) Biochemistry 26,3556-3564 [Medline] [Order article via Infotrieve]
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