Identification of the Individual Residues That Determine
Human CD59 Species Selective Activity*
Hui-fen
Zhang,
Jinghua
Yu,
Shaohua
Chen,
B. Paul
Morgan
,
Ruben
Abagyan§, and
Stephen
Tomlinson¶
From the Department of Pathology, New York University Medical
Center, § Department of Biochemistry, The Skirball
Institute, New York, New York 10016, and
Department of
Medical Biochemistry, University of Wales College of Medicine, Heath
Park, Cardiff, C4F 4XN United Kingdom
 |
ABSTRACT |
Formation of the cytolytic membrane attack
complex of complement on host cells is inhibited by the membrane-bound
glycoprotein, CD59. The inhibitory activity of CD59 is species
restricted, and human CD59 is not effective against rat complement.
Previous functional analysis of chimeric human/rat CD59 proteins
indicated that the residues responsible for the species selective
function of human CD59 map to a region contained between positions 40 and 66 in the primary structure. By comparative analysis of rat and
human CD59 models and by mutational analysis of candidate residues, we
now identify the individual residues within the 40-66 region that
confer species selective function on human CD59. All nonconserved residues within the 40-66 sequence were substituted from human to rat
residues in a series of chimeric human/rat CD59 mutant proteins.
Functional analysis revealed that the individual human to rat residue
substitutions F47A, T51L, R55E, and K65Q each produced a mutant human
CD59 protein with enhanced rat complement inhibitory activity with the
single F47A substitution having the most significant effect.
Interestingly, the side chains of the residues at positions 47, 51, and
55 are all located on the short single helix (residues 47-55) of CD59
and form an exposed continuous strip parallel to the helix axis. A
single human CD59 mutant protein containing rat residue substitutions
at all three helix residues produced a protein with species selective
activity comparable to that of rat CD59. We further found that
synthetic peptides spanning the human CD59 helix sequence were able to
inhibit the binding of human CD59 to human C8, but had little effect on
the binding of rat CD59 to rat C8.
 |
INTRODUCTION |
Complement activation can lead to the formation of the
proinflammatory and cytolytic complement membrane attack complex
(MAC)1 (4) (or C5b-9) on cell
membranes, and inappropriate MAC formation on host cell membranes has
been implicated in the pathogenesis of various autoimmune and
inflammatory diseases. Host cells are normally protected from the
effects of the MAC by CD59, a widely distributed membrane-bound glycoprotein.
The mature CD59 protein consists of 77 amino acids arranged in a single
compact cysteine-rich domain composed of two antiparallel
-sheets,
five protruding surface loops, and a short helix (1, 2). CD59 functions
by binding the terminal complement proteins C8 and C9 in the assembling
MAC and interfering with its membrane insertion (3-6). Because of
species selective recognition of C8 and/or C9 (3, 7), the activity of
CD59 is species restricted. However, species restriction is not
absolute, and the effectiveness of CD59 from different species against
heterologous complement varies.
Mutational analysis of CD59 has begun to define residues important for
its complement inhibitory function. Two basic strategies have been
used. In one approach, mutagenesis of human CD59 was used to determine
protein regions and amino acids essential for its inhibitory function
against human complement (8-10). These studies have putatively mapped
the human CD59 active site to one side of the protein that contains the
short helix. Most of the identified functionally important human
residues are well conserved between species and are located in the
vicinity of a hydrophobic cleft on the membrane-distal face of the
protein (8). In a second related approach, residues important for
species selective function have been identified by functional analysis
of chimeric human/animal CD59 proteins (11, 12). It is not clear
whether CD59 from different species share a common ligand binding site with species selective binding determined by other residues via indirect or allosteric mechanisms, or whether the residues involved in
CD59 species selectivity are directly involved in ligand binding.
In a quantitative study on the species selectivity of human and rat
CD59, it has been shown that human CD59 is not effective against rat
complement, but that rat CD59 is equally effective against rat and
human complement (11). Functional analysis of human/rat CD59 chimeric
proteins has indicated that the residues responsible for the species
selective activity of human CD59 lie between positions 40 and 66 in the
primary structure (11). Consistent with this conclusion, a more recent
study using chimeric human/rabbit CD59 indicated that sequence between
residues 42 and 58 determine human CD59 species selectivity (12). In
the current study, we identify individual residues involved in the
species selective function of human CD59.
 |
EXPERIMENTAL PROCEDURES |
Materials--
Human CD59 cDNA was a gift from H. Okada
(Nagoya City University, Nagoya, Japan) and the isolation of rat CD59
was described previously (13). The mammalian expression vector
pCDNA3 containing the G418 selection marker (Invitrogen, Carlsbad,
CA) was used for all DNA manipulation and recombinant protein
expression. All DNA primers used in PCR-based mutagenesis procedures
were synthesized by Integrated DNA Technologies. Inc. (Coralville, IA).
Recombinant CD59 proteins were expressed in Chinese hamster ovary cells
(CHO) that were maintained in Dulbecco's modified essential medium
containing 10% heat-inactivated fetal calf serum. Rabbit antiserum to
CHO cell membranes (14) was prepared as described (15). Anti-tag monoclonal antibody 2A10 directed against NANPNANPNA, a repeat domain
of Plasmodium falciparum circumsporozoite protein, was described previously (4). Fluorescein isothiocyanate-conjugated antibodies used for flow cytometry were from Sigma. Rat C8 was purified
as described (16). Recombinant soluble rat and human CD59 was expressed
in CHO cells and purified by affinity chromatography as described (2).
Human C8 was purchased from Advanced Research Technologies (San Diego,
CA). Four CD59 sequence specific peptides were synthesized and high
pressure liquid chromatography-purified (>80%) by Genemed (South San
Francisco, CA); peptide 1, RLRENELTY; peptide 2, FNDVTTRLRENELTY;
peptide 3, WKFEHCNFNDVTTRLRENELTY; and peptide 4, NFNDVTTRLRE. Normal
human serum was obtained from the blood of healthy volunteers in the
laboratory. Rat serum was purchased from Cocalico Biologicals
(Reamstown, PA).
Construction of Mutant CD59 Proteins--
Residue substitutions
in human CD59 were prepared by standard PCR mutagenesis techniques as
described (8, 11). In the first PCR amplification, 5' and 3' primers
matching an untranslated region of human CD59 and containing a
HindIII and ApaI site, respectively, were paired
with primers spanning the target site in which a rat amino acid codon
was substituted. Each final PCR product was digested with
HindIII and ApaI and was cloned into pCDNA3
expression vector for sequencing and expression. To quantitate the
relative expression of recombinant proteins, an oligonucleotide
encoding the tag-peptide sequence NANPNANPNA was inserted after the
human CD59 N-terminal Leu codon as described (8).
Expression of Recombinant Proteins--
CHO cells were
transfected with pCDNA3 constructs using
LipofectAMINETM according to the manufacturer's
instructions (Life Technologies, Inc.). Stable transfectants were
selected by the addition of G418 (400 µg/ml) 3 days after
transfection. After 14 days of selection, stable populations of CHO
cells each expressing similar levels of tagged recombinant protein were
sorted by flow cytometry by means of anti-tag monoclonal antibody 2A10
as described (8). At least three rounds of cell sorting were required
to obtain homogeneous cell populations expressing similar levels of
recombinant protein.
Flow Cytometry--
For quantitative analysis of tagged
recombinant protein expression, stably transfected detached CHO cells
were incubated with monoclonal antibody 2A10 (10 µg/ml) for 30 min at
4 °C. Cells were then washed, and incubated with
fluorescein-conjugated anti-mouse IgG for 30 min at 4 °C. Cells were
then washed again, fixed with 2% paraformaldehyde in
phosphate-buffered saline, and analyzed using a Becton Dickinson
FACScan. All incubations and washing were carried out in Dulbecco's
modified essential medium, 10% fetal calf serum. Cells for sorting
were fluorescently labeled as above but were not fixed. Sorting was
done in a Coulter Epics Elite with EPS sort module (Coulter Corp.,
Miami, FL).
Cell Lysis Assay--
Complement-mediated CHO cell lysis assays
were performed as described previously (8). Briefly, cells were
incubated in 20% heat-treated anti-CHO antiserum, washed once, and
exposed to 20% human or rat serum (either active or heat-inactivated). Cell lysis was determined by both trypan blue exclusion and by measuring the release of a preloaded fluorescent probe, calcein-AM (8).
Both methods gave similar results. Lysis was determined using sets of
homogenous cell populations expressing similar levels of rat CD59,
human CD59, or chimeric CD59 on their surface (Ref. 11 and also see above).
CD59 Binding Assay--
The ability of synthetic human CD59
peptides to inhibit the binding of CD59 to its ligand C8 was determined
using a previously described microtiter plate binding assay (4).
Briefly, human or rat C8 was coated onto microtiter wells, and the
respective binding of biotinylated human or rat CD59 was determined in
the presence of varying concentrations of peptide. CD59 (at final concentration of 20 µg/ml in phosphate-buffered saline containing 0.1% bovine serum albumin) and different concentrations of peptide were mixed before addition to C8 coated wells. All peptides were prepared as a 4 mg/ml stock solution in phosphate-buffered saline. Peptide 3 (see above) required a short sonication for solubilization. Binding of biotinylated CD59 was determined by means of
Extravidin-peroxidase soluble o-phenylenediamine substrate
system (Sigma). CD59 was biotinylated using EZ-link LC-biotin as
described by the manufacturer for the biotinylation of IgG, using the
same protein:biotin ratios (Pierce). Ratios were calculated based on
molecular weights of 18,000 and 155,000 for CD59 and IgG, respectively
(these ratios were determined to be important).
Molecular Modeling--
Modeling by homology and subsequent
analyses were performed with the ICM program developed for molecular
modeling and structure predictions by global restrained energy
optimization of arbitrarily constrained molecules (17, 18). The energy
is calculated with ECEPP/3 force field (19) extended by recently
developed solvation and side-chain entropic terms (18). The following
terms were included in the energy function: van der Waals and 1-4 nonbonded interactions, hydrogen bonding, torsion, electrostatic,
disulfide bond restraints, solvation energy, and side-chain entropy.
Cut off distance for truncation of van der Waals and electrostatic interactions was set to 7.5, and for hydrogen bond interactions it was
set to 3.0. The side-chain torsion angles were predicted by
simultaneous global optimization of the energy for all residues that
were different in the rat and human sequences. The biased probability
Monte Carlo-minimization method (18) was used for global optimization.
A region around insertion at the C terminus was predicted with the loop
prediction procedure described earlier (20). The final root mean square
deviation of the backbone atoms between the human and rat coordinate
sets was 0.64A. The molecular surface was built with the fast
analytical "contour buildup" algorithm (21).
 |
RESULTS |
Molecular Modeling and Comparative Analysis of Human and Rat
CD59--
Human CD59 is not an effective inhibitor of rat complement,
and previous functional analysis of chimeric human/rat CD59 proteins demonstrated that residues important for the species selective function
of human CD59 lie between positions 40-66 in the primary structure
(11). To identify the individual residues involved in human CD59
species selective function, we first built a model of rat CD59 on the
basis of the known structure of human CD59 and determined the location
and the distribution of nonconserved surface patches within the 40-66
amino acid region of human and rat CD59. The CD59 sequences can be
aligned with 45% sequence identity and only a single residue insertion
at the C terminus of the protein (Fig.
1). A model by homology was built and
refined using the ICM global energy optimization procedure (see
"Experimental Procedures"). Analytical molecular surfaces (21) for
both human and rat proteins were then built, and the surface shape and
distribution of surface patches of human and rat CD59 were analyzed
(see Fig. 2, A and
B). Residues 40-66 are shown in color in Fig. 2, and as
previously shown (11), they all map to one side of the molecule. The
region of CD59 shown in white represents the three-dimensional location
of the regions that do not appear to be involved in species selective
function (i.e. residues 1-39 and 66-77).

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Fig. 1.
Sequence alignment of human and rat
CD59. The 40-66 sequence previously determined to contain the
residues important for conferring species selective function (11) are
shown in bold. Identical residues are indicated by
vertical bars. Mature protein sequences are shown and the
C-terminal end of rat CD59 is predicted.
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Fig. 2.
Diagrams of human and rat CD59.
Panels A and B show a comparison of molecular
surfaces in the region identified as important for species selective
function (residues 40-66). The surface of regions 1-39 and 66-77
that are not important for species selectivity is colored
white. Conserved residues previously identified as
functionally important in human CD59 (8, 9) are colored
magenta and are not numbered. Side chains of all
nonidentical residues within the 40-66 sequence (and potentially
responsible for species selective activity) are colored red
(negatively charged residues), blue (positively charged
residues), yellow (hydrophobic residues), and
green (other residues). Backbone atoms of other residues, as
well as side chains of residues that are identical in human and rat
CD59 and therefore not important for species selectivity, are shown in
white. Panel C is a ribbon diagram of human CD59
showing the residues experimentally determined to influence species
selectivity.
|
|
Comparison of the models revealed pronounced differences between groups
of clustered residues within the 40-66 region. The two largest and
most conspicuously different groupings were six clustered residues at
positions 48, 52, 55, 56, 57, and 58 that will produce very different
surface patterns of electrostatic potential and hydrophobicity in the
human and rat proteins, and three residues at positions 41, 43, and 44 that form a cluster of difference in shape and electrostatic
properties. Other nonconserved groupings include residues 47 and 51, and residues 60, 62, and 66 that occur in a linear arrangement across
the membrane proximal face of CD59 (this arrangement is not apparent in
the views of CD59 shown in Fig. 2).
Mutational Analysis of CD59--
Human to rat amino acid
substitutions that result in acquisition of rat complement inhibitory
activity will identify functionally important residues. To determine
whether the candidate residue groups identified by model comparison
above are involved in the species selective function of human CD59, the
groups of residues were substituted for corresponding rat residues.
Some additional residues were also substituted so that all nonidentical
residues within the 40-66 sequence were accounted for. Further, some
substitutions were made for residues that are outside of the 40-66
sequence, but that neighbor human residues previously identified as
important for CD59 activity (8, 9). The mutant human CD59 proteins containing groups of substituted rat residues that were initially prepared and tested are shown in Fig. 3
(mutant series A). The proteins were recombinantly expressed on the
surface of CHO cells, and cell populations expressing similar levels of
protein were isolated (see "Experimental Procedures") and then
assayed for their susceptibility to human and rat serum. CD59
expression levels were quantitated using flow cytometry by means of an
epitope tag inserted at the N terminus of all recombinantly expressed
proteins as described previously (8, 11).

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Fig. 3.
Human to rat amino acid substitutions made in
chimeric CD59 proteins. The individual residue substitutions shown
in series B were selected based on functional data obtained from series
A mutant proteins.
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The data in Fig. 4 show that human CD59,
rat CD59, and all chimeric CD59 proteins are equally effective against
human complement, indicating that none of the substitutions had any
adverse effect on protein conformation and activity. When compared with
the activity of human CD59, the A1, A3, A6, and A7 chimeric proteins
provided enhanced protection against rat complement. The A6 and A7
proteins were about 25 and 70% as effective as rat CD59 against rat
complement, respectively (calculated based on the difference between
rat complement-mediated lysis of CHO cells expressing either human or
rat CD59) (Fig. 4). The A6 and A7 proteins were significantly more
effective against rat complement than the A1 and A3 proteins. Each of
the A6 and A7 chimeras contained only two substituted residues (Fig.
3), putatively identifying one or more human residues from a total of
four that primarily determine the species selective activity of human
CD59, i.e. Phe-47, Thr-51, Arg-55, and Lys-65. The human to
rat R55E substitution is common to the A1, A3, and A6 proteins, suggesting that this substitution is responsible for the slightly increased inhibitory activity against rat complement of the A1 and A3
proteins.

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Fig. 4.
Complement resistance of CHO cells expressing
human and rat CD59 and human-rat chimeric CD59 proteins. Stable
CHO cell populations expressing similar levels of recombinant protein
were exposed to 20% human serum (panel A) or rat serum
(panel B) and lysis percentage determined. A1-A7 represent
mutant human CD59 proteins containing groups of rat residue
substitutions (refer to Fig. 3). An N-terminal epitope tag that does
not effect CD59 function was used to measure cell surface expression of
CD59 (8, 11) (also see "Experimental Procedures"). Mean ± S.D. (n = 6).
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In a second series of mutations, each of the four candidate
functionally important human residues, and a residue not expected to
effect species selectivity (Lys-41), were individually substituted for
corresponding rat residues (see Fig. 3, series B). The data in Fig. 5 show that the individual
substitution of each candidate human residues with the corresponding
rat residue produced a protein with enhanced rat complement inhibitory
activity. The F47A substitution (B4 mutant) was by far the most
effective at enhancing the activity of human CD59 against rat
complement. This single rat residue substitution in human CD59 resulted
in a protein that was about 65% as effective as rat CD59 against rat
complement (Fig. 5). The T51L and R55E substitutions resulted in
proteins that each possessed close to 20% of rat CD59 inhibitory
activity. The K65Q substitution also appeared to display a small (about
10%), but statistically insignificant increase in activity against rat
complement. Nevertheless, the A6 protein that contains both an R55E and
K65Q substitution (Fig. 4) was slightly more effective against rat complement than an R55E substitution alone (Fig. 5). The single human
to rat residue substitution at position 41 (K41R) did not alter the
functional characteristics of human CD59, as predicted from functional
data obtained with the A2 protein (contains a K41R substitution). To
further confirm an important role for residue 47 in determining the
species selective function of human CD59, an additional mutant protein
was prepared containing a human to mouse substitution at residue
position 47 (protein B5 (F47G), see Fig. 3). We have shown previously
that human CD59 is not effective against mouse complement (22), and the
single F47G substitution produced a mutant protein possessing species
selective function that was quantitatively similar to the F47A (human
to rat) substitution (Fig. 5). None of the residue substitutions had
any effect on human complement inhibitory activity, indicating that all
recombinant proteins were correctly folded (Fig. 5).

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Fig. 5.
Complement resistance of CHO cells expressing
human CD59 containing single rat residue substitutions. Stable CHO
cell populations expressing similar levels of recombinant protein were
exposed to 20% human serum (panel A) or rat serum
(panel B) and lysis percentage determined. The B7 mutant
protein contained three human to rat residue substitutions (F47A, T51L,
and R55E, see Fig. 3.) Mean ± S.D. (n = 4).
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The positions and side-chain characteristics of the identified
functionally important human residues on the CD59 protein are shown in
Fig. 2. Interestingly, the side chains of the Phe-47, Thr-51, and
Arg-55 residues are all located in a strip on the same face of the CD59
helix (Fig. 2C). In a final mutant CD59 protein, each of the
three human helix residues that individually affected species selective
function were substituted with rat residues (mutant B7, Fig. 5). The
rat complement inhibitory activity of this mutant protein approached
that of rat CD59 (about 80% as effective) (Fig. 5), further indicating
that the identified helix residues, and in particular Phe-47, are the
principal determinants of human CD59 species selective function.
Effect of Synthetic CD59 Peptides on the Binding of CD59 to
C8--
We used a previously characterized microtiter plate binding
assay to determine whether synthetic human CD59 peptides from the
vicinity of the helix region could interfere with the binding of CD59
to its ligand, C8. We found two peptides, both spanning the helix
residue sequence, that modestly inhibited the binding of human CD59 to
human C8; a peptide to CD59 ratio of 320:1 inhibited binding by
30-40% (peptides 2 and 3, Fig. 6). In
contrast, the same peptides were much less effective at inhibiting the
binding of rat CD59 to rat C8. This data is consistent with the above mutagenesis data and suggests that the identified CD59 helix residue(s) are directly involved in the species selective binding of C8. A shorter
peptide containing the helix residues (peptide 4, Fig. 6), as well as a
peptide containing sequence C-terminal to the helix residues (peptide
1, Fig. 6), had little effect on the binding of CD59 to C8. A possible
explanation for the lack of inhibition by the short helix peptide is
that the N- and/or C-terminal helix residue extensions of the longer
peptides stabilize a structure that is more favorable for binding. In a
functional assay, the CD59 peptides were also tested for their effect
on human C5b-9-mediated hemolysis of human erythrocytes (as described
in Ref. 23). Peptide binding to C8 in the assembling C5b-9 complex at a
cell surface might interfere with the inhibitory effect of CD59, but
the peptides had no effect on C5b-9-mediated hemolysis (not shown).

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Fig. 6.
The effect of human CD59-specific peptides on
the binding of human and rat CD59 to their autologous C8 ligand.
Purified human (a) or rat (b) C8 was adsorbed
onto plastic (microtiter plates), and the binding of biotinylated human
(a) or rat (b) CD59 was determined in the
presence of various synthetic human CD59 sequence-specific peptides.
The amino acid sequences of the peptides are shown in the top
panel. The sequences span, or are in the close vicinity of, the
helix (residues 47-55) (representative data shown: panel a,
n = 3; panel b, n = 2).
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 |
DISCUSSION |
By measuring the inhibitory activity of chimeric human-rat CD59
proteins against human and rat complement, it was previously determined
that the species divergent 40-66 residue sequence contains the
residues important for the species restricted function of human CD59
(11). We have now identified individual residues within this region
that are responsible for human CD59 species selectivity. Individual
substitutions of the human residues Phe-47, Thr-51, and Arg-55 for
corresponding rat residues, each produced proteins with enhanced
activity against rat complement. The substitution of all three residues
in a single protein resulted in a CD59 protein (termed B7) with a
species selective activity that was quantitatively similar to that of
rat CD59; compared with the negligible activity of human CD59 against
rat complement, the B7 mutant was about 80% as effective as rat CD59
at inhibiting rat complement. A fourth residue, Lys-65, also appears to
contribute to the selectivity of human CD59 function, albeit to a
lesser degree than the three helix residues, and may at least partly
account for the slightly reduced activity of the B7 protein against rat
complement as compared with rat CD59. No other nonconserved residue
within the 40-66 sequence had any detectable effect on the species
selective function of human CD59.
The residues identified here as determinants of species selectivity are
distinct from previously identified human CD59 active site residues.
Site-directed mutagenesis of human CD59 (nonconservative substitution)
has indicated that residues Phe-23, Asp-24, Trp-40, Arg-53, Leu-54,
Glu-56, and Tyr-62 are important for human CD59 function (8-10). With
the exception of Tyr-62, these residues are located on the
membrane-distal face of CD59 in the vicinity of a hydrophobic cleft,
and with the further exception of residues Phe-23 and Glu-56, they are
conserved in human and rat CD59 (refer to Fig. 1). It is possible that
CD59 from different species possess a conserved ligand binding site,
and that nonconserved residues in CD59 proteins influence the
specificity of ligand binding via indirect or allosteric
mechanisms. Alternatively, CD59 residues involved in determining the
species selectivity may participate directly in ligand binding. We
identify Phe-47, Thr-51, and Arg-55 as being the residues primarily
involved in restricting human CD59 activity. In rat CD59, these
residues are replaced by alanine, leucine, and glutamic acid,
respectively. Interestingly, these three residues form a continuous
strip parallel to the axis of the CD59 helix and are exposed to the
solvent. It is therefore considered unlikely that the side chains of
residues Phe-47, Thr-51, and Arg-55 influence specificity through
affecting the relative position of the helix with respect to other
binding pocket residues. Rather, it seems more likely that these
residues are directly involved in ligand binding. The side-chain
differences between the three human and rat CD59 residues are such that
all three substitutions can potentially contribute to specificity,
provided that this phase of the short helix is involved in the direct
interaction. The phenylalanine and alanine side chains are both
hydrophobic but differ in size, the threonine and leucine differ by a
polar group and hydrophobic character, and arginine and glutamic acid have different charges although they share the same hydrophobic stem.
The single residue that contributes by far the most to human CD59
species selective function is Phe-47. A key role for Phe-47 in species
selectivity was further indicated by functional analysis of a human to
mouse F47G substitution (see "Results"). Perhaps a binding pocket
on the rodent C8/C9 ligands that can accommodate the rodent alanine and
glycine residues cannot accommodate the large phenylalanine residue in
the corresponding location on human CD59. Such an explanation is
compatible with the fact that rat (11) and mouse (22) CD59 are both
effective against human complement, whereas human CD59 does not
function effectively against rodent complement. Also compatible with
this "docking" concept is the previous result that a
nonconservative F47E mutation resulted in a human CD59 protein with
only a weak protective effect against human complement (9). It was
suggested from this finding that Phe-47 may be at the periphery of the
human CD59 active site. The positively charged residue Lys-65 in human
CD59 that is replaced by a polar Gln-65 in rat CD59 also had a small
effect on species selectivity. Lys-65 is positioned next to the
conserved and functionally important residue Asp-24 (9) and is located
at one end of the hydrophobic cleft that may be important for
complement ligand binding (see above). The current data does not
exclude the possibility that the location of residues that determine
species selectivity may differ in different CD59 proteins.
Nevertheless, consistent with the current data, a recent analysis of
the species selectivity of chimeric human/rabbit CD59 proteins
indicated that species selectivity is determined solely by sequence
contained between residues 42 and 58 of human CD59 (12).
Analysis of the patterns of species selectivity of various cloned CD59
proteins is consistent with functional contributions from residues at
positions 47, 51, and 55. Mouse and rat CD59 display similarities in
their pattern of species selectivity with regard to homologous and
human complement, and both have a small side chain in position 47 (glycine and alanine, respectively), a long hydrophobic side chain in
position 51 (leucine and methionine, respectively), and a conserved
residue at position 55 (glutamic acid) (refer to Fig. 1). In contrast,
human, primate, and pig CD59 are all effective against human but not
rodent complement. In CD59 from these species, the Phe-47 is conserved,
the residue substitution threonine to serine in position 51 preserves
the side-chain hydroxyl group, whereas human Arg-55 is replaced in primate and pig CD59 by a smaller side chain, rather than to an oppositely charged residue (as in rodent CD59). Rabbit CD59, however, which does not provide effective protection from human complement (12),
contains an oppositely charged residue at position 55 (glutamic
acid instead of arginine) and may account for its incompatibility with human CD59, despite similarities in positions 47 and 51.
Recombinant soluble complement inhibitors based on membrane regulators
of complement activation are effective at suppressing inflammation and
disease pathology in a variety of animal models, and an understanding
of the molecular basis for CD59 function may provide the rationale for
the design of efficient soluble MAC inhibitory constructs for clinical
application. Inhibiting the terminal pathway of complement but leaving
the activation pathway intact may offer significant clinical advantages
in diseases in which the MAC plays an important role. This is because
products of the complement activation pathway play important roles in
immunity to infection and in immune complex catabolism. Evidence
indicates that an effective CD59-based inhibitor will also provide
efficient protection from complement-mediated hyperacute rejection of
xenotransplanted tissue (24, 25). Transgenic pig organs expressing high
levels of human CD59 are protected from human complement and show
prolonged survival when transplanted into primates (26, 27). The
identification here of the residues important for species selectivity
and the conservation of these residues in human and pig CD59 support
the view that the level of CD59 expression will be more important than
the species of CD59 in prolonging pig to human graft survival (28, 29).
Finally, defining the functional site(s) of CD59 may also assist in the
design of inhibitors of CD59. Inhibiting CD59 function on the surface
of tumor cells may prove effective in anti-tumor
complement-dependent immunotherapy.
 |
ACKNOWLEDGEMENTS |
We thank Dr. John Hirst for performing flow cytometry.
 |
FOOTNOTES |
*
This work was supported by Grants from the National
Institutes of Health (AI 34451), the American Heart Association, and
Department of the Army (DAMD179717273) (to S. T.), and by the
Wellcome Trust (to B, P. M.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
To whom correspondence should be addressed: New York University
Medical Center, Dept. of Pathology, MSB 126, 550 First Ave., New York,
NY 10016. Tel.: 212-263-8514; Fax: 212-263-8179; E-mail: tomlis01{at}popmail.med.nyu.edu.
 |
ABBREVIATIONS |
The abbreviations used are:
MAC, membrane attack
complex;
CHO, Chinese hamster ovary;
PCR, polymerase chain
reaction.
 |
REFERENCES |
-
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