Immunoglobulins are composed of light chains and heavy chains. The light chain (λ or κ) is a protein of ~220 amino acids, composed of a variable domain, VL (a segment of approximately 110 amino acids), and a constant domain, CL (also approximately 110 amino acids long). The μ heavy chain of IgM is a protein of ~576 amino acids, includes a variable domain (VH ~110 amino acids), four distinct constant region domains (Cμ1, Cμ2, Cμ3, Cμ4, each ~110 amino acids) and a "tailpiece" of ~20 amino acids. The μ heavy chain bears oligosaccharides at five asparagine residues. The oligosaccharides on mouse and human IgM have been partially characterized by a variety of techniques, including NMR, lectin binding, various chromatographic systems, and enzymatic sensitivity (reviewed in). The structure of the oligosaccharides at each site varies in detail, and the predominant oligosaccharides—biantennary, triantennary, and high mannose—differ among the sites. The multimeric structure of IgM is shown schematically in Figure 1. Figure 1A shows the "heterodimer" composed of one light chain, denoted L, and one heavy chain, denoted μ. The heavy and light chains are held together both by disulfide bonds (depicted as red triangles) and by non-covalent interactions. Figure 1B shows two μL units linked by a disulfide bond in the Cμ2 domains; this (μL)2 structure is often referred to as the IgM "monomer", as it is analogous in some ways to the structure of
immunoglobulin G (IgG). On the basis of its sedimentation velocity and appearance in electron micrographs, it was inferred that IgM usually occurs as a "pentamer", i.e., a polymer composed of five “monomers” [(μL)2]5, and was originally depicted by the models in Figures 1C and 1D, with disulfide bonds between the Cμ3 domains and between the tail pieces. Also shown is that pentameric IgM includes a third protein, the J chain. J chain (J for joining) was discovered as a covalently bonded component of polymeric IgA and IgM. The J chain is a small (~137 amino acids), acidic protein. As shown, the J chain joins two μ chains via
disulfide bonds involving cysteines in the tailpieces.
Molecular requirements for forming polymeric IgM It was initially expected that the J chain would be important for forming the polymeric immunoglobulins, and indeed polymerization of IgA depends strongly (but not absolutely) on the J chain. In contrast, polymeric IgM forms efficiently in the absence of the J chain. The predominant form of human and mouse IgM is the pentamer. By way of comparison, the structure of IgM from frogs (Xenopus) is predominantly hexameric, IgM from bony fish is predominantly tetrameric, and IgM from cartilaginous fish (mainly sharks) is predominantly pentameric. Although the pentameric form predominates in mice and humans, the hexameric form has also been observed. Subsequent studies using recombinant DNA expression systems indicated that a hexamer is a major form of mouse IgM when the IgM is produced under conditions where the incorporation of the J chain is prevented, either by producing IgM in cells that lack the J chain In summary, hexameric IgM never contains the J chain; pentameric IgM can be formed so as to include or not include the J chain. An important difference between the μ and γ heavy chains is the availability of cysteines for forming disulfide bonds between heavy chains. In the case of the γ heavy chain, the only inter-γ bonds are formed by cysteines in the hinge, and accordingly, each γ chain binds to only one other γ chain. By contrast, the Cμ2 and Cμ3 domains and the tailpiece each include a cysteine that form a disulfide bond with another μ chain. The cysteines in the Cμ2 domains mediate the formation of monomeric IgM (μL)2. The tailpiece along with the included cysteine is necessary and sufficient for the formation of polymeric immunoglobulins. That is, deleting the tailpiece from the μ heavy chain prevents the formation of polymeric IgM. Conversely, cells expressing a γ heavy chain that has been modified to include the tailpiece produce polymeric IgG. The role of the cysteine in the Cμ3 domain is more subtle. Figures 1C and 1D represent possible models for pentameric IgM. In these models, each μ chain is envisaged to bind two other μ chains. However, neither model alone can fully account for the structure of polymeric IgM. For example, the model in Figure 1C predicts that the disulfide bond between the Cμ2 domains is essential for making disulfide-bonded polymeric IgM. The model in Figure 1D predicts that the disulfide bond between the Cμ3 domains is essential. Disulfide bonded, polymeric, IgM can still be made if any one of the three cysteines is absent. In the context of models in which each μ chain interacts with only two other μ chains, these results suggest that some molecules are like Figure 1C and some like Figure 1D. However, the availability of three cysteines for inter-μ chain bonding suggests that the μ chains might each bind three other μ chains, as illustrated in Figure 2. In the same spirit, Figure 2C presents a model for a J chain-containing pentamer that reflects evidence that the J chain joins μ chains that are not joined to other μ chains by the cysteines in the Cμ3 domains. These and other models, both regular and irregular are discussed elsewhere. File:Some alternative ways of linking µ chains.jpg|thumb|Figure 2. Some alternative ways of linking μ chainsA, B) These figures depict two of many possible models of inter-μ chain disulfide bonding in hexameric IgM. As in Figure 1, the Cμ2 disulfide bonds and the Cμ4tp disulfide bonds are represented by a red double arrowhead, and the Cμ3 disulfide bonds are represented by the long double-headed arrows. In both models A and B each type of disulfide bond (Cμ2-Cμ2; Cμ3-Cμ3; Cμ4tp-Cμ4tp) joins μ chains eries with each of the others. Methods for distinguishing these and other models are discussed in reference [28].C) This representation of pentameric IgM illustrates how the J chain might be bonded to μ chains that are not linked via Cμ3 disulfide bonds Pentameric IgM is typically represented as containing a single J chain per polymer, but in actuality the measurements of J chain stoichiometry have ranged from one J molecule per polymer to three J molecules per polymer. The wide range might be due to technical problems, such as incomplete
radiolabeling or imprecisely quantitating an Ouchterlony line. However, the variation might also be due to heterogeneity in the IgM preparations, i.e., the various preparations might have differed substantially in their content of J-containing and J-deficient polymers.
Tertiary and quaternary structure of the μ constant region Individual C2, C3, and C4tp domains were generated independently in
E. coli and then studied using a range of approaches, including sedimentation rate,
X-ray crystallography, and
NMR spectroscopy, to obtain insight into the detailed three-dimensional structure of the chain. The domains of the heavy chain, like those of other immunoglobulins, have the distinctive overlaying -sheets of seven strands, which are stabilized by intra-domain disulfide linkages. Overall, the IgM constant region has a "mushroom-like" shape, with the C2-C3 domains forming a disk similar to the mushroom's head and the C4tp domains protruding like a short stem. ==Function==