©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Structure and Function of Several Anti-Dansyl Chimeric Antibodies Formed by Domain Interchanges between Human IgM and Mouse IgG2b (*)

Pak H. Poon (1) (3)(§), Sherie L. Morrison (3) (2), Verne N. Schumaker (1) (3)

From the (1) Departments of Chemistry and Biochemistry and (2) Microbiology and Molecular Genetics and the (3) Molecular Biology Institute, University of California, Los Angeles, California 90024

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

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 µµµ and µ was generated by switching the Cµ3 and C2 domains between IgM and IgG2b. The second pair of chimeras µµ and µµ were formed by switching both Cµ3 and Cµ4 with C2 and C3. 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 µµµ 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 Cµand Cµfor full activity.


INTRODUCTION

Complement fixation by IgG and IgM antibodies involve different mechanisms. IgG antibodies have an exposed complement binding site located in the C2 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.

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 (µL) 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 C2 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.

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 Fabare 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).

The technique of domain switching has been profitably employed to examine the role of the hinge and C2 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 FcRI (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.


EXPERIMENTAL PROCEDURES

Construction of Plasmids and Production of Antibodies The plasmid pUC was constructed by subcloning into pUC19 (New England BioLabs) a 3.7-kilobase pair XbaI- BglII fragment of genomic DNA containing C1, hinge, C2, and C3 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 C2, and an MluI was introduced at the PpuMI site in the intron 3` to C2. (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, C2, and C3 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 C1-hinge-Cµ3-C3.



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() Vfrom 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.

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

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 10ml), 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.


RESULTS

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 ().

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 Cysof Cµ2, forming an intrasubunit disulfide, and at Cysof Cµ3 and Cysof 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; µµµ 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 Cysand Cys, it was nevertheless defective in polymer assembly, since 29% was secreted as monomer. In summary, only the mAb's containing Cµ4 with Cyspresent in the tail piece gave rise to appreciable amounts of polymer, with µµµµ > µµ > µµµ.


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.

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).


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; , µµµ; , 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.

For the chimeric antibodies containing C2, the data show µµµ 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.

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 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, µµµ 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 µµµ 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; , µµµ; , 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-Lyswas 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, µµµ 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 C1rC1ssubcomponents. 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 µµµ, 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; µµµ and µµ, 0.16 mg/ml. Vertical bars indicate, from left to right, 7, 18, and 27 S.




DISCUSSION

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`)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.

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 µ was approximately 95% monomer and only 5% dimer (), even though the Cµ3 domain contained Cysused for one of the two intersubunit disulfide bridges found in wild-type IgM polymer. In the mutant IgM-Ser, Cyswas 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 Cysin Cµ3), although secreted predominately as monomer, formed a mixed population of oligomeric antibodies ranging in size from monomer to hexamer (Smith and Morrison, 1994).

Mutant 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.

Chimera µµµ which lacked Cysof 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-Sersecreted only 20-40% polymer (Davis et al., 1989b). Mutant µµµ 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 C2 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 µµµ was distorted upon multivalent interaction with the dansylated polylysine.

In contrast to murine IgM, which requires the intersubunit disulfide bonds linking Cysresidues 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 µµµ. 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.

Chimera µµ which lacked the tail piece and both Cysand 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 C2 as the monomeric IgG2b and presumably the polymeric form of µµµ, 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.

The high polymer content (71%) of µµ probably reflected the availability of both Cysand Cysfor 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).

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 µµµ, 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 µµµ and C1 probably represents a dynamic equilibrium between the unassociated species with predominately 1:1 and 2:1 complexes of C1:µµµ. 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).

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 C2 presented by chimera µµµ 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 C2 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 µµµ 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 C1 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 Cysdisulfide 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.

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 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.

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.

  
Table: 0p4in Less than 5%.


FOOTNOTES

*
This work was supported by Research Grants GM 13914 (to V. N. S.) and CA 16858 and AI 29470 (to S. L. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 310-825-3531; Fax: 310-206-7286.

The abbreviations used are: dansyl (or DNS), 5-dimethylaminonaphthalene-1-sulfonyl; PAGE, polyacrylamide gel electrophoresis; mAb, monoclonal antibody; SRBC, sheep red blood cells; TBS, Tris-buffered saline; gpS, guinea pig serum; gpSC, guinea pig serum complement; NHS, normal human serum; NHSC, normal human serum complement; EA, sheep red blood cells optimally sensitized with hemolysin.


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