From the
Two pairs of chimeric, domain-switched immunoglobulins with
identical murine, anti-dansyl (5-dimethylaminonaphthalene-1-sulfonyl)
variable domains have been generated, employing as parent antibodies a
human IgM and a mouse IgG2b. The first pair of chimeric antibodies
µµ
Complement fixation by IgG and IgM antibodies involve different
mechanisms. IgG antibodies have an exposed complement binding site
located in the C
IgM is a naturally occurring
polymer synthesized in two forms containing either five IgM monomers
and a J chain, or six IgM monomers and no J chain, arranged in a ring
with the Fab`s pointing outward. Each IgM monomer
(µ
Since multiple binding sites for C1 should be present on
pentameric and hexameric IgM, a single IgM attached to a red blood cell
is sufficient for complement binding and activation (Borsos and Rapp,
1965a, 1965b; Ishizaka et al., 1968). However, since unbound
IgM binds C1q and C1 weakly (Poon et al., 1985; Poon and
Schumaker, 1991) and does not consume complement, it is generally held
that efficient interaction with C1 requires conformational changes
among the IgM monomers which only occur upon interaction with antigen.
A ``distortional'' model for complement activation in which
the Fab
The technique of domain switching has been
profitably employed to examine the role of the hinge and C
Construction of Plasmids and Production of Antibodies The plasmid pUC
SDS-PAGE was performed according to Laemmli (1970)
using a Protean II minigel apparatus (Bio-Rad). Western blots performed
as described (Burnette, 1981; Towbin et al., 1979) were
blotted with goat anti-human IgM (Sigma) or rabbit anti-mouse IgG2b
(Miles Scientific, Naperville, IL) followed by horseradish
peroxidase-conjugated anti-goat or anti-rabbit IgG antibodies, and
bands were detected using the chemiluminescence ECL reagents (Amersham
Corp.).
Analytical ultracentrifugation was performed in the model E
analytical ultracentrifuge (Beckman) with an UV optical system
interfaced to an IBM PC computer. Sedimentation coefficients were
calculated from measurements of the boundary positions at different
times. The data are displayed as sedimentation distribution curves in
which migrating boundaries were converted to ``peaks''
(Schumaker and Schachman, 1957). Complement Assays
Sedimentation coefficients and the relative amounts
of monomer and polymer formed by each chimera were determined by
analytical ultracentrifugation () and confirmed by
nonreducing SDS-PAGE (Fig. 2). The correspondence between the
results obtained with the two methods indicated that the 7 S monomers
and the 18 S polymers were formed by covalent inter-heavy chain
disulfide bonds. In murine IgG2b, these inter-heavy chain disulfide
bonds are located in the hinge, whereas in the human IgM, they were
located at Cys
Direct lysis of dansyl-coupled
sheep red blood cells employed a large excess of NHSC and gpSC of
comparable hemolytic activity. Results shown in Fig. 3indicate
that recombinant human IgM was equally active with NHSC and gpSC,
whereas recombinant mouse IgG2b was more active with gpSC. Polymeric
IgM also activated complement at much lower concentrations than
monomeric IgG as had been reported previously using polyclonal
antibodies (Ishizaka et al., 1968; van der Zee et
al., 1986).
For the chimeric
antibodies containing C
The chimeric antibodies were also examined in an indirect assay
which measured complement consumption by antibody-antigen complexes. In
this assay, diluted NHSC or gpSC containing nanomolar C1 and an optimal
amount (4-8 n
M) of dansyl polylysine were incubated with
varying concentrations (1-100 n
M) of mAb. The amount of
complement remaining at the end of the incubation period was then
determined using a separate hemolytic assay. The results of these
complement consumption assays are shown for NHSC (Fig. 4) and gpSC
(Fig. 5). These data show
Murine IgG2b and the human IgM were employed as the parent
genes for generating through exon exchange the four constructs encoding
the chimeric immunoglobulins. Within experimental error, the two
parental IgG2b and IgM proteins were entirely 6.9 S monomer and 18.8 S
polymer, respectively, as shown by analytical ultracentrifugation and
gel electrophoresis (Fig. 2). Both of these antibodies, purified
by affinity chromatography, appeared uncontaminated by significant
quantities of other proteins. For the parent IgM, the two intersubunit
disulfides were probably formed, because mutant IgM lacking either of
these have been reported partially defective in assembly (Davis et
al., 1989b).
The parental immunoglobulins behaved as expected
in the direct lysis experiments employing sheep red blood cells coated
with dansyl-coupled, anti-sheep (Fab`)
The parental immunoglobulins also behaved as
expected in the consumption assays employing soluble dansylated
polylysine as antigen. The mechanisms involved in consumption have not
been fully clarified; however, in contrast to the direct lysis
experiments where only a small fraction of the added complement need be
activated to cause cell lysis, the complement consumption experiments
required that a significant fraction of the added complement be
consumed for the difference to be measured. Roughly equivalent amounts
of IgM antibody and complement C1 were added in these experiments, that
is, 100 µl of a 1/100 dilution of serum contained about 2 pmol of
C1, and 100 µl of 1 µg/ml IgM contained about 1.1 pmol. Thus,
if each IgM tightly bound a single molecule of C1 rendering it
unavailable for the subsequent lysis experiments, about 50% of the
complement would have been ``fixed'' or consumed; C1 binding
may have been the principle mechanism of complement fixation at 4
°C where activation of C1 is expected to be minimal. Alternatively,
and especially at 37 °C, the C1 could have been activated, leading
to activation and depletion of essential components of the classical
pathway subsequently employed in the lytic step of the assay. It has
been observed previously that 7 S antibodies react much more
effectively at 4 °C than at 37 °C, whereas 19 S antibodies are
relatively more reactive at 37 °C than at 4 °C (Cunniff and
Stollar, 1968). Our results for the parental immunoglobulins are in
complete agreement with these earlier studies, for it was found that
the IgM parent consumed complement much more effectively at 37 °C
than at 4 °C, whereas for the IgG2b parent, the converse was true,
(Figs. 4 and 5).
Monoclonal antibody
Mutant
Chimera µµ
In contrast to murine
IgM, which requires the intersubunit disulfide bonds linking
Cys
Chimera µµ
The high polymer
content (71%) of
Binding affinities between the polymeric chimeras and
C1 were reflected by the enhanced sedimentation coefficients of the
leading peaks observed during the ultracentrifuge analyses
(Fig. 7), the affinity for C1 being greatest for
µµ
What conclusions may be drawn from these studies
concerning the mechanism of complement binding and activation and the
functions of the various domains? First, the data support an
associative model for complement activation for IgG, for the cluster of
C
The results presented here
coupled with previous studies allow us to formulate a model for
complement binding by IgM. In the absence of antigen, IgM has been
shown to bind two molecules of C1q, one on each side of the symmetrical
IgM disk, in a noncooperative manner (Poon et al., 1985). That
the binding was noncooperative would seem to rule out a two-state model
in which the IgM flickers between a binding and a nonbinding
conformation, since in that case, the first bound C1q would assist in
maintaining the binding conformation for the second C1q, yielding a
cooperative interaction. Noncooperative binding implies an alternative
model in which weak, independent binding sites for C1q were always
exposed on both sides of the IgM disk.
If weak binding sites were
always present, then the required conformational change probably
aligned additional residues to form a tight binding site. Residues on
both Cµ3 and Cµ4 may be required to form a binding site, since
chimera
Although this low resolution model
contains speculative elements, it may prove useful in the design of
further experiments to locate the complement binding site and elucidate
the conformational change required for the activation of complement by
IgM.
µ and
µ
was generated by
switching the Cµ3 and C
2 domains between IgM and IgG2b. The
second pair of chimeras µµ
and
µµ
were formed by switching both Cµ3 and Cµ4 with C
2 and
C
3. SDS-polyacrylamide gel electrophoresis and analytical
ultracentrifugation showed that over half (57 and 71%) of the two
chimeric antibodies possessing the Cµ4 domain and tail piece formed
disulfide-linked IgM-like polymers. In contrast, the two chimeric
antibodies lacking the Cµ4 domain were almost entirely monomeric.
Both monomeric chimeras had reduced ability to activate complement. The
chimera
µ
had no activity under any of the assay
conditions, whereas µµ
caused only a small amount of
cell lysis but was fully active in consuming complement at 4 °C.
The polymeric chimera
µµ was much less active than
IgM, bound C1 weakly and caused some cell lysis but consumed little
complement with soluble antigen. The polymeric chimera
µµ
µ bound C1strongly and was the most active antibody
in all assays, even more active than the parental IgG2b and IgM
antibodies; it was the only antibody that exhibited antigen-independent
activity. The results suggest that Cµ3 alone does not constitute
the complement binding site in IgM but requires both
Cµ
and Cµ
for full activity.
2 domain. The participating amino acid residues,
highly conserved among human, rat, mouse, guinea pig, and rabbit IgGs,
include Glu
, Lys
, and Ly
(Duncan and Winter, 1988), located on the fy3 strand of
sheet on the Fy face of the domain (Beale and Feinstein, 1976;
Deisenhofer et al., 1976; Edmundson et al., 1975).
Although C1q binds weakly to monomeric IgG (Schumaker et al.,
1976), strong multivalent binding to a cluster of IgG molecules
attached to an antigen array is believed to be responsible for C1
activation (Hanson et al., 1985; Hoekzema et al.,
1988; Hughes-Jones et al., 1983; Metzger, 1974, 1978). This
requirement for multivalent binding is referred to as the
``associative'' model.
L
) is homologous in architecture to IgG,
although instead of a hinge, the IgM possesses an extra domain,
designated Cµ2, and is much less flexible than IgG. Although older
evidence implicated the Cµ4 domain in complement fixation (Bubb and
Conradie, 1978; Hurst et al., 1975; Johnson and Thames, 1976),
more recent studies (Arya et al., 1994; Wright et
al., 1988) have identified residues on Cµ3 that participate in
C1 binding (Arya et al., 1994), and the evidence suggests that
the C1 binding site is not homologous to the site on C
2 of IgG.
Although the folding of the polypeptide backbone in Cµ3 may be
modeled (Arya et al., 1994), no crystallographic data are
available, and the orientation of Cµ3 in the IgM molecule is
unknown.
are bent toward the cell surface when IgM binds
multivalently to a cluster of epitopes, converting the IgM
``starfish'' to the ``table'' or
``staple'' conformation with exposed C1 binding sites has
been proposed (Feinstein and Munn, 1966, 1969; Feinstein et
al., 1983).
2 domain
in complement fixation (Norderhaug et al., 1991; Tan et
al., 1990; Tao et al., 1991), to map the site of
interaction of mouse IgE with murine Fc
RI (Weetall et
al., 1990), and to dissect the interaction of individual Ig
domains with human Fc receptors (Shopes et al., 1990). In the
present study, domain exchange between a human IgM and a mouse IgG2b
has been employed in a study of complement fixation and activation. A
preliminary report of a similar study using mouse IgM has appeared
(Chen et al., 1994). The present studies indicate that the
context of the C1 binding site is important in determining the
efficiency of complement activation.
was constructed by subcloning into pUC19 (New
England BioLabs) a 3.7-kilobase pair XbaI- BglII
fragment of genomic DNA containing C
1, hinge, C
2, and C
3
of mouse IgG2b. The plasmid pUCµ was generated by subcloning the
3.6-kilobase pair XbaI fragment of genomic DNA containing
Cµ1, Cµ2, Cµ3, and Cµ4 of human IgM from a plasmid
derived from pN-X-µTNP (Boulianne et al., 1984) into
pUC19. Unique NotI and MluI sites were generated by
partial digestion of these plasmids with the appropriate restriction
enzymes, isolation of singly cut plasmids by agarose gel
electrophoresis, and ligation with NotI or MluI
linkers, containing the appropriate adaptor and about 15 base pairs of
the intron sequence (synthesized using the Gene Assembler, Pharmacia
Biotech Inc.). A NotI site was introduced at the SacI
site in the intron 5` to C
2, and an MluI was introduced
at the PpuMI site in the intron 3` to C
2. (Fig. 1,
construct i). NotI and MluI sites were
introduced at the PpuMI sites flanking the Cµ3 domain.
(Fig. 1, construct ii).
Figure 1:
Construction of the heavy
chain constant region genes for the production of chimeric antibodies.
The heavy chain exons of mouse IgG2b are represented by empty boxes and of human IgM by filled boxes. The four exons in their
respective positions from left to right represent
C1, hinge, C
2, and C
3 of IgG2b and Cµ1, Cµ2,
Cµ3, and Cµ4 of IgM. Restriction sites used for domain
switching are indicated in the appropriate introns: NotI
( N), MluI ( M), and SalI
( S). To aid identification, a four Greek letter nomenclature
has been used; for example,
µ
represents the H-chain
of mAb iii consisting of
C
1-hinge-Cµ3-C
3.
The NotI and
MluI sites were used together with the unique SalI
sites to generate four additional constructs, labeled iii, iv,
v, and vi in Fig. 1. Each of these constructs was
then joined to the mouse anti-dansyl(
)
V
from hybridoma 27-44 (Oi et al., 1984) in an
expression vector pSV2Hgpt-DNS-VH. Using electroporation (Shin and
Morrison, 1989), the resultant vector was transfected into TSW2, an
immunoglobulin nonproducing mouse myeloma cell line previously
transfected with a light chain construct incorporating the
anti-dansyl variable domain from hybridoma 27-44 joined to a
human C
constant domain. Transfectants were selected with
mycophenolic acid (Life Technologies, Inc.), and clones were screened
by enzyme-linked immunosorbent assay using DNS-bovine serum
albumin-coated plates and alkaline phosphatase-conjugated goat
anti-human C
(Sigma). After subcloning, high titer
antibody-producing clones were expanded in 1-liter roller bottles in
Iscove's modified Dulbecco's medium containing 1%
immunoglobulin-depleted calf serum (Hyclone Laboratories, Logan, UT). Purification and Physical Analyses of Antibodies Anti-dansyl antibodies were purified by affinity chromatography (Dangl,
1986; Schneider et al., 1987) using a 1-ml column of
EAH-Sepharose 4B (Pharmacia) coupled to a dansyl analog via
2-dimethylaminonaphthyl-5-sulfonyl chloride (Molecular Probes, Eugene,
OR). The elution hapten, 2-dimethylaminonaphthyl-5-sulfonyl caproate,
was made by overnight incubation of equal volumes of 200 m
M 2-dimethylaminonaphthyl-5-sulfonyl chloride in acetone and 250
m
M
-aminocaproic acid (Sigma) in 250 m
M carbonate, pH 12, and used directly at a dilution of 3 m
M with phosphate-buffered saline. Eluted antibodies were collected
in 1-ml fractions and the peak fractions, as judged by reducing
SDS-PAGE, pooled and dialyzed in the cold with at least 24 changes of
100
volume of TBS, pH 7.8. Antibody concentrations were
calculated from A
, using E (1 cm, 1
mg/ml) = 1.4 and 1.2 for mAb i and ii, respectively, and 1.3 for
all chimeras.
Direct Lysis of Dansyl-coupled SRBC
SRBC were coupled
with dansylated rabbit anti-SRBC IgG(Fab`)according to
Aase and Michaelsen (1991). (Fab`)
prepared by digesting
anti-SRBC antibodies (Cordis, Miami, FL) with pepsin was treated with
dansyl-chloride according to Weber (1952) at an input of 23 mol/mol
(Fab`)
, and a dansyl
-(Fab`)
was
obtained based on spectral analysis (Hardy, 1986). The assay procedure
was similar to that of Davis et al. (1988): 100 µl of
SRBC, coupled with an optimal amount of the dansylated antibody
fragments, at 1.5
10
/ml in TBS
(TBS containing 0.5 m
M MgCl
, 0.15 m
M CaCl
, 1 mg/ml bovine serum albumin) was incubated for
15 min at 37 °C with the indicated concentration of monoclonal
antibody and then 20 µl of guinea pig serum (gpS) diluted 1/20 or
normal human serum (NHS) diluted 1/10, both preadsorbed with SRBC, were
added and the mixture incubated for an additional 40 min. The mixture
was then diluted to 1 ml with cold TBS
,
centrifuged, and assayed for cell lysis by absorbance at 412 nm.
Controls without mAb included tubes without serum, tubes with serum,
and tubes with serum plus water as diluent.
Complement Consumption
For the soluble antigen,
polylysine with an average of 560 monomer units (Sigma) was dansylated
with an input of 28 mol of dansyl-chloride/mol polylysine, and
approximately 17 mol of dansyl were incorporated. Consumption assays
were performed according to Stollar (1978), with minor modifications.
For the complement consumption step, 100 µl of TBScontaining the indicated concentrations of mAb and antigen, and
gpS (1/100) or NHS (1/50) was incubated either at 37 °C for 1 h or
at 4 °C for 18-21 h. Then SRBC optimally sensitized with
hemolysin, EA, were added, the mixture diluted to 0.7 ml (final EA
concentration, 1
10
ml
), and
further incubated at 37 °C for 40 min, centrifuged, and assayed for
cell lysis by absorbance at 412 nm. The amounts of EA and complement
had been predetermined such that, in the absence of consumption, cell
lysis was 80-90% complete. Controls included tubes without mAb
and tubes without antigen. Results reported for complement consumption
employed a single concentration of polylysine, predetermined using
fixed concentrations of mAb i and mAb ii.
Purification and Production of Chimeric
Antibodies
Each of the gene constructs shown in Fig. 1was
used to transfect TSW2 cells, a cell line producing a chimeric light
chain composed of the murine anti-dansyl variable domain and human
C. Antibodies were isolated from the cell culture medium by
affinity chromatography, as described under ``Experimental
Procedures,'' yielding in each case 1-2 µg of purified
protein/ml of culture medium. On reducing SDS-PAGE, each antibody
exhibited only two bands: a 23-kDa light chain and a heavy chain whose
relative size was consistent with the expected product. Western blots
with anti-human-µ and anti-mouse-
2b also indicated that domain
switching had occurred in each of the chimeric antibodies
().
of Cµ2, forming an intrasubunit
disulfide, and at Cys
of Cµ3 and Cys
of
the Cµ4 tail piece, forming intersubunit disulfides (Davis et
al., 1989b). Thus, the parental IgG2b (
) and
IgM (µµµµ) were assembled as monomer and polymer,
respectively, as expected. For the different chimeras,
µ
yielded practically all monomer, although it
contained Cys
; µµ
µ which lacked
Cys
, but contained Cys
, yielded about 43%
monomer and 57% polymer; µµ
, possessing only the
intrasubunit Cys
, produced only monomer, as expected, and
although
µµ contained both Cys
and
Cys
, it was nevertheless defective in polymer assembly,
since 29% was secreted as monomer. In summary, only the mAb's
containing Cµ4 with Cys
present in the tail piece
gave rise to appreciable amounts of polymer, with µµµµ
>
µµ > µµ
µ.
Figure 2:
Analytical ultracentrifugation and
SDS-PAGE. A, sedimentation distribution patterns of
affinity-purified mAb i-vi in TBS; protein concentrations ranged
between 0.1 and 0.2 mg/ml. In each pattern, the left and
right vertical bars denote 7 and 18 S, respectively.
B, Coomassie Blue staining of 4% nonreducing SDS-PAGE; 15
µg of each recombinant mAb were loaded along with a nonspecific
mouse IgG2b ( G) and human IgM ( M) and molecular mass
markers of 200 and 116 kDa (flanking lanes). C, distribution
patterns of sedimentation at high speed showing the 10.2 S component in
µ
. In each pattern, the left and right
vertical bars denote 7 and 10 S.
Activation of Complement by Chimeric
Antibodies
Since the expressed chimeric antibodies possessed
identical anti-dansyl Fab, any differences in complement activation
should reflect the differences in the constant regions. Differences in
complement activation were studied employing both human (NHSC) and
guinea pig (gpSC) complement, and complement consumption assays were
performed at both 37 and 4 °C.
Figure 3:
Complement activating ability of
recombinant antibodies assayed by direct lysis of DNS-coupled SRBC.
A, using normal human serum complement; B, using
guinea pig serum complement. 100 µl of mixtures containing 1.5
10
/ml DNS-coupled SRBC and the indicated
concentrations of antibodies were incubated 15 min at 37 °C, then
20 µl of diluted serum were added to a final dilution of 1/10 of
NHSC or 1/20 of gpSC, and lysis was allowed to proceed for 40 min at 37
°C. The affinity-purified antibodies were mixtures of monomers and
polymers, as shown in Fig. 2 and Table I:
, IgG2b;
, IgM ;
,
µ
;
, µµ
µ;
,
µµ
;
,
µµ.
For the chimeric antibodies containing Cµ3,
µ
was inactive in the cell lysis assay, whereas
µµ was more effective than the parental IgG2b with
NHSC, but less effective than parental IgM.
2, the data show µµ
µ to be
more hemolytically active with both NHSC and gpSC than either parental
antibody. This monoclonal antibody was 57% polymer, and presumably it
was the polymer which possessed the strong lytic activity, but this was
not determined. In marked contrast, the ability of the monomeric
µµ
to promote cell lysis was barely detectable.
µ
to be inactive in
all assays, whereas
µµ exhibited slight activity only
with NHSC at 37 °C. It is noteworthy that compared with IgM,
µµ was much less active in this consumption assay
than in promoting lysis at the cell surface. µµ
,
barely active in the direct assay with both NHSC and gpSC, also was
barely active in the indirect assay at 37 °C with NHSC, but showed
no activity with gpSC; however, its activity was comparable with IgG2b
when assayed at 4 °C. In the indirect assay, µµ
µ
was again the most active of all of the antibodies. Recombinant human
IgM was most active with NHSC at 37 °C; in contrast, recombinant
mouse IgG2b was most active with gpSC at the lower temperature. These
differences were reported previously for polyclonal IgM (Cunniff and
Stollar, 1968; van der Zee et al., 1986) and for monoclonal
IgG2b (Dangl, 1986; Gee et al., 1981). Over the range of
antibody concentrations employed in these assays, only
µµ
µ was capable of complement consumption in the
absence of antigen (Fig. 6).
Figure 5:
Complement consumption ability assayed
employing DNS-Lys
as soluble antigen with
guinea pig serum as the source of complement. A, complement
consumption at 37°; B, complement consumption at 4 °C.
The assay conditions were identical to those described in the legend to
Fig. 4, except that a 1/100 dilution of the gpSC was used.
,
IgG2b;
, IgM;
,
µ
;
,
µµ
µ;
, µµ
;
,
µµ.
Figure 6:
Complement consumption ability of
µµµ assayed in the absence of antigens. A,
NHSC; B, gpSC.
, complement consumption at 37 °C;
▾, complement consumption at 4 °C. Conditions were identical
to those described in the legends to Figs. 4 and 5 except that
DNS
-Lys
was
omitted.
Ultracentrifuge Studies of the Binding of C1 to the
Polymeric Chimeras in the Absence of Antigen
Solutions
containing human C1 (0.17 mg/ml or 0.22 µ
M) and
recombinant IgM (0.1 mg/ml or 0.11 µ
M polymer) or
µµµ (0.16 mg/ml or about 0.09 µ
M polymer)
or
µµ (0.16 mg/ml or about 0.14 µ
M polymer), at two ionic strengths, were studied by analytical
ultracentrifugation (Fig. 7). At 0.12
M ionic strength,
binding of 18.0 S C1 by the 18.8 S parental IgM gave rise to a faster,
broader 22 S (median) boundary, probably representing a mixture of the
unassociated species reversibly equilibrating with weakly associated
1:1 and 2:1 (C1:IgM) complexes (Poon and Schumaker, 1991). When mixed
with C1, µµ
µ showed three peaks in the
ultracentrifuge, probably representing the 7.8 S unassociated
(trailing) monomer, the excess 18 S C1, and a 30.2 S boundary probably
representing a mixture of strongly associated 1:1 and 2:1 C1:polymer
complexes. The mixture of
µµ and C1 yielded two
peaks, probably the 7.2 S unassociated monomer and a 19 S peak
representing unassociated species in equilibrium with very weakly
associated complexes. When the ionic strength was increased only
moderately from 0.12 to 0.15 (Fig. 7 B), the binding was
practically abolished with
µµ, and much diminished
with IgM, probably reflecting the 6th power dependence on ionic
strength previously reported for the binding between C1q and IgM (Poon
et al., 1985). In addition, the decreased sedimentation
coefficient for C1 at the higher ionic strength (16.6 S versus 18.0 S) and the pronounced trailing boundary indicated that the C1
complex was less stable at the higher ionic strength, probably
dissociating reversibly into its 10 S C1q and 8.5 S
C1r
C1s
subcomponents. This enhanced
dissociation of C1 would have contributed to the decreased binding of
C1 by weakly binding IgM and
µµ. In contrast, for the
mixture of C1 and µµ
µ, complex formation was almost
unaffected by the increase in ionic strength, reflecting a much tighter
binding between this chimera and C1.
Figure 7:
Analytical ultracentrifugation of mixtures
of human C1 and IgM, µµµ, and
µµ at
ionic strengths of 0.120
M ( A) and 0.150
M ( B). The top panels show C1 without addition of
antibody; the remaining panels represent mixtures of the indicated mAb
with C1 ( solid lines) and without C1 ( dotted lines).
The concentrations employed were: C1, 0.17 mg/ml (0.22
µ
M); IgM, 0.09 mg/ml; µµ
µ and
µµ, 0.16 mg/ml. Vertical bars indicate, from
left to right, 7, 18, and 27
S.
and guinea pig
complement. The IgM parent achieved 50% cell lysis at 0.2 µg/ml
IgM, whereas the IgG2b parent reached 50% at about 3 µg/ml of
protein. The greater efficiency of IgM compared with IgG on a weight
basis in cell lysis probably reflects the requirement for only a single
IgM to establish a complement activation site, whereas at least two IgG
must be bound in close proximity (Borsos and Rapp, 1965a, 1965b;
Ishizaka et al., 1968). In cell lysis experiments, not only
must complement be bound and activated, but additional interactions
between antibody and C4b appear to be critical as well (Ziccardi,
1986); clearly both parental antibodies were capable of supporting all
the required reactions.
µ
was
approximately 95% monomer and only 5% dimer (), even though
the Cµ3 domain contained Cys
used for one of the two
intersubunit disulfide bridges found in wild-type IgM polymer. In the
mutant IgM-Ser
, Cys
was used to produce the
20-40% pentamer/hexamer observed (Davis et al., 1989a).
Absence of polymer formation with
µ
may reflect the
lack of the tail piece, which appears to play an essential role in
polymer formation (Baker et al., 1986; Davis et al.,
1988, 1989a). However, IgG3 lacking a tail piece but mutated to contain
Cys
(corresponding by sequence homology to Cys
in Cµ3), although secreted predominately as monomer, formed a
mixed population of oligomeric antibodies ranging in size from monomer
to hexamer (Smith and Morrison, 1994).
µ
was the only one of the six antibodies studied that was completely
unable to activate or consume complement in either the direct or
indirect assays (Figs. 3-5) as had previously been observed with
a similar
µ
chimera (Chen et al., 1994).
Upon aggregation by antigen, this monomeric antibody would be expected
to bind and activate complement if a complement binding site were
exposed on the Fc. It would appear that the failure to activate
complement reflected the lack of a functional complement binding site
on the Cµ3 domain.
µ which lacked
Cys
of IgM which participates in the intersubunit bonds
was secreted as 57% polymer and 43% monomer. The amount of polymer was
somewhat higher than might have been expected given that the mutant
IgM-Ser
secreted only 20-40% polymer (Davis et
al., 1989b). Mutant µµ
µ was more active than
either parental antibody in both the direct and indirect complement
assays and was the only monoclonal that was active in the absence of
antigen (Fig. 6). This last observation implies that the
complement binding site on C
2 must have been exposed in the
absence of antigen on the IgM-like polymer. Moreover, comparison of
Figs. 4 and 5 with Fig. 6shows that the activity was increased
4-20-fold upon addition of dansylated polylysine. It seems
possible that the IgM architecture, especially Cµ2 and Cµ4,
partially concealed the complement binding site, and that full exposure
was restored when µµ
µ was distorted upon multivalent
interaction with the dansylated polylysine.
residues for significant complement activity (Davis
et al., 1989b), the presence of these disulfides clearly was
not essential for the strong complement activity displayed by chimera
µµ
µ. This result is similar to what is observed for
polymeric IgG3 containing the IgM tail piece (Smith and Morrison,
1994), in which introduction of the analogous disulfides neither
enhanced nor inhibited activity.
which
lacked the tail piece and both Cys
and Cys
,
and was secreted only as a 7.2 S monomer, did not cause significant
cell lysis unless high concentrations were employed (Fig. 3).
However at 4 °C, this antibody consumed complement almost as well
as IgG2b. These results were surprising, since µµ
contained the same preformed complement binding site on C
2 as the
monomeric IgG2b and presumably the polymeric form of
µµ
µ, both of which effectively lysed red blood cells.
Perhaps monomeric chimera µµ
bound C1 but failed to
promote other steps essential for the lytic chain.
µµ probably reflected the
availability of both Cys
and Cys
for
intersubunit disulfide bridges. However, since the remaining 29% of
this protein was secreted as monomer, it may be concluded that domain
interactions involving Cµ1 and Cµ2 in addition to those
involving Cµ3 and Cµ4 were involved in the complete assembly of
the IgM polymer. Chimera
µµ promoted cell lysis
(Fig. 3) in the presence of normal human serum, but the curve
reached a maximum at 50% lysis, then leveled off and finally dropped
toward zero, probably reflecting the transition between polyvalent and
monovalent binding expected to occur at high antibody concentrations
(Pruul and Leon, 1978). This phenomenon was also observed for
complement consumption by the parental IgM (Figs. 4 A and
5 A). Chimera
µµ was about an order of
magnitude less effective than the parental IgM in promoting cell lysis
with human serum, and much less complement consumption was found for
this chimera with the soluble dansylated polylysine antigen, possibly
reflecting a defect in the stabilization of an activating conformation
because of the substitution of an intact Cµ2 domain by an IgG
hinge. A similar conclusion was reached in a study of the effects of
limited denaturation of Cµ2 in equine IgM by heat (Siegel and
Cathou, 1981).
µ, followed by IgM and weakest for
µµ. It is interesting that this ranking also
corresponds to their abilities to activate complement (Figs. 3 and 4).
The sedimentation data also give information about the size of the
complexes. In a previous report (Poon and Schumaker, 1991) the 1:1
C1:IgM complex had a measured sedimentation coefficient of 27 S.
Therefore, the 22 and 19 S peaks observed when C1 was added to IgM and
µµ, respectively, probably represented a dynamic
equilibrium between the unassociated species and the predominantly 1:1
complexes. A 2:1 complex of C1:IgM should have a sedimentation rate of
35-36 S. Therefore, the 30 S complex shown in Fig. 7 A for the leading peak in mixtures of chimera µµ
µ
and C1 probably represents a dynamic equilibrium between the
unassociated species with predominately 1:1 and 2:1 complexes of
C1:µµ
µ. Previously it was shown that 2:1 complexes
were formed when high concentrations of C1q were added to IgM, and when
these were cross-linked and visualized with the electron microscope,
C1q was seen to be bound to opposite sides of the IgM disk (Poon et
al., 1985).
2 presented by chimera µµ
µ was sufficient to
bind C1 strongly and activate complement even in the absence of
antigen. As a corollary, it follows that the complement binding site
must be at least partially exposed when C
2 is introduced into the
IgM architecture. Moreover, the data also show that full exposure or
proper orientation of the C1 binding sites was induced by multivalent
binding of chimera µµ
µ to dansyl-polylysine, which can
support the distortive model for complement activation by IgM, although
additional aggregation may contribute to the enhanced activation.
Cµ1 and Cµ2 probably transmitted this distortion in wild-type
IgM; however, when they were replaced by C
1 and the hinge in
µµ, activity was greatly diminished. Although
assembly of IgM monomer to form polymer required the tail piece and at
least one of the two intersubunit disulfides, Cµ1 and Cµ2 must
also play some role in assembly, because 29% of the protein was
secreted as monomer in their absence. In addition, Cµ3 did not
appear to contain a complete complement binding site, for chimera
µ
was inactive in all of the assays. Probably
Cµ4 must act together with Cµ3 to form the binding site;
alternatively, it is possible that adjacent Cµ3 domains joined by
the Cys
disulfide form the site, or the
domains
render a site on Cµ3 inaccessible. Finally, we should point out
that these antibodies were chimeras not only of Cµ and C
domains, but also of human and mouse proteins. Interpretation of these
data are probably not seriously limited by the cross-species
composition, for the divergence in primary sequence between IgM and IgG
class antibodies is greater than the cross-species divergence between
immunoglobulins of the same class.
µµ was capable of erythrocyte lysis, whereas
chimera
µ
was totally inactive. Cµ2 probably
transmits the distortion in IgM occasioned by multivalent binding,
possibly rotating Cµ3 with respect to Cµ4 to convert a weak to
a strong complement binding site.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.