Structural requirements for incorporation of J chain into human IgM and IgA
Vigdis Sørensen,
Ingunn B. Rasmussen,
Vibeke Sundvold1,
Terje E. Michaelsen2 and
Inger Sandlie
Department of Molecular Cell Biology, Institute of Biology, University of Oslo, PO Box 1050, 0316 Oslo, Norway
1 Institute of Immunology, The National Hospital, 0027 Oslo, Norway
2 Department of Vaccinology, National Institute of Public Health, 0403 Oslo, Norway
Correspondence to:
I. Sandlie
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Abstract
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J chain is associated with pentameric IgM and dimeric IgA via disulfide bonds involving the penultimate cysteine residue in the secretory tailpiece of the µ or the
heavy chain. We have investigated the structural basis for incorporation of J chain by analyzing several IgM mutants, IgA mutants and IgG/IgM hybrid molecules. IgM mutants with the µ secretory tailpiece replaced by the
secretory tailpiece and/or Cys414 replaced by serine incorporated J chain, although in reduced amounts correlating with reduced pentamer/polymer formation. In addition to pentamers, tetramers of IgMC414S contained J chain, while no J chain was associated with smaller polymers or hexamers of IgM. An IgA/IgM hybrid tailpiece abolished J chain incorporation to pentameric IgM. Analysis of IgG molecules that have added a secretory tailpiece and/or have IgM domain replacements showed that J chain incorporation depends on regions of the Cµ4 domain in addition to the tailpiece. Features of the Cµ3 domain other than Cys414 also play a role in efficient formation of pentamers and J chain incorporation, while the Cµ2 domain is not specifically required. By analysis of two IgA mutants that formed larger polymers than IgAwt, we found J chain equally incorporated into dimers, trimers, tetramers and pentamers. Thus, the results show that J chain incorporation into IgA does not depend on the polymeric structure, while J chain incorporation into IgM is restricted to certain polymeric conformations.
Keywords: antibody, IgG, protein assembly, structure, polymer, SDSPAGE, immunoblotting
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Introduction
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J chain is a 15 kDa protein covalently associated with polymeric IgM and IgA. J chain is required for binding of IgM and IgA to the poly-Ig receptor, which mediates epithelial transcytosis of the Ig (14). J chain also plays a role in the intracellular assembly of IgM and IgA by modulating their structure and thereby effector functions.
J chain possesses eight cysteine residues of which two, Cys14 and Cys68, form disulfide bonds to the penultimate cysteine residue, Cys495 or Cys575, in the secretory tailpiece in the
or µ heavy chain respectively (58). The J chain joins two
chains of different monomers in the IgA dimer. The two remaining
chains in the dimer are linked directly to each other by a Cys495Cys495 disulfide bond (9,10). The J chain probably also joins two monomers in the IgM pentamer (11), while further connections between µ chains in the polymer may occur at three sites: Cys575, Cys414 and Cys337 (1214). Probably, Cys337 residues form intramonomeric Cys337Cys337 bonds, while Cys575 and Cys414 form intermonomeric Cys575Cys575 and Cys414Cys414 bonds (14). The homologue to Cys414 in the
chain, Cys309, can form a disulfide bond to the poly-Ig receptor (15). Several reports indicate that there is approximately one J chain per IgM polymer (11,16,17) or IgA polymer (18), but other reports are at variance with this (1922).
It has been suggested that J chain is necessary for the intracellular polymerization of IgM and IgA (9,11,18,23). However, large amounts of polymeric IgM can be secreted from various cell types that do not express J chain (2428). Although IgM polymerization can occur in the absence of J chain, J chain influence which polymers are formed. Formation of pentameric IgM is preferred in the presence of J chain, while more hexamers are formed when J chain is absent or in limiting amounts (2426,29,30). J chain seems to insert in the IgM polymer at a late stage in the polymerization process and J chain incorporation is suggested to be thermodynamically favored over incorporation of a sixth monomer (17,25,29). The hexamer has never been found to contain J chain (3033), but is shown to activate complement more efficiently than pentameric IgM and may thus have a specialized function in the immune system (27,30,31).
Contrary to IgM, IgA shows a strong dependence on J chain for polymerization. CHO cells (34), plant cells (35) and insect cells (36), which do not produce J chain, are reported to produce monomeric but no covalently linked dimeric IgA when transfected with genes encoding light chain and
heavy chain. However, upon co-transfection of a J chain gene dimer production was restored (3537). Also, mutation of Cys14 or Cys68 in J chain precludes formation of IgA dimers (37). On the other hand, in J chain knockout mice some dimeric IgA is formed, although the dimer/monomer ratio is strongly reduced (3). IgA containing less than one J chain per polymer has also been reported (19,20,38).
Although IgM and IgA share several features, such as similar locations of cysteine residues in the heavy chains and the abilities to form polymers and bind J chain, crucial differences must exist in the heavy chains, which make IgM and IgA polymerize and incorporate J chain in different manners. In this paper we investigate the structural requirements for J chain incorporation to IgM and IgA by analyzing several IgM and IgA mutants that have alterations in the tailpiece and Cys414/Cys309 regions, and form various types of polymers (39). We also investigate which regions in IgM are important for efficient J chain incorporation by construction and analysis of several IgG/IgM hybrid molecules. The results demonstrate the importance of the tailpiece, the Cµ4 domain, but also the Cµ3 domain for J chain incorporation into IgM. Furthermore, whereas J chain incorporation into IgM is restricted to certain polymeric structures, J chain equally incorporates into many different polymers of IgA.
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Methods
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Construction of mutant heavy chain genes
Cloning of the human µ,
1 and
3 constant heavy chain genes into pUC19, and construction of IgM
tp, IgM-VAEVD, IgGµtp and IgGL309Cµtp have been reported in (39). Construction of IgMC414S, IgMC414S
tp, IgAµtp and IgAalma has been reported in (40). IgM-VAEVD contains the mutations L566V, S569A, D570E, T571V and A572D. IgAalma contains the mutations P307S, G308I, A310E, E311A and P312D. Four of these five amino acid replacements introduce the amino acid found in the homologous position in IgM (positions 412, 413, 415 and 417). Domain swap mutants between IgGL309Cµtp and IgM were constructed by introduction of unique restriction enzyme sites in introns followed by exon shuffling. A 1.2 kb PstI fragment containing the fourth hinge exon and the C
2 and C
3 exons from the IgGL309Cµtp construct, was cloned in M13mp19. Also, a 2.0 kb SmaI fragment containing part of Cµ1 and Cµ2, Cµ3 and Cµ4 exons, was cloned in M13mp19. A XhoI site was introduced between hinge and C
2 exons, and between Cµ2 and Cµ3 using the primers 5'-gtcctgcctcgagctggag-3' and 5'-gaatcgagactcgagtggac-3' respectively. A NcoI site was introduced between C
2 and C
3 and between Cµ3 and Cµ4 using the primers 5'-gcctctgtccccatggccctcata-3' and 5'-gaggtc- ccatggagtgcag-3' respectively. Mutagenesis was performed according to the protocol by Kunkel (41) using reagents purchased from BioRad (Richmond, CA). Exon shuffling was performed in the pUC19 vector. C
2 was exchanged with Cµ3 using XhoI and NcoI restriction enzyme cutting, leading to the mutant heavy chain of IgG-Cµ3-µtp. C
3 was exchanged with Cµ4 using NcoI and BamHI cutting, leading to the mutant heavy chain of IgG-L309C-Cµ4. C
2 and C
3 was exchanged with Cµ3 and Cµ4 by XhoI and BamHI cutting, giving IgG-Cµ3-Cµ4. Constructs are shown schematically in Fig. 1
. The constant heavy chain gene constructs were cloned on HindIII and BamHI sites, downstream a of VNP gene, in the pSV2gptVNP vector (a gift from Dr M. S. Neuberger, Medical Research Council Laboratory of Molecular Biology, Cambridge, UK) for expression in J558L cells. The VNP gene encodes a variable region with specificity for the hapten 3-nitro-4-hydroxy-5-iodophenylacetic acid (NIP).
Cell culture, transfections and metabolic labeling
J558L cells (a gift from Dr S. L. Morrison, Department of Microbiology, Molecular Biology Institute, University of CaliforniaLos Angeles), which express endogenous
light chains and J chain, were maintained and transfected as previously described (39). Clones producing IgG/IgM hybrid proteins were identified by ELISA using NIP-BSA-coated microtiter wells, locally produced sheep anti-human IgM and anti-human IgG antibodies, and as previously described (42). IgG/IgM hybrid antibodies produced by transfected J558L were metabolically labeled for 5 h and precipitated from cell supernatant by rabbit anti-human IgM (µ chain specific) (Sigma, St Louis, MO), rabbit anti-human IgG (Fc specific) (Sigma) and Dynabeads sheep anti-rabbit IgG (Dynal, Oslo, Norway) as previously described (39).
Adsorption of antibodies to NIPSepharose
For preparation of NIPSepharose, 10 mg NIP-caproate-O-succinimide (NIP-CAP-OSu; Genosys Biotechnologies, The Woodlands, TX) dissolved in 1 ml dimethylformamide and 10 ml EAH Sepharose 4B (Amersham Pharmacia Biotech, Uppsala, Sweden) were incubated in 0.1 M NaHCO3 and 0.5 M NaCl, pH8.5 overnight, and excess of free NIP was removed by extensive washing in PBS. Antibodies secreted from transfected J558L were adsorbed to NIPSepharose by incubating 20 ml cell supernatant with 100 µl NIPSepharose overnight. The Sepharose was then washed in several rounds of PBS/Tween, PBS and distilled water before elution of the adsorbed antibodies in gel loading buffer for SDSPAGE.
SDSPAGE and immunoblotting
Antibodies were analyzed unreduced by SDSPAGE in 4% acrylamide0.5% agarose phosphate buffer gels as previously described (39) or reduced in 9% gels according to the method of Laemmli (43). For metabolically labeled antibodies, the gels were dried, exposed to BIOMAX film (Eastman Kodak, Rochester, NY) and scanned by a phosphorimager (GS-250 BioRad Molecular Imager) for quantification of assembly products. Unlabeled antibodies were blotted onto Immobilon-P transfer membranes (Millipore, Bedford, MA) for immunodetection. Laemmli gels were blotted in 25 mM Tris, 192 mM glycine and 20% methanol. Phosphate-buffered gels were blotted in 25 mM Tris, 192 mM glycine and 0.02% SDS, after equilibration in blotting buffer for 20 min. Membranes were blocked in PBS/5% dried milk powder. Primary antiserum used for immunodetection was one or two of the following: rabbit anti-human IgM (µ chain specific) (Sigma), rabbit anti-human IgG (Fc specific) (Sigma), rabbit anti-human IgA (
chain specific) (Sigma) or rabbit anti-mouse J chain (gift from Dr R. M. E. Parkhouse, Division of Immunology, Institute for Animal Health, Pirbright, UK), which was diluted ~1:10,000 in PBS/Tween. The secondary detection antibody was horseradish peroxidase linked anti-rabbit Ig (Amersham Pharmacia Biotech). Chemiluminescent signals were obtained by incubating membranes in Super Signal (Pierce, Rockford, IL) and detected by XOMAT-AR film (Kodak). Membranes were first detected by anti-J chain antiserum and then by anti-heavy chain antiserum. The membrane was rinsed in PBS/Tween and/or stripped in buffer containing 125 mM Tris, 1.25M glycine and 0.5% SDS, pH 8.3 by briefly heating to boiling between the two rounds of detection. The anti-J chain antiserum showed some cross-reaction with heavy chains and light chains; however, conditions during J chain detections were chosen so that no signal was evident from internal negative controls present in each blot. One or several of the following were considered as negative controls: IgM hexamers, monomers, IgG or heavy chains. IgG/IgM domain swap antibodies were detected in blots by incubation of the blot first in anti-IgM antiserum (1 h) and then in anti-IgG antiserum (1 h), before continuing with secondary detection antibody. For reducing gels, the relative J chain content in wild-type and mutant antibodies was estimated by phosphorimager scanning of blots from three or four repeated experiments and by comparison of the relative signal intensities obtained by J chain detection with the signal intensities obtained by heavy chain detection. For non-reducing gels, the identity of each band in the gel (i.e. monomer, dimer, trimer, etc.) was determined by reference to migration of mol. wt standards and pentameric IgMwt, and by correlating the distances migrated in the gel with estimated mol. wt (assuming multiples of HL or H2L2 subunits) that give a linear relationship in semilogaritmic plots, as has been described previously for several of the antibodies studied in this report (39,40).
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Results
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Analysis of J chain incorporation into IgM mutants
To investigate how structural variations in IgM polymers affect incorporation of J chain, we analyzed the mutants IgM
tp, IgMC414S and IgMC414S
tp (see Fig. 1
for overview of constructs). These antibodies were secreted from transfected J558L cells as pentamers, but also as other polymeric structures as reported previously (39,40) and summarized in Table 1
. IgM
tp, which is IgA-like in the secretory tailpiece, forms more hexamers than IgMwt, while IgMC414S and IgMC414S
tp form no hexamers, but pentamers, tetramers and smaller assembly products. To investigate if J chain is incorporated into these antibodies, secreted antibodies were analyzed by reducing SDSPAGE and immunoblotting as described in Methods. J chain was incorporated into IgM
tp, IgMC414S and IgMC414S
tp (Fig. 2
). The relative J chain content in IgMwt and the mutants was estimated as described in Methods. IgM
tp was found to have a slightly reduced J chain content compared to IgMwt (~70%), while IgMC414S and IgMC414S
tp had even less J chain (~2550%), which seem to correlated with a reduced pentamer fraction for each mutant antibody (Table 1
). To investigate whether J chain is incorporated into pentamers or possibly other polymeric structures as well, the antibodies were analyzed by non-reducing SDSPAGE and immunoblotting (Fig. 3
). IgMC414S consists of monomers and several polymeric structures. The two major bands, which represent polymers, migrate slightly slower in the gel than the IgMwt polymers. However, we and others have shown previously that they correspond to pentamers and tetramers of IgMC414S (13,40). J chain was detected in pentameric but not hexameric IgMwt, and in pentameric and tetrameric IgMC414S (Fig. 3
). Similarly, J chain was found only in the pentameric fraction of IgM
tp, and in pentamers and tetramers of IgMC414S
tp (results not shown). Thus, J chain can be incorporated into two different polymeric structures of IgM, i.e. pentamers and tetramers, but not into monomers or hexamers. To further investigate if J chain can be incorporated into IgM structures other than pentamers and tetramers, we analyzed an IgM-VAEVD mutant for J chain. This mutant contains five IgA-like mutations in the tailpiece and we have previously reported that it forms several polymeric structures, i.e. dimers, trimers, tetramers, pentamers and hexamers (39) (Fig. 1
, Table 1
and Fig. 4
). Surprisingly, no J chain was associated with this mutant (Fig. 4
). This result was repeated for several individual clones of the IgM-VAEVD mutant. Thus, the structure of the C-terminal part of the µ tailpiece is of critical importance for J chain incorporation into IgM.

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Fig. 3. Analysis of J chain incorporation to polymeric IgMwt and IgMC414S. Antibodies secreted by transfected J558L cells were adsorbed to NIPSepharose, fractionated unreduced by SDSPAGE in a 4% acrylamide0.5% agarose gel and blotted onto an Immobilon-P membrane. Polymers containing J chain were detected using J chain-specific antiserum (right panel), then the membrane was stripped and the antibodies were detected by µ heavy chain-specific antiserum (left panel).
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Fig. 4. Analysis of J chain incorporation into IgM-VAEVD. Antibodies secreted by transfected J558L cells were adsorbed to NIPSepharose. The samples were split in two and fractionated unreduced (upper panel) or reduced (middle and lower panel) by SDSPAGE and blotted onto Immobilon-P membranes. The unreduced samples were detected by µ heavy chain-specific antiserum (upper panel), and the reduced samples were detected by µ heavy chain-specific antiserum (middle panel) and J chain-specific antiserum (lower panel).
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Analysis of J chain incorporation into IgG/IgM hybrids
We, and others, have previously shown that addition of the µ tailpiece to an IgG molecule (mutant IgGµtp) induces polymer formation and that introduction of cysteine in position 309 in combination with the tailpiece leads to a polymerization pattern for the IgGL309Cµtp mutant that is very similar to that of IgM, except that more hexamers than pentamers are formed (39,44) (see Figs 1 and 5
, and Table 1
). Analysis of IgGµtp and IgGL309Cµtp by reducing SDSPAGE and immunoblotting, revealed that no, or at best minute amounts of, J chain was incorporated into these antibodies (Fig. 6
).

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Fig. 5. Analysis of the polymerization pattern of IgG/IgM hybrid antibodies by SDSPAGE. Metabolically labeled antibodies were immunoprecipitated from supernatant of transfected J558L cells, fractionated unreduced in a 4% acrylamide0.5% agarose gel and visualized by autoradiography. The distributions of assembly products were quantitated and are reported in Table 1 .
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To investigate which parts of the µ chain in addition to the tailpiece are important for efficient pentamer formation and J chain incorporation, we constructed mutants based on IgGL309Cµtp which have C
2, or C
3, or both, replaced by the corresponding IgM domains, Cµ3 and Cµ4. The resulting IgG-Cµ3-µtp, IgG-L309C-Cµ4 and IgG-Cµ3-Cµ4 (see Fig. 1
) were expressed in J558L, and the secreted antibodies were analyzed by metabolical labeling and non-reducing SDSPAGE (Fig. 5
), and by reducing SDSPAGE and immunoblotting (Fig. 6
) as described in Methods. The distribution of assembly products was quantitated by phosphorimager analysis and is reported in Table 1
.
An IgG that had the entire Fc region replaced by Cµ3 and Cµ4 domains, showed a polymerization pattern and J chain incorporation practically identical to that of IgMwt. The polymers did, however, migrate faster in the gel than the corresponding IgMwt polymers because the IgG/IgM hybrid molecules have a short hinge region of 15 amino acids [the fourth hinge region of IgG3 (39,45)] compared to the Cµ2 domain in IgM. This shows that the hinge/Cµ2 and CH1 domains do not significantly influence IgM polymerization or J chain incorporation. The mutant with C
2 replaced by Cµ3 (mutant IgG-Cµ3-µtp) formed considerably less polymers than the IgGL309Cµtp mutant (Fig. 5
and Table 1
). Apparently, a combination of C
3 and Cµ3 domains in the Fc impedes polymerization. Replacement of C
3 in IgGL309µtp with Cµ4 (mutant IgG-L309C-Cµ4) allowed formation of mainly large polymers, but hexamers were still the dominant species (Fig. 5
and Table 1
). Thus, motifs from both Cµ3 and Cµ4 are required for formation of mainly pentamers.
No, or only minute amounts of, J chain was associated with IgG-Cµ3-µtp, while J chain was positively detected in IgG-L309C-Cµ4 (Fig. 6
). Since IgG-L309C-Cµ4 formed approximately the same amount of pentamers as IgGL309Cµtp, but incorporated more J chain, motifs in Cµ4 outside the tailpiece must have contributed to J chain incorporation. However, considerably less J chain was incorporated into IgG-L309C-Cµ4 than into IgMwt.
Analysis of J chain incorporation into IgA mutants
To investigate whether J chain incorporation into IgA is influenced by variations in the polymer structure, we analyzed J chain content in human IgA1 and two mutants, IgAµtp and IgAalma, which have polymer distributions deviating from that of IgAwt, as reported previously (40) and summarized in Table 1
. IgAµtp, which is IgM-like in the secretory tailpiece, formed mostly monomers, but also polymers ranging in size from dimers to hexamers. IgAalma, which has four IgM-like mutations in amino acids flanking Cys309, had a higher proportion of trimers and tetramers than IgAwt, and also formed some pentamers and hexamers (see Fig. 8
). IgAwt, IgAµtp and IgAalma secreted from J558L cells were analyzed by reducing SDSPAGE and immunoblotting. J chain was incorporated into IgAwt, IgAµtp and IgAalma (Fig. 7
). After repeated experiments and analysis of several parallel clones, we were not able to determine significant differences in J chain content for these antibodies. We investigated which of the polymers have incorporated J chain by non-reducing SDSPAGE and immunoblotting (Fig. 8
). For IgAwt, J chain was detected in trimers, dimers and H3L3 species, and we also often detected J chain in some monomers. The monomeric fraction of IgAwt was resolved in the gel as a double band (Fig. 8
, left panel), but only the slower migrating monomer band was positive for J chain (Fig. 8
, right panel). Possibly, this J chain-positive monomer resulted from depolymerization in the IgA preparation during analysis. The monomeric fraction of neither IgAµtp nor IgAalma was positive for J chain. On the other hand, dimers, trimers, tetramers and pentamers of both IgAµtp and IgAalma contained J chain, while J chain seemed to be absent in the hexameric fractions of IgAalma and IgAµtp. By comparing the relative signal intensities of bands in the blot obtained by
heavy chain detection or J chain detection respectively (Fig. 8
), J chain seemed to be equally incorporated into dimers, trimers, tetramers and pentamers of IgA, IgAµtp and IgAalma.

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Fig. 8. Analysis of J chain incorporation into polymeric IgAwt, IgAµtp and IgAalma. Antibodies secreted by transfected J558L cells were adsorbed to NIPSepharose, fractionated unreduced by SDSPAGE in a 4% acrylamide0.5% agarose gel and blotted onto an Immobilon-P membrane. Polymers containing J chain were detected using J chain-specific antiserum (right panel), then the membrane was stripped and the antibodies were detected by heavy chain-specific antiserum (left panel).
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Fig. 7. Analysis of J chain incorporation to IgAwt, IgAµtp and IgAalma. Antibodies secreted by transfected J558L cells were adsorbed to NIPSepharose, reduced, fractionated by SDSPAGE and blotted onto an Immobilon-P membrane. The heavy chains (upper panel) and J chain (lower panel) were detected using specific antisera.
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Discussion
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We report here on J chain content in several mutants of human IgM, human IgA1 and hybrid IgG/IgM molecules, which vary in their polymer composition.
We found J chain in pentameric IgM
tp, IgMC414S and IgMC414S
tp, and in tetrameric IgMC414S and IgMC414S
tp. No J chain was found in hexamers or in any polymeric IgM species smaller than tetramers and no J chain was associated with any IgM-VAEVD polymers. These results imply that J chain can be incorporated into a few polymeric structures of IgM only. This is consistent with the hypothesis that J chain adds to the IgM polymer at a late stage in the polymerization process and that a binding motif for J chain in IgM is established after formation of a specific polymeric conformation (17). That a hexameric IgM structure prohibits J chain incorporation is well established and explained by a model where IgM subunits form a polymer by fitting either six monomers or five monomers and one J chain into a ring-like structure (17,25,29,46).
IgMC414S and IgMC414S
tp mutants consist of considerable amounts of pentamers and tetramers, and both of these species contained J chain. The effect of the C414S mutation on polymerization has been discussed previously (13,40,47) and the incorporation of J chain into IgMC414S has also previously been reported (47). Although the presence of both tetramers and monomers in the IgMC414S preparation, as revealed by SDSPAGE, might be due to dissociation of non-covalently assembled pentamers, analysis performed by Fazel et al. (47) indicated that some J chain-containing tetrameric IgMC414S was secreted as such from the cells. It was also shown that similar amounts of tetrameric and pentameric IgMC414S were formed in cells lacking J chain (47), indicating that the polymerization pattern of IgMC414S is largely unaffected by J chain incorporation. Thus, Cys414Cys414 bonds are required for hexamer formation and enhances, but are not strictly required for, pentamer formation and J chain incorporation.
The lack of J chain incorporation into IgM-VAEVD, despite the occurrence of both pentameric and tetrameric IgM-VAEVD, indicates that the fine structure of the µ secretory tailpiece is of critical importance for J chain incorporation to IgM. The 18 C-terminal amino acids in the µ and
chains is a structure unique to the µ and
heavy chains, and it is not surprising that this region is important for J chain interactions since it contains the cysteine residue employed for covalent binding of J chain to both IgM and IgA. It has been shown that mutation of Cys575 in the µ tailpiece abolishes J chain incorporation into IgM, while some hexamers are formed, due to other cysteine residues in the µ heavy chain available for inter-monomeric bonds (13,31). A carbohydrate chain attached to a conserved asparagine in the tailpiece also seems to play a role in J chain incorporation; mutation of this site in IgA or IgM is reported to reduce J chain incorporation (48,49). IgM-VAEVD has five IgA-like mutations in the tailpiece and shows a polymerization pattern deviating from that of IgMwt (Figs 1 and 4
) (39). IgM
tp on the other hand, which has two additional IgA-like mutations, is very similar to IgMwt. Furthermore, IgM
tp incorporated J chain, but had slightly more hexamers and less J chain than IgMwt. It is unclear at this point whether formation of hexameric IgM
tp precludes J chain incorporation or a reduced J chain incorporation allows formation of more hexamers. According to the theory by Brewer et al. (17), however, incorporation of J chain into assembling IgM is thermodynamically favored over incorporation of a sixth monomer. Thus, it is possible that the tailpiece structure of IgM
tp deviates from the wild-type sufficiently to reduce J chain incorporation and thereby indirectly affects polymerization.
Although it is generally believed that pentameric IgM and dimeric IgA contain one J chain per polymeric molecule, Brandtzaeg (20) have reported two J chains for IgA and three or four J chains for IgM. Our quantitation of J chain content in the various mutant antibodies (Table 1
) does not give an indication as to whether the number of J chains per polymer varies for different mutants or for different polymeric structures; however, it should be considered a possibility that mutations in Ig heavy chains may affect the number of J chains that can be incorporated into a polymer and that this in turn affects the final polymeric structure of the antibody.
The µ tailpiece in itself does not contain sufficient structural information for J chain incorporation into polymeric antibodies. We show here that human IgG3-derived mutants with the µ tailpiece added to their C-terminal (mutants IgGµtp and IgGL309Cµtp), lack bound J chain despite formation of 7090% polymers. This is in agreement with results by Smith et al. (50), who showed that IgG of the four human subclasses with the µ tailpiece added to their C-terminals formed polymers, but did not incorporate J chain. The IgGL309Cµtp mutant resembles IgM in polymerizing mainly to hexamers and pentamers. However, it consists of ~3 times more hexamers than pentamers. Possibly, this is a consequence of poor J chain incorporation. Taken together, the results from IgM
tp/IgM-VAEVD and IgGL309Cµtp suggest that the µ tailpiece in combination with other regions of the µ heavy chain form a J chain binding motif.
To further investigate which regions in IgM govern J chain incorporation and pentamer formation we replaced domains in IgGL309Cµtp with corresponding domains from the human µ heavy chain. When both C
2 and C
3 were replaced by Cµ3 and Cµ4 (mutant IgG-Cµ3-Cµ4), formation of pentamers and J chain incorporation similar to that of IgMwt was achieved. This is in agreement with results by Poon et al. (51) who observed mainly pentamers when C
2 and C
3 of mouse IgG2b were replaced by Cµ3 and Cµ4 of human IgM. Thus, the Cµ1 and Cµ2 domains are not decisive for IgM pentamer assembly.
We could not directly determine the role of the Cµ3 domain in IgM polymerization, since the mutant with C
2 replaced by Cµ3 (IgG-Cµ3-µtp) gave poor polymerization. Obstruction of efficient polymerization when Cµ3 and C
3 domains are combined in the Fc has also previously been observed by Wiersma et al. (49) who replaced Cµ4 in mouse IgM with C
3 from mouse IgG2b with the µtp added to its C-terminal. Observations of aberrant polymerization when domains from different antibody classes are combined in an Fc suggest that the connecting amino acids and/or contact sites between domains might be important for the structure or stability of the Fc and therefore important for polymerization.
Our results show that regions in Cµ4 outside the tailpiece contribute to J chain incorporation. IgG-L309C-Cµ4, which has C
3-µtp replaced by Cµ4, bound significantly more J chain than IgGL309Cµtp. It still contained less than one-third the amount of J chain found in IgMwt and formed more hexamers than pentamers. Thus, both Cµ3 and Cµ4 are required for efficient IgM pentamer assembly and J chain incorporation. There are three previous reports of antibody constructs that have a combination of C
2 and Cµ4 domains in the Fc region, but neither of these constructs does also have the L309C mutation in C
2, which can enhance polymerization. Poon et al. (51) replaced Cµ3 in human IgM with C
2 from mouse IgG2b and obtained mainly hexamers and monomers. Chen et al. (52) replaced C
3 in mouse IgG2b with mouse Cµ4, and reported production of pentamers and other assembly products. Wiersma et al. (49) replaced Cµ2 and Cµ3 in mouse IgM with the hinge and C
2 from mouse IgG2b, and reported production of pentamers and hexamers or hexamers and heptamers in addition to other polymers, and also identified J chain incorporation into this antibody. Thus, the C
2 + Cµ4 Fc structure generally induces formation of polymers such as hexamers and pentamers, and also binds J chain, but the polymerization deviates somewhat from that of IgM, demonstrating a role for the Cµ3 domain. Our results show that the role of Cµ3 is not solely related to Cys414. The rather complex requirements observed for J chain incorporation into IgM suggest that the part of IgM that constitutes J chain binding motif is disguised or unavailable in the monomeric conformation. Possibly the tailpiece and other parts of Cµ4 confine the actual J chain binding site, while Cµ3 has a modulating function that allows establishment of the appropriate polymeric conformation that reveals J chain binding site.
In contrast to the restricted J chain incorporation to higher polymers of IgM, it is known that dimers, trimers and tetramers of IgA can contain bound J chain (18,20,53). We show here that mutants of human IgA1 (IgAµtp and IgAalma) that produce more or larger polymers than IgAwt, incorporate J chain in amounts similar to IgAwt. J chain was incorporated into dimers, trimers, tetramers and pentamers of IgAµtp and IgAalma, with no detectable preference for any of these species. Thus, a J chain binding site seems to exist in IgA independent of the state of polymerization, with the exception of hexamers, which as for IgM, lack J chain. It has previously been shown that when Cys14 or Cys68 in J chain was mutated, monomeric IgA bound to J chain could be secreted from cells (37), indicating that interaction with only one IgA monomer may in fact be sufficient for J chain binding. Such an easily accessible J chain binding site may be determined by regions of the
chain outside the tailpiece since J chain is efficiently incorporated into various polymeric forms of the IgAµtp mutant. It also offers a possible explanation for our observation of H3L3 and monomeric subunits of IgAwt with bound J chain.
The observation that most IgA polymers contain bound J chain and the previous reports that absence of J chain precludes IgA polymerization (3,3436) could suggest that J chain is required for IgA inter-monomeric association, possibly by bridging all monomers in a polymer (which for large polymers would require more than one J chain per polymeric molecule). It seems, however, that direct IgA inter-monomeric contact to some extent can occur since IgA polymers lacking J chain have been observed (3,19,20,38). Furthermore, it has been reported that limited reduction of tetrameric IgA released two monomers and one dimer that contained J chain (9,54), indicating that only two of the four monomers were bridged by J chain. Theoretically, monomerJ chain interactions could be greatly enhanced over direct monomer interactions and thereby explain why IgA polymerization is ineffective in cells lacking J chain. The J chain-containing dimer possibly also serves as the substrate for formation of larger IgA polymers. Since the four Cys495 residues in dimeric IgA normally are engaged in intradimeric disulfide bonds (9,10), formation of trimeric/tetrameric IgA may rely on formation of intermonomeric Cys309Cys309 bonds. Such bonds have been reported for polymeric IgA (55), while it is shown that mutation of Cys495 in IgA precludes formation of covalent IgA dimers (48), indicating that formation of intermonomeric Cys309Cys309 bonds normally is inefficient. Intermonomeric Cys309Cys309 bonds may, however, be increased in the IgAalma mutant, which is IgM-like in four amino acids flanking Cys309 and formed more trimers and tetramers than IgAwt.
The strong propensity for IgA dimer formation is not shared by the IgAµtp mutant, which is IgM-like in the tailpiece, indicating that the preference for dimer formation relies on the
tailpiece. The IgAµtp mutant may have inter-monomeric interactions resembling IgM (although inefficient). Furthermore, since we were not able to detect J chain in hexamers of IgAµtp or IgAalma, it is possible that these hexamers, like hexameric IgM, are formed by a closed circular arrangement of monomers that excludes J chain. IgAalma and IgAµtp may thus be sufficiently similar to IgM to form some IgM-type polymers.
The observed formation of several different polymeric forms from identical subunits of IgM or IgA suggests that there is considerable structural flexibility in the monomermonomer interactions. On the other hand, J chain is a small protein containing several internal disulfide bonds and, although its three-dimensional structure has not been solved, it is likely that it has a rather rigid one-domain structure (7,56). Therefore, the orientation of Cys14 and Cys68 should determine the conformation of the two interacting IgA/IgM monomers, and it is probably required that quite similar J chain binding sites exist in the different IgA and IgM polymers. The dimeric structure of IgA is from electron microscopy images known to have a `tail-to-tail' shape (57,58). Since this is a constellation of two monomers which contains bound J chain, it is possible that a similar constellation constitutes J chain binding site(s) in larger IgA polymers as well as in the appropriate IgM polymers. This would probably require that J chain is not positioned between adjacent monomers in the IgM polymer as suggested by Perkins et al. (59), but rather bridges non-adjacent monomers, as recently suggested and discussed by Wiersma et al. (27) and Fazel et al. (47).
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Acknowledgments
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This study was supported by grants nos 107257/310 and 107256/310 from the Research Council of Norway, and B95078/001 from the Norwegian Cancer Association.
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Abbreviations
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NIP 3-nitro-4-hydroxy-5-iodo-phenylacetic acid |
tp tailpiece |
wt wild-type |
 |
Notes
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Transmitting editor: H. Bazin
Received 11 June 1999,
accepted 17 September 1999.
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References
|
---|
-
Brandtzaeg, P. and Prydz, H. 1984. Direct evidence for an integrated function of J chain and secretory component in epithelial transport of immunoglobulins. Nature 311:71.[ISI][Medline]
-
Hendrickson, B. A., Rindisbacher, L., Corthesy, B., Kendall, D., Waltz, D. A., Neutra, M. R. and Seidman, J. G. 1996. Lack of association of secretory component with IgA in J chain-deficient mice. J. Immunol. 157:750.[Abstract]
-
Hendrickson, B. A., Conner, D. A., Ladd, D. J., Kendall, D., Casanova, J. E., Corthesy, B., Max, E. E., Neutra, M. R., Seidman, C. E. and Seidman, J. G. 1995. Altered hepatic transport of immunoglobulin A in mice lacking the J chain. J. Exp. Med. 182:1905.[Abstract]
-
Vaerman, J. P., Langendries, A. E., Giffroy, D. A., Kaetzel, C. S., Fiani, C. M., Moro, I., Brandtzaeg, P. and Kobayashi, K. 1998. Antibody against the human J chain inhibits polymeric Ig receptor-mediated biliary and epithelial transport of human polymeric IgA. Eur. J. Immunol. 28:171.[ISI][Medline]
-
Mestecky, J., Schrohenloher, R. E., Kulhavy, R., Wright, G. P. and Tomana, M. 1974. Site of J chain attachment to human polymeric IgA. Proc. Natl Acad. Sci. USA 71:544.[Abstract]
-
Mestecky, J. and Schrohenloher, R. E. 1974. Site of attachment of J chain to human immunoglobulin M. Nature 249:650.[ISI][Medline]
-
Bastian, A., Kratzin, H., Fallgren-Gebauer, E., Eckart, K. and Hilschmann, N. 1995. Intra- and inter-chain disulfide bridges of J chain in human S-IgA. Adv. Exp. Med. Biol. 371A:581.
-
Frutiger, S., Hughes, G. J., Paquet, N., Luthy, R. and Jaton, J. C. 1992. Disulfide bond assignment in human J chain and its covalent pairing with immunoglobulin M. Biochemistry 31:12643.[ISI][Medline]
-
Chapuis, R. M. and Koshland, M. E. 1975. Linkage and assembly of polymeric IgA immunoglobulins. Biochemistry 14:1320.[ISI][Medline]
-
Bastian, A., Kratzin, H., Eckart, K. and Hilschmann, N. 1992. Intra- and interchain disulfide bridges of the human J chain in secretory immunoglobulin A. Biol. Chem. Hoppe Seyler 373:1255.[ISI][Medline]
-
Chapuis, R. M. and Koshland, M. E. 1974. Mechanism of IgM polymerization. Proc. Natl Acad. Sci. USA 71:657.[Abstract]
-
Beale, D. and Buttress, N. 1969. Studies on a human 19-S immunoglobulin M. The arrangement of inter-chain disulphide bridges and carbohydrate sites. Biochim. Biophys. Acta 181:250.[ISI][Medline]
-
Davis, A. C., Roux, K. H., Pursey, J. and Shulman, M. J. 1989. Intermolecular disulfide bonding in IgM: effects of replacing cysteine residues in the mu heavy chain. EMBO J. 8:2519.[Abstract]
-
Wiersma, E. J. and Shulman, M. J. 1995. Assembly of IgM. Role of disulfide bonding and noncovalent interactions. J. Immunol. 154:5265.[Abstract/Free Full Text]
-
Fallgren-Gebauer, E., Gebauer, W., Bastian, A., Kratzin, H., Eiffert, H., Zimmerman, B., Karas, M. and Hilschmann, N. 1995. The covalent linkage of the secretory component to IgA. Adv. Exp. Med. Biol. 371A:625.
-
Mihaesco, C., Mihaesco, E. and Metzger, H. 1973. Variable J-chain content in human IgM. FEBS Lett. 37:303.[ISI][Medline]
-
Brewer, J. W. and Corley, R. B. 1997. Late events in assembly determine the polymeric structure and biological activity of secretory IgM. Mol. Immunol. 34:323.[ISI][Medline]
-
Halpern, M. S. and Koshland, M. E. 1973. The stoichiometry of J chain in human secretory IgA. J. Immunol. 111:1653.[ISI][Medline]
-
Tomasi, T. B. and Czerwinski, D. S. 1976. Naturally occurring polymers of IgA lacking J chain. Scand. J. Immunol. 5:647.[Medline]
-
Brandtzaeg, P. 1976. Complex formation between secretory component and human immunoglobulins related to their content of J chain. Scand. J. Immunol. 5:411.[ISI][Medline]
-
Brandtzaeg, P. 1975. Immunochemical studies on free and bound J chain of human IgA and IgM. Scand. J. Immunol. 4:439.[ISI][Medline]
-
Grubb, A. O. 1978. Quantitation of J chain in human biological fluids by a simple immunochemical procedure. Acta Med. Scand. 204:453.[ISI][Medline]
-
Della Corte, E. and Parkhouse, R. M. 1973. Biosynthesis of immunoglobulin A (IgA) and immunoglobulin M (IgM). Requirement for J chain and a disulphide-exchanging enzyme for polymerization. Biochem. J. 136:597.[ISI][Medline]
-
Cattaneo, A. and Neuberger, M. S. 1987. Polymeric immunoglobulin M is secreted by transfectants of non-lymphoid cells in the absence of immunoglobulin J chain. EMBO J. 6:2753.[Abstract]
-
Randall, T. D., Brewer, J. W. and Corley, R. B. 1992. Direct evidence that J chain regulates the polymeric structure of IgM in antibody-secreting B cells. J. Biol. Chem. 267:18002.[Abstract/Free Full Text]
-
Niles, M. J., Matsuuchi, L. and Koshland, M. E. 1995. Polymer IgM assembly and secretion in lymphoid and nonlymphoid cell lines: evidence that J chain is required for pentamer IgM synthesis. Proc. Natl Acad. Sci. USA 92:2884.[Abstract]
-
Wiersma, E. J., Collins, C., Fazel, S. and Shulman, M. J. 1998. Structural and functional analysis of J chain-deficient IgM. J. Immunol. 160:5979.[Abstract/Free Full Text]
-
Wood, C. R., Dorner, A. J., Morris, G. E., Alderman, E. M., Wilson, D., O'Hara, R. M., Jr and Kaufman, R. J. 1990. High level synthesis of immunoglobulins in Chinese hamster ovary cells. J. Immunol. 145:3011.[Abstract/Free Full Text]
-
Randall, T. D., Parkhouse, R. M. and Corley, R. B. 1992. J chain synthesis and secretion of hexameric IgM is differentially regulated by lipopolysaccharide and interleukin 5. Proc. Natl Acad. Sci. USA 89:962.[Abstract]
-
Randall, T. D., King, L. B. and Corley, R. B. 1990. The biological effects of IgM hexamer formation. Eur. J. Immunol. 20:1971.[ISI][Medline]
-
Davis, A. C., Roux, K. H. and Shulman, M. J. 1988. On the structure of polymeric IgM. Eur. J. Immunol. 18:1001.[ISI][Medline]
-
Meng, Y. G., Criss, A. B. and Georgiadis, K. E. 1990. J chain deficiency in human IgM monoclonal antibodies produced by EpsteinBarr virus-transformed B lymphocytes. Eur. J. Immunol. 20:2505.[ISI][Medline]
-
Hughey, C. T., Brewer, J. W., Colosia, A. D., Rosse, W. F. and Corley, R. B. 1998. Production of IgM hexamers by normal and autoimmune B cells: implications for the physiologic role of hexameric IgM. J. Immunol. 161:4091.[Abstract/Free Full Text]
-
Morton, H. C., Atkin, J. D., Owens, R. J. and Woof, J. M. 1993. Purification and characterization of chimeric human IgA1 and IgA2 expressed in COS and Chinese hamster ovary cells. J. Immunol. 151:4743.[Abstract/Free Full Text]
-
Ma, J. K., Hiatt, A., Hein, M., Vine, N. D., Wang, F., Stabila, P., van Dolleweerd, C., Mostov, K. and Lehner, T. 1995. Generation and assembly of secretory antibodies in plants [see Comments]. Science 268:716.[ISI][Medline]
-
Carayannopoulos, L., Max, E. E. and Capra, J. D. 1994. Recombinant human IgA expressed in insect cells. Proc. Natl Acad. Sci. USA 91:8348.[Abstract]
-
Krugmann, S., Pleass, R. J., Atkin, J. D. and Woof, J. M. 1997. Structural requirements for assembly of dimeric IgA probed by site-directed mutagenesis of J chain and a cysteine residue of the alpha-chain CH2 domain. J. Immunol. 159:244.[Abstract]
-
Vaerman, J. P., Langendries, A., Giffroy, D., Brandtzaeg, P. and Kobayashi, K. 1998. Lack of SC/pIgR-mediated epithelial transport of a human polymeric IgA devoid of J chain: in vitro and in vivo studies. Immunology 95:90.[ISI][Medline]
-
Sørensen, V., Rasmussen, I. B., Norderhaug, L., Natvig, I., Michaelsen, T. E. and Sandlie, I. 1996. Effect of the IgM and IgA secretory tailpieces on polymerization and secretion of IgM and IgG. J. Immunol. 156:2858.[Abstract]
-
Sørensen, V., Sundvold, V., Michaelsen, T. E. and Sandlie, I. 1999. Polymerization of IgA and IgM: roles of Cys309/Cys414 and the secretory tailpiece. J. Immunol. 162:3448.[Abstract/Free Full Text]
-
Kunkel, T. A., Roberts, J. D. and Zakour, R. A. 1987. Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol. 154:367.[ISI][Medline]
-
Michaelsen, T. E., Aase, A., Westby, C. and Sandlie, I. 1990. Enhancement of complement activation and cytolysis of human IgG3 by deletion of hinge exons. Scand. J. Immunol. 32:517.[ISI][Medline]
-
Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680.[ISI][Medline]
-
Smith, R. I. and Morrison, S. L. 1994. Recombinant polymeric IgG: an approach to engineering more potent antibodies. Biotechnology 12:683.[ISI][Medline]
-
Sandlie, I., Aase, A., Westby, C. and Michaelsen, T. E. 1989. C1q binding to chimeric monoclonal IgG3 antibodies consisting of mouse variable regions and human constant regions with shortened hinge containing 15 to 47 amino acids. Eur. J. Immunol. 19:1599.[ISI][Medline]
-
Davis, A. C. and Shulman M. J. 1989. IgMmolecular requirements for its assembly and function [Review]. Immunol. Today 10:118.[ISI][Medline]
-
Fazel, S., Wiersma, E. J. and Shulman, M. J. 1997. Interplay of J chain and disulfide bonding in assembly of polymeric IgM. Int. Immunol. 9:1149.[Abstract]
-
Atkin, J. D., Pleass, R. J., Owens, R. J. and Woof, J. M. 1996. Mutagenesis of the human IgA1 heavy chain tailpiece that prevents dimer assembly. J. Immunol. 157:156.[Abstract]
-
Wiersma, E. J., Chen, F., Bazin, R., Collins, C., Painter, R. H., Lemieux, R. and Shulman, M. J. 1997. Analysis of IgM structures involved in J chain incorporation. J. Immunol. 158:1719.[Abstract]
-
Smith, R. I., Coloma, M. J. and Morrison, S. L. 1995. Addition of a mu-tailpiece to IgG results in polymeric antibodies with enhanced effector functions including complement-mediated cytolysis by IgG4. J. Immunol. 154:2226.[Abstract/Free Full Text]
-
Poon, P. H., Morrison, S. L. and Schumaker, V. N. 1995. Structure and function of several anti-dansyl chimeric antibodies formed by domain interchanges between human IgM and mouse IgG2b. J. Biol. Chem. 270:8571.[Abstract/Free Full Text]
-
Chen, F. H., Arya, S. K., Rinfret, A., Isenman, D. E., Shulman, M. J. and Painter, R. H. 1997. Domain-switched mouse IgM/IgG2b hybrids indicate individual roles for C mu 2, C mu 3 and C mu 4 domains in the regulation of the interaction of IgM with complement C1q. J. Immunol. 159:3354.[Abstract]
-
Vaerman, J. P., Langendries, A. and Vander Maelen, C. 1995. Homogenous IgA monomers, dimers, trimers and tetramers from the same IgA myeloma serum. Immunol. Invest. 24:631.[ISI][Medline]
-
Hauptman, S. P. and Tomasi, T. B., Jr. 1975. Mechanism of immunoglobulin A polymerization. J. Biol. Chem. 250:3891.[Abstract]
-
Yang, C., Kratzin, H., Gotz, H. and Hilschmann, N. 1979. [Rule of antibody structure. Primary structure of a human monoclonal IgA-immunoglobulin (myeloma protein Tro). VII. Purification and characterization of the disulfide bridges] [in German]. Hoppe Seylers Z. Physiol. Chem. 360:1919.[ISI][Medline]
-
Zikan, J., Novotny, J., Trapane, T. L., Koshland, M. E., Urry, D. W., Bennett, J. C. and Mestecky, J. 1985. Secondary structure of the immunoglobulin J chain. Proc. Natl Acad. Sci. USA 82:5905.[Abstract]
-
Dourmashkin, R. R., Virella, G. and Parkhouse, R. M. 1971. Electron microscopy of human and mouse myeloma serum IgA. J. Mol. Biol. 56:207.[ISI][Medline]
-
Feinstein, A., Munn, E. A. and Richardson, N. E. 1971. The three-dimensional conformation of M and A globulin molecules. Ann. NY Acad. Sci. 190:104.[ISI][Medline]
-
Perkins, S. J., Nealis, A. S., Sutton, B. J. and Feinstein, A. 1991. Solution structure of human and mouse immunoglobulin M by synchrotron X-ray scattering and molecular graphics modelling. A possible mechanism for complement activation. J. Mol. Biol. 221:1345.[ISI][Medline]