By
From the * Department of Pathology, New York University Medical Center, New York 10016 and Department of Biochemistry, The Skirball Institute, New York 10016
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.
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
Several studies have demonstrated the potential usefulness of recombinant soluble complement regulatory proteins for therapy of autoimmune and inflammatory disease
(7). 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). 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.
Cells and DNA.
All DNA manipulations were carried out in
the mammalian expression vector pCDNA3 (Invitrogen, San Diego, CA) containing either CD59 or Ly6E cDNA subcloned into
its multiple cloning site. pCDNA3 contains a G418 resistance
marker. CD59 and Ly6E cDNA were kind gifts from Drs. H. Okada (Nagoya City University, Nagoya, Japan) and U. Hämmerling (Memorial SloanKettering Cancer Center, New York),
respectively. Chinese hamster ovary cells (CHO) were used for
protein expression and were maintained in Dulbecco's modified essential medium (DMEM) supplemented with 10% FCS. Stably
transfected CHO cell lines were selected after the cultivation of
cells in the presence of G418 (GIBCO BRL, Gaithersburg, MD).
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 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
CHO Cell Transfection and Selection.
pCDNA3 constructs were
transfected into CHO cells that were 50-75% confluent. Transfections were carried out with lipofectamine according to the
manufacturer's instructions (GIBCO BRL). 3 d after transfection,
recombinant protein expression was confirmed, and G418 was
added to the medium for selection of stably transfected populations. After 2 wk in selection medium, cells were fluorescently labeled by means of appropriate Abs and sorted by flow cytometry. Stable populations of CHO cells each expressing similar relative levels of recombinant proteins on their surface were isolated by
repeated rounds of cell sorting using either the tag-specific mAb
2A10 (for CD59, CD-Ly chimeras, and point mutants) or antipeptide Ab (for Ly-CD proteins). Three rounds of sorting were
usually sufficient to obtain uniform cell populations that were stable for several weeks. Stably transfected CHO cell clones expressing CD59, unglycosylated CD59 and NH2 terminus tagged CD59
were prepared according to instructions supplied by GIBCO BRL
with lipofectamine reagent.
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 × 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 × 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 × 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.
Native
CD59 is glycosylated at Asn18, and there are conflicting
reports concerning its functional importance (28). We
substituted Asn18 with Gln, and expressed the mutant protein in CHO cells. Fig. 3 shows that CHO cells expressing
recombinant wild-type and unglycosylated CD59 are much
more resistant to lysis by complement than mock transfected controls. Two stable CHO cell clones expressing unglycosylated CD59 were isolated, and each was matched
with a CHO cell clone expressing a similar level of native
CD59 for functional assays (Table 1). In both cases, cells
expressing unglycosylated CD59 were more resistant to complement than cells expressing similar levels of native CD59.
The difference was small but significant (P <0.05) over a
range of serum concentrations. The expected reduction in
MW of unglycosylated CD59 was confirmed by Western
blotting of CHO cell membrane preparations (not shown).
Table 1.
Flow Cytometric Analysis of Stable CHO Cell Clones and Sorted CHO Cell Populations Expressing CD59 and Mutant Constructs
-sheets, 5 protruding surface loops and a short helix (1, 2).
CD59 functions by binding to C8
and/or C9 in the assembling MAC and interfering with C9 membrane insertion and polymerization (3).
Fig. 1.
CD59 and Ly6E sequence comparison. Mature protein sequences are shown with
the conserved cysteine residues
marked (:). The amino acids
forming the surface loops and
helix of CD59 are shown. The
COOH-terminal end of mature
Ly6E is predicted.
[View Larger Version of this Image (9K GIF file)]
70°C.
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).
Fig. 2.
Diagram of CD59Ly6E chimeric constructs. All
numbers refer to amino acid positions of CD59.
[View Larger Version of this Image (23K GIF file)]
Role of N-linked Carbohydrate in CD59 Function.
Fig. 3.
Complement inhibitory activity of unglycosylated CD59.
Stable CHO cell clones expressing similar levels of CD59 or unglycosylated CD59 (N18Q) were assayed for their susceptibility to complement. Controls were mock transfected CHO cells. Each point represents the
mean of at least four determinations.
[View Larger Version of this Image (17K GIF file)]
Transfectant
Mean Relative Fluorescence
Anti-CD59
polyclonal
Anti-tag
mAb 2A10
Anti-CD59 peptide
polyclonal
Anti-CD59
mAb YTH53.1
Anti-CD59
mAb MEM43/5
Anti-CD59
mAb HC1
CD59
1246
1218
CD59-tagged
1207
695
1119
CD59 (1)
1314
1299
N18Q (1)
1196
1201
CD59 (2)
289
N18Q (2)
221
CD59 (CD-Ly set)
820
CD-Ly 24
1220
CD-Ly 47
894
CD-Ly 57
798
CD-Ly 64
980
CD59 (Ly-CD set)
632
Ly-CD 16
561
Ly-CD 31
516
Ly-CD 39
616
CD59 (1)
986
766
460
826
CD59 (F23D)
780
727
448
770
CD59 (Y36F)
812
686
404
770
CD59 (2)
305
480
242
434
CD59 (L54N)
240
316
165
376
CD59 (W40Y)
23
12
Mean relative fluorescence of mock transfected CHO cells was below 20 with all Abs. Figures are relative within each grouped set.
To identify regions of CD59 important for function, a series of CD59/Ly6E chimeric proteins containing either CD59 or Ly6E at their NH2-terminus were expressed on CHO cells. We reasoned that the residues involved in ligand binding were probably located in surface loops. For this reason, most of the chosen switch over points between CD59 and Ly6E were at selected loop boundaries (see Fig. 2).
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.
To quantitate the relative surface expression of CD59
and CD-Ly chimeric proteins, a 10-amino acid epitope tag
was engineered onto the NH2 termini of the constructs.
The tag did not affect the expression (Table 1) or the function (Fig. 5) of CD59. However, when the tag was placed
at the NH2 terminus of Ly6E in the Ly-CD constructs, it
interfered with expression and function of the chimeras.
Therefore, surface expression of Ly-CD chimeras was quantitated using a rabbit Ab against a peptide corresponding to a sequence from the COOH terminus of CD59 (Table 1). This Ab reacted quantitatively with CHO cell clones
stably expressing different levels of CD59 (not shown). Cell
populations used in these experiments were obtained by
several rounds of cell sorting, and separate CD-Ly and LyCD sets were prepared, each with a corresponding CHO
cell population expressing a similar level of CD59.
Modeling the Molecular Surface of CD59.
To reveal the
hypothetical location and extent of the functional site, we
built a model of the molecular surface of CD59 and analyzed: (a) the three-dimensional location of the fragments that can be replaced by the Ly6E fragments, (b) the distribution of surface patches that are conserved between CD59
of seven different species that are either closely related to
human, or that have been shown to inhibit human complement (Fig. 6), and finally, (c) the surface shape. A very clear
picture emerged from this analysis. Fig. 7 a displays the
molecule in an orientation that shows that the regions of
CD59 that can be replaced by Ly6E without loss of function (1-16 and 57-77) are located at the backside of the
molecule (in yellow, Fig. 7 a). From the position of residue 77, to which the GPI membrane anchor is attached, it can
be predicted that the active site is located on the exposed
face of the molecule.
Comparison of CD59 sequences within the region required for function (residues 16-57, Fig. 6) reveal a number of evolutionary conserved residues (Fig. 6). However, of these residues, only five are located on the surface of the shown face of the molecule, and three (Y36, W40, and L54) are conspicuously grouped together in a deep groove (Fig. 7 b, conserved residues colored green). Furthermore, conserved residue classes (Fig. 7 b, colored blue) are located above and below the conserved residues within this groove. From top to bottom these residue positions are (refer to Fig. 7 b): no. 31: Ala or Ser, a small residue, presumably important to keep the hypothetical binding groove open; no. 59: Leu or Val, a hydrophobic residue, outside the CD59 16-57 sequence, but conserved in Ly6E; no. 36: Tyr, no. 54 Leu and no. 40 Trp, conserved residues; no. 23: Phe or Leu, a hydrophobic residue, and no. 41: Lys or Arg, the only charged residues. The only addition to this list of conserved residues at the shown face of the molecule are Val 35 and two conserved Cys-Cys bridges (Cys19Cys39, and Cys6-Cys13, shown in yellow). Val 35 and Cys6-Cys13 are located at the inner surface of a tunnel, an interesting structural feature of the molecule (these residues are obscured by the tunnel in Fig. 7 b). The conserved cysteines are most likely necessary for conservation of the topology of the molecule.
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).
The results in this report represent a first approximation at defining the active site of CD59. We have shown that: (a) glycosylation of CD59 at Asn18 is not required for function; (b) replacement of CD59 sequences 1-16 and 5777 with those of non-functional Ly6E did not affect CD59 activity. These regions are therefore not required for CD59 activity, and they map to the backside of the molecule that is closer to the cell surface (Fig. 7 a); (c) a comparison of sequences of human CD59 and CD59 homologues from other species within the identified functional region (amino acids 16-57), reveal an almost linear arrangement of conserved residues and residue classes in the three dimensional structure. These residues were predominantly hydrophobic in character, and were not contiguous in the primary structure. Together they marked a groove across the molecule (Fig. 7 b); (d) mutation of residues within this groove disrupted the function of the expressed mutant proteins.
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.
Address correspondence to Stephen Tomlinson, New York University Medical Center, Department of Pathology, MSB 127, 550 First Ave., New York, NY 10016. Dr. Dong's present address is Ohio State University, Department of Molecular Genetics, Biological Science Building, 484 West 12th St., Columbus, OH 43210.
Received for publication 8 July 1996
1Abbreviations used in this paper: CHO, Chinese hamster ovary cells; GPI, glycosylphosphatidylinositol; MAC, membrane attack complex; NHS, normal human serum.We thank Dr. Michael Whitlow for critical discussion, Dr. John Hirst for performing flow cytometry and Dr. Jonathon Weider for the generation of photographs.
This work was supported by National Institutes of Health grants AI34451 and AI08499, and by a Grant in Aid from the American Heart Association.
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