Mapping of the binding site for FcµR in human IgM-Fc

Fc µ R is a high-affinity receptor for the Fc portion of human IgM. It participates in B cell activation, cell survival and proliferation, but the full range of its functions remains to be elucidated. The receptor has an extracellular immunoglobulin (Ig)-like domain homologous to those in Fc α / µ R 1 and pIgR, but unlike these two other IgM receptors which also bind IgA, Fc µ R exhibits a binding specificity for only IgM-Fc. Previous studies have suggested that the IgM/Fc µ R interaction mainly involves the C µ 4 domains with possible contributions from either C µ 3 or C µ 2. To define the binding site more precisely, we generated three recombinant IgM-Fc proteins with specific mutations in the C µ 3 and C µ 4 domains, as well as a construct lacking the C µ 2 domains, and analyzed their interaction with the extracellular Ig-like domain of FcµR using surface plasmon resonance analysis. There is a binding site for Fc µ R in each IgM heavy chain. Neither the absence of the C µ 2 domains nor the quadruple mutant D340S/Q341G/D342S/T343S (in C µ 3 adjacent to C µ 2) affected FcµR binding, whereas double mutant K361D/D416R (in C µ 3 at the C µ 4 interface) substantially decreased binding, and a single mutation Q510R (in C µ 4) completely abolished FcµR binding. We conclude that glutamine at position 510 in C µ 4 is critical for IgM binding to FcµR. This will facilitate discrimination between the distinct effects of Fc µ R interactions with soluble IgM and with the IgM BCR.


Introduction
The binding of immunoglobulins to their receptors via the Fc domains is key to expressing effector functions that are essential in host defense. Identification of the binding sites for these receptors on immunoglobulin Fc regions is therefore critical for understanding the molecular pathways through which they act.
Until the year 2000, the only known human IgM-Fc receptor was polymeric immunoglobulin receptor (pIgR), which binds both IgM and IgA and is expressed on basolateral surfaces of mucus epithelium and ducts of secretory glands [1]. A second receptor, designated Fcα/µR and expressed on follicular dendritic cells, macrophages and lymphocytes in humans [2,3], also binds IgM and IgA [4,5]. The recently discovered human FcµR [6] also referred to as FAIM3 or TOSO, is a high-affinity human IgM-Fc receptor expressed on B cells, T cells and a subset of NK cells [7][8][9]. The functions of FcµR are yet to be fully explored [10,11] but the receptor is thought to be involved in tonic signalling, early B cell activation and regulation of B cell-mediated T cell immunity [12][13][14][15][16].
FcµR is a 390-amino acid (aa) polypeptide consisting of a 17-aa signal peptide and 107-aa Ig-like domain, followed by a further 127-aa extracellular region, a 21-aa transmembrane portion that has a charged histidine residue and a 118-aa cytoplasmic tail. The receptor has no N-linked glycosylation site [7,17], however, O-linked glycosylation in the stalk region has been reported [18].
The present study focuses on the FcµR binding properties of IgM-Fc. We generated the recombinant extracellular Ig-like domain of human FcµR (sIgFcµR), IgM-Fc with and without the Cµ2 domains, and IgM-Fc with site-directed mutations, for binding analysis by surface plasmon resonance (SPR), to identify the structural determinants of IgM-Fc responsible for FcµR binding.
Previous work has shown that the Cµ3 and Cµ4 domains of polymeric IgM are involved not only in binding FcµR [6], but also the human pIgR and Fcα/µR receptors [1,19,20]. Using a panel of domainswapped antibodies, a recent study [21] identified the Cµ4 domain as the dominant region of IgM-Fc for FcµR binding, with a minor contribution from the Cµ2 and/or the Cµ3 domains; molecular dynamics simulations of models of this interaction favoured involvement of Cµ2 residues together with Cµ4 [21]. We now report studies using site-directed mutagenesis and fragments of IgM-Fc to map more precisely the FcµR binding site and assess the contributions of the Cµ2, Cµ3 and Cµ4 domains.

Cloning and expression of sIgFcµR
The cloned receptor in Zero Blunt TOPO was kindly provided by Prof. H. Kubagawa. The coding sequence for the extracellular Ig-like domain was cloned into the plasmid expression vector pET24+ and expressed in BL21 (DE3) competent cells at 37 °C under the control of the T7 promoter. The oligonucleotides used were 5'-TGAGATCCGGCTGCTAACAAAG-3' and 3'-TAAAACAAATTGAAATTCTTCCTCTATATGTA-5'. Cells were cultured in 1 L of ampicillinsupplemented (50 µg/mL) LB broth and grown at 37 ºC with orbital shaking at 225 rpm. At an OD600 between 0.6 and 0.8, isopropyl β-D-1-thiogalactopyranoside (IPTG) was added to a final concentration of 1 mM to induce protein expression. The cells were grown overnight at 37 °C and pelleted by centrifugation at 4000 g for 20 min at 4 °C. The supernatant was discarded, and the cell pellets were stored at -80 °C.

Purification of sIgFcµR protein
The cell pellets were thawed on ice and re-suspended in 50 mL lysis buffer (1M NaCl, 50mM Tris-HCL, pH 7.5 and 1 tablet of Complete EDTA-free Protease Inhibitor (Roche)). The cells were then passed through a pre-chilled cell disrupter twice, under a pressure of 1000 psi. The disrupted cells were centrifuged at 16000 g for 15 min at 4 °C. The pellets were washed 5 times in 30 mL of 1 M NaCl, 50 mM Tris-HCL, pH 7.5 and 2% Triton-X. After the final centrifugation, the inclusion bodies were dissolved with solubilization buffer (6 M guanidine hydrochloride in 0.5 M Tris acetate buffer, pH 8.6 containing 2 mM β-mercaptoethanol), for 1 hr at 37 ºC. Insoluble contaminants were pelleted by centrifuging at 14000 rpm for 15 min at 4 °C, and the supernatant containing the protein collected. The sIgFcµR protein was slowly diluted 50-fold into ice-cold refolding buffer (0.1M Tris acetate pH 8.6, 0.4 mM oxidized glutathione and 2 mM reduced glutathione), with rapid stirring at 4 ºC and left to stand for 72 hrs. Before purification, NaCl and imidazole were added to the refold solution to a final concentration of 0.5 M and 10 mM, respectively and the pH adjusted to between 7 and 8 using 1 M acetic acid. The solution was filtered with 0.45 µM membrane filter (Millipore) before purification by nickel column chromatography. The purity of the refolded sIgFcµR was analyzed by 18% SDS-PAGE.

Stable expression of IgM-Fc (Fcµ2-4) mutants
All IgM proteins were stably transfected into human cells. HEK293 cells were seeded in 96-well plates to 2 x 10 5 cells/well and cultured overnight in a 5% CO2 incubator at 37 ºC using the transfection medium (Dulbecco's modified Eagle's medium supplemented with 10% of fetal bovine serum and 5% penicillin/streptomycin). The appropriate DNA was co-transfected with expression vector pOG44 (100 ng) using Fugene tranfection reagent (Promega) following the protocol supplied by the manufacturer. After 48 hours, the cells were split into 24 well plates in transfection medium containing hygromycin B at a final concentration of 100 µg/mL. Once the cells were confluent, they were trypsinised and seeded into 1 L of Freestyleä serum-free medium containing 5% penicillin-streptomycin and hygromycin B at a final concentration of 100 µg/mL. The cells were grown in spinners for 3-4 weeks and then harvested. Supernatants were centrifuged at 4000 g for 30 minutes and then filtered through a 0.45 µM filter (Millipore). 0.1% sodium azide was added and the supernatants were stored at 4 °C. Proteins were first purified on an AKTA Prime system (Amersham) using nickel column chromatography and then on the Gilson HPLC using a Superdex 200, 10/300 column (GE Healthcare).

Multi-angle Laser Light Scattering (MALLS)
MALLS studies were performed in-line with SEC on mutant IgM protein samples to assess monodispersity and the molecular mass of the protein samples. The peaks corresponding to the homodimeric Fcµ2-4 mutants from SEC were run on the Superdex 200 Increase 5/150 column (GE Healthcare) using an in-line miniDAWN multi-angle light scattering detector and an Optilab DSP Interferometric Refractometer (Wyatt Technology). The data were analysed using the ASTRA 4.9 software (Wyatt Technology).

Circular dichroism (CD) analysis
Far-UV (190-280) CD spectra were acquired on a Chirascan Plus spectropolarimeter (Applied Photophysics Ltd) flushed continuously with pure evaporated nitrogen throughout the experiments.
Measurements were recorded in a 0.5 mm strain-free rectangular cell using a 2 nm spectral bandwidth, 1 nm step-size and 1 sec measurement time-per-point. The concentrations of the protein samples used ranged between 0.1 mg/mL -0.4 mg/mL and buffer used was 10 mM HEPES and 150 mM NaCl at pH 7.4. All spectra were acquired at 25 °C and buffer baseline corrected before analysis.

Surface plasmon resonance (SPR) analysis
All SPR assays were performed on a Biacore T200 instrument (Biacore, Uppsala, Sweden) at 25 °C in running buffer (0.01 M HEPES, 0.15 M NaCl and 0.005% Surfactant P20, pH 7.4). Serum IgM, IgM-Fc, Fcµ3-4 and IgM-Fc (Fcµ2-4) mutants were immobilized on Biacore CM5 chips in 10 mM sodium acetate, pH 4.5, using standard amine coupling chemistry. The analyte was sIgFcµR in concentrations ranging from 0.25 µM -12 µM. In each experiment, the binding curves were corrected by subtracting the signal obtained from the control flow cell. Non-specific binding was less than 30%. Steady state binding curves were analyzed using Biacore T200 evaluation software. The errors in the KD values represent the fitting of the 1:1 binding model to the experimental data.
To investigate the stoichiometry of sIgFcµR binding to IgM or its homodimeric fragments, the following equation was used to calculate the activity of the immobilized IgM or fragments (which can be up to 100%, depending on whether any is damaged or misoriented by immobilization): % Activity = 100 x (Mol. Wt. of IgM or homodimeric fragment x Rmax of sIgFcµR bound)/ (Mol. Wt. of sIgFcµR x RU of IgM or homodimeric fragment immobilized).

Results
The extracellular Ig-like domain of human FcµR (sIgFcµR) was expressed as described in Material and Methods. After affinity purification, SEC was conducted to ensure the purity of the protein ( Figure 1A). Fractions from the main peak were collected and further checked by analytical SEC before SPR analysis; the fraction used co-migrated in SEC with cytochrome c (molecular weight 12.4 kDa). The far-UV CD spectrum showed a characteristic β-sheet signature of a folded polypeptide with a broad minimum near 218 nm ( Figure 1B). SPR analysis of sIgFcµR binding to IgM, IgM-Fc (also termed Fcµ2-4, consisting of two each of the Cµ2, Cµ3 and Cµ4 domains) and Fcµ3-4 (lacking the Cµ2 domains), yielded KD values of 2.49 ± 0.54 µM, 1.15 ± 0.07 µM and 1.09 ± 0.09 µM respectively ( Figure 2). This showed that neither pentamerisation, presence of the Fabs nor the Cµ2 domains substantially affected binding of sIgFcµR.  SPR has limitaitons as a method to determine the absolute stoichiometry between, in this case, an antibody or fragment and its receptor, due to inevitable loss of activity caused by chemical coupling of the antibody or fragment to the matrix. However, since the initial baseline signal in RU is proportional to the mass of antibody or fragment immobilizsed, and the additional signal in RU is proportional to the mass of receptor bound, it is possible to calculate the theoretical maximum expected signal assuimg a particular stoichiometry of binding. For each of the proteins, IgM, IgM-Fc and Fcµ3-4, the ratio of Rmax (the extrapolated maximum SPR signal for sIgFcµR binding in resonance units, RU) to the number of RU immobilized on the sensor chip, taking into account the relative molecular weights (900 kDa, 76 kD and 51 kDa respectively), indicated that each IgM heavy chain has a binding site for sIgFcµR.  [23,24], as well as other immunoglobulin-Fc/receptor structures [25,26], were used to predict likely binding sites for FcµR in IgM-Fc. Furthermore, residues in IgA Ca3 have been implicated in pIgR binding [27][28][29], and there is an homologous loop in IgM Cµ4. Thus IgM-Fc residues structurally homologous to those involved in IgE-Fc/FcεRIα, IgE-Fc/CD23, IgA-Fc/FcαR and IgA-Fc/pIgR interactions were targeted with mutations. A striking similarity between the nature and location of the binding sites in IgG and IgE for their Fc receptors has been observed [26,30], and thus the proposition that the IgM-Fc/FcµR binding site might be structurally homologous to one of these interactions is not unreasonable.   The KD values were generated from the steady state binding curves using Biacore T200 evaluation software.

Discussion
When the receptor FcµR was first identified, the binding region in IgM was shown to lie (principally) within the Cµ3 and Cµ4 domains since an Fcµ5 fragment, consisting largely of these domains, inhibited the interaction [6,17]. A later study with "domain-swapped" antibody Fc regions confirmed the involvement of the Cµ4 domain and implicated a possible contribution from either Cµ2 or Cµ3 [21]. In order to locate more precisely the binding site for FcµR in IgM-Fc, we produced for the first time the recombinant extracellular Ig-like domain of FcµR (sIgFcµR) in E. coli, and studied its binding to IgM, IgM-Fc, a series of IgM-Fc mutants and a sub-fragment of IgM-Fc lacking the Cµ2 domains.
The binding of purified sIgFcµR to whole serum IgM (largely pentameric), IgM-Fc (Fcµ2-4), and the Fc sub-fragment Fcµ3-4, was investigated by SPR ( Figure 2). It is clear that sIgFcµR interacts with both whole IgM and the two Fc fragments with similar binding affinities, indicating that the Cµ2 domains do not contribute to FcµR binding. These results also show that unlike pIgR and Fca/µR [19,20], FcµR can bind to both homodimeric and polymeric IgM. Assuming a 1:1 stoichiometry of each heavy chain to sIgFcµR, a binding affinity of KD ~ 1-2.5 µΜ was determined by SPR. This is ~ 100 fold weaker than cell surface FcµR binding to pentameric serum IgM, which is reported as KD ~ 10 nM [6]; the difference is presumably due to the very high effective concentration of FcµR on the cell surface and the possibility of simultaneously engaging more than one receptor domain (i.e. an avidity effect).
In order to determine the FcµR binding site on IgM, we generated three IgM-Fc mutants, M1, M2 and M3 ( Figure 3) and studied their interaction with the receptor sIgFcµR by SPR ( Figure 6). Mutations in Cµ3 (M3, Figure 3) corresponding to the location of the high-affinity receptor FceRI in IgE-Fc, had no effect upon sIgFcµR binding. In contrast, mutation Q510R in Cµ4 (M2, Figure 3) abrogated receptor binding completely. A glutamine residue lies within the structurally homologous loop in the Ca3 domain of IgA-Fc, which has been implicated in the IgA-Fc/pIgR interaction [27]. A double mutation (K361D/D416R) in Cµ3 (M1, Figure 3), adjacent to Cµ4 in the region to which CD23 binds in IgE, and FcaR in IgA-Fc, also substantially reduces sIgFcµR binding. These results establish that residue Q510, located in an exposed loop region of Cµ4, is a key component of the binding site for FcµR that may also encompass an adjacent part of Cµ3.
Lloyd et al. proposed a model of the interaction between IgM-Fc and FcµR, based upon molecular dynamics simulations, that involved several residues from Cµ4, but not Q510, together with a contribution from Cµ2 [21]. Their experimental data implicated involvement of Cµ4 with minor contributions from either Cµ2 or Cµ3. Our data suggest that it is Cµ3, but not Cµ2, that may be involved together with Cµ4. Q510 is however adjacent to Cµ4 residues in the model of Lloyd et al., and taken together they may define an extended binding region ( Figure 7A) that could be encompassed by the (modelled) Ig-like domain of FcµR ( Figure 7B).
In cryoEM structures and models of pentameric and hexameric IgM [32][33][34], the Q510 loop and adjacent Cµ4 residues are accessible. Residues 361 and 416 however, lie at the interface between subunits in these polymeric structures, although they would presumably be exposed in membrane IgM expressed on B cells as part of the B cell receptor (BCR) for antigen. The FcµR binding site very likely overlaps with part of that of pIgR, by analogy to the location identified in IgA Ca3 [27][28][29] and the fact that Cµ4 is involved in binding to IgM [19]; furthermore, overlap with the Fca/µR binding site is also likely since residues identified as involved in Fca/µR binding to IgA are located in adjacent loop regions [20].
The functional significance of this clustering of IgM receptor binding sites in Cµ4 is unclear, although some different cell types which express these receptors do co-localise (e.g. FDCs and B cells in germinal centres or epithelial cells and B cells) and could lead to competition for IgM. However, the Cµ4 domain has also been identified as the binding site for the erythrocyte membrane protein pfEMP1 expressed by cells infected with Plasmodium falciparum [35], and interference with FcµR binding has been proposed as a possible means by which this malarial parasite evades the host immune system [36].

Conclusions
There have been contradictory findings in relation to the function of FcµR [10]. The identification of a critical amino-acid residue substitution in Cµ4 (Q510R) now permits the preparation of pentameric IgM which cannot bind FcµR; this could be used to investigate B cell reactions in which FcµR can only interact with the IgM BCR. Indeed, if this mutation could be effected in the IgM BCR, then the proposed  [21] are coloured in blue and light purple, respectively. The residues K361D, D416R and Q510R reported in our study are coloured in orange, cyan and pink, respectively. Panel B: model of sIgFcµR. The models of Fcµ3-4 and sIgFcµR were predicted using the Swiss Model server [31]. The template for Fcµ3-4 was IgE-Fc (PDB ID: 2WQR) and for sIgFcµR the template was camelid VHH fragment (PDB ID: 5JMR).
interaction with FcµR and its role in B cell survival, IgM BCR expression and tonic signalling [13,15] could be tested directly.