The IgM pentamer is an asymmetric pentagon with an open groove that binds the AIM protein

Abstract

Soluble immunoglobulin M (IgM) forms a pentamer containing a joining (J) chain polypeptide. While IgM pentamer has various immune functions, it also behaves as a carrier of circulating apoptosis inhibitor of macrophage (AIM; also called CD5L) protein that facilitates repair during different diseases. AIM binds to the IgM pentamer solely in the presence of the J chain. Here, using a single-particle negative-stain electron microscopy, we found that the IgM pentamer exhibits an asymmetric pentagon containing one large gap, which is markedly different from the textbook symmetric pentagon model. A single AIM molecule specifically fits into the gap, cross-bridging two IgM-Fc that form the edges of the gap through a disulfide bond at one side and a charge-based interaction at the other side. The discovery of the bona fide shape of the IgM pentamer advances our structural understanding of the pentameric IgM and its binding mode with AIM.

Fig. 1. The 2D structure of IgM pentamer.

(A) Western blotting (WB). FLAG-tagged mouse IgM-Fc and HA-tagged J chain were expressed in HEK293T cells, and the supernatant was analyzed for IgM-Fc (using an anti-FLAG antibody) or the J chain (using an anti-HA antibody) in reducing and nonreducing conditions. As controls, supernatants from cells expressing either or neither molecule were also blotted. Note that the J chain exhibited several bands in a reducing condition with unknown reason. Figure S2 shows the whole picture of the Western blotting in a reducing condition. (B) The negative-stain EM image for the mouse IgM-Fc (plus J chain) and a scheme for the image. Scale bar, 5 nm. Each Ig-like polypeptide (Cμ domain; yellow), J chain (green), and the cysteine residues involved in pentameric formation of Fc monomers (red, Cys⁴¹⁴; blue, Cys⁵⁷⁵) as well as those in the J chain (green) are presented. Black line depicts the disulfide bond. Arrow indicates the specific gap. The central white spot is likely to be an assembly of the tail peptide of each Fc chain (19 amino acids by 10 pieces). (C) The negative-stain EM image for human IgM-Fc pentamer. Scale bar, 5 nm. (D) The negative-stain EM image of monoclonal mouse IgM pentamer and a scheme for the image. Scale bar, 5 nm. The peripheral region corresponds to Fab (gated by dotted lines), which appears to move flexibly and structurally unlocked and thus could not be observed clearly. In (B) to (D), all particles were picked up in a reference free fashion using Gautomatch.

Fig. 2. The structure of oligomeric IgM in the absence of J chain.

(A) The negative-stain EM images for the hexamer and pentamer composed of FLAG-tagged mouse IgM-Fc monomers in the absence of the J chain and their schemes. The frequency of hexamer and pentamer observed in the analysis is described (relative to hexamer as 100). Scale bars, 5 nm. In the scheme, unlike in the presence of the J chain, the disulfide bonds between Cys⁴¹⁴ residues may not be partly developed (21, 25), and they are drawn by shadowed lines. (B) The negative-stain EM images for the pentamer composed by the FLAG-tagged mouse IgM-Fc Cys⁴¹⁴Ser variant in the presence of the J chain, which include asymmetric pentamers, tetramers, and symmetric-like pentamers. Scale bars, 5 nm. The frequency of observed each item is described (relative to asymmetric pentamer as 100). The size of the gap in the asymmetric pentamer was comparable to that in the wild-type IgM-Fc pentamer. In (A) and (B), all particles were picked up in a reference-free fashion using Gautomatch. (C) Western blotting (WB). FLAG-tagged wild type (WT) of Cys⁴¹⁴Ser (C414S) mouse IgM-Fc and HA-tagged J chain were expressed in HEK293T cells, and the supernatant was analyzed for by an anti-FLAG antibody in nonreducing (top) and reducing (bottom) conditions. Cys⁴¹⁴Ser IgM-Fc formed both pentamer and tetramer as judged by size as indicated.

Fig. 3. The defined mode of AIM association with pentameric IgM.

(A) The negative-stain EM image for the mouse IgM-Fc pentamer (with the J chain) associated with AIM. Scale bars, 5 nm. The gap area of the Fc-pentamer carrying a broad bean–like structure (that represents the associated AIM) is also presented at a higher magnification with identification of different Cμ domains by colors. All particles were picked up in a reference-free fashion using Gautomatch. (B) An image subtraction between Fc pentamer with and without AIM association. The associating AIM is a broad bean–like structure. Scale bar, 5 nm. (C) The negative-stain EM image for the FLAG-tagged human IgM-Fc pentamer with the J chain, associated with the human AIM. Scale bar, 5 nm. All particles were picked up in a reference-free fashion using Gautomatch. (D) The number of AIM molecule associated with an Fc pentamer. The Fc-pentamer/J-HA/AIM-HA complex protein was analyzed by Western blotting using an anti-HA antibody in a reducing condition. The AIM-HA band was observed at around 50 kDa in size, whereas the J chain was at around 20 kDa in size. Although the J chain exhibited several bands in a reducing condition, as also described in the legend for Fig. 1A, the number of the band decreased after the column purification. The signal intensity of the bands for AIM-HA and J-HA was quantified using the National Institutes of Health (NIH) ImageJ software. We performed five experiments. The representative blot and the average of the ratio of signal levels are presented.

Fig. 4. Involvement of a disulfide bond and the charge distribution in AIM-IgM association.

(A) A homology-based structural model of AIM. Yellow circles indicate cysteine residues. All cysteines except the solitary cysteine at the SRCR2 domain (Cys¹⁹⁴) form internal disulfide bonds within each SRCR domain. The structural model of AIM and IgM-Fc were built on the basis of Protein Data Bank codes 5a2e and 1o0V. (B) Western blotting of the supernatant from cocultured HEK293 cells expressing FLAG-tagged wild-type (WT) or Cys⁴¹⁴Ser (C414S) variant mouse Fc (with the Myc-tagged J chain) and those expressing wild-type or Cys¹⁹⁴Ser (C194S) variant AIM as indicated combinations, for Fc (using an anti-FLAG antibody) and AIM (using an anti-AIM antibody), in a nonreducing and reducing conditions. The Cys⁴¹⁴Ser Fc variant formed various sizes of oligomers (lanes 3 and 5 in the nonreduced IgM blotting). The loading amount per lane was identical (10 μl of supernatant). (C) The supernatant from cocultured HEK293 cells expressing FLAG-tagged wild-type (with the Myc-tagged J chain) and those expressing AIM of wild-type, AIM-deleted SRCR1 (Δ1) or AIM-deleted SRCR3 (Δ3), was immunoblotted for AIM using an anti-AIM antibody in a nonreducing and reducing conditions. Note that the expression of Δ1 in the supernatant was lower than others, as observed in the reduced blots. Nevertheless, the binding to Fc pentamer was obvious in contrast to Δ3 that exhibited no binding to IgM-Fc pentamer. (D) 3D mapping of the charge distribution of amino acids for mouse IgM-Fc monomer and mouse AIM on the homology-based models. Scale bar, 2 nm. For IgM-Fc, the homology-based model in which each Cμ domain is identified by different color is also shown for obvious orientation. The simplified schemes for IgM-Fc and AIM are also presented. Blue, positively charged; red, negatively charged; white, neutrally charged. The cluster of negatively charged amino acids in IgM Cμ4 domain(s) and positively charged amino acids in AIM SRCR3 domain (including His²⁹⁴, Lys²⁹⁸, Arg³⁰⁰, Lys³⁰¹, and Lys³⁴⁰) are gated. Note that the tail chain of the Fc is not included in the model. (E) Hypothetic schema of how AIM associates with the IgM-Fc pentamer. Left: A simplified scheme. The AIM containing three SRCR domains is depicted schematically (pink). Yellow circle indicates the solitary cysteine residue at the SRCR2 domain (Cys¹⁹⁴) of AIM. The SRCR1 domain may move flexibly. Right: A model depicted by reflecting the information of homology modeling and the charge distribution. The charge distribution is reflected only in the AIM molecule and the associating Fc-monomer, whereas other Fc units are colored for different Cμ domains as in (D). Red circle indicates the disulfide bond between the Cys¹⁹⁴ of AIM and the Cys⁴¹⁴ of the Fc Cμ3, and black circle indicates the charge interaction between AIM-SRCR3 and the Fc Cμ4. Note that the tail chain of the Fc and the J chain are not included in the model. Scale bar, 5 nm. The size and the structure of the homology model matches the averaged EM images presented in Figs. 1 and 3. In (D) and (E), all molecular graphics representations were created using PyMOL Molecular Graphics System (www.pymol.org).