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Immunoglobulin M (IgM) is one of several forms of antibody that are produced by vertebrates. IgM is the largest antibody, and it is the first antibody to appear in the response to initial exposure to an antigen. In the case of humans and other mammals that have been studied, the spleen, where plasmablasts responsible for antibody production reside, is the major site of specific IgM production.
Immunoglobulins, also known as antibodies, comprise a family of proteins that occur in five major forms, also termed classes or isotypes – IgM, IgD, IgG, IgE and IgA. Immunoglobulins are produced by vertebrate animals as part of the normal immune response to microbial, e.g., bacterial or viral, infection. Binding of the immunoglobulins to a microbe can have immediate effects such as blocking invasion and it can mobilize other components of the immune system to destroy or otherwise inactivate the microbes, thereby providing protection against infectious diseases. The molecular structures that microbes present to the immune system and elicit immunoglobulin (antibody) production are collectively denoted as antigens.
Although all vertebrates that have been studied – from fish to human – produce IgM, there are significant differences among the IgMs of different species. This article focuses on human and mouse IgM, which are both well studied and have very similar properties. For the most part IgM is produced by plasma cells in the spleen and lymph nodes and secreted into serum, where it is typically at a concentration of ~1.5 mg/mL. This article describes the structure of serum IgM, as opposed to other forms, such as the IgM membrane receptor.
Immunoglobulins are composed of two types of protein chain—the heavy chain and the light chain. The heavy chains, which determine the immunoglobulin class, are of five different types, denoted by the Greek letters, µ, δ, γ, ε and α, corresponding to the classes, IgM, IgD, IgG, IgE, and IgA, respectively. Light chains are of two types, denoted λ and κ. Each chain is itself divided into two functional parts, the variable (V) domain and the constant (C) domain. The V domains of the light and heavy chain are folded together to form a structure that binds antigen; different V domains bind different antigens, i.e., binding of antigen by immunoglobulin is antigen specific. Inasmuch as the V domains can occur in a nearly unlimited variety of amino acid sequences, immunoglobulins collectively have the potential of binding to virtually any molecular structure. The immunoglobulin C domains interact with other physiological components, e.g. cellular immunoglobulin receptors and the complement system. Thus, the constant domain of an immunoglobulin can help mobilize phagocytic cells or the complement system to act on an antigen to which it is bound by via variable domain.
The study of IgM began with the report in 1937 that horses hyperimmunized with pneumococcus polysaccharide produced antibody that was much larger than the typical rabbit γ-globulin, with a molecular weight of 990,000 daltons. In accordance with its large size, the new antibody was originally referred to as γ-macroglobulin, and then in subsequent terminology as IgM—M for “macro”. The V domains of normal immunoglobulin are highly heterogeneous, reflecting their role in protecting against the great variety of infectious microbes, and this heterogeneity impeded detailed structural analysis of IgM. Two sources of homogeneous IgM were subsequently discovered. First, the high molecular weight protein produced by some multiple myeloma patients was recognized to be a tumor-produced γ-macroglobulin, and we now know that because the tumor is a clone the IgM it produces is homogeneous. In the 1960s, methods were developed for inducing immunoglobulin-producing tumors (plasmacytomas) in mice, thus also providing a source of homogeneous immunoglobulins of various isotypes, including IgM (reviewed in). More recently, expression of engineered immunoglobulin genes in tissue culture can be used to produce IgM with specific alternations and thus to identify the molecular requirements for features of interest.
Immunoglobulins include 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, and 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, 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 is mostly 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. J chain is a small (~137 amino acids), acidic protein. As shown, 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 J chain would be important for forming the polymeric immunoglobulins, and indeed polymerization of IgA depends strongly (but not absolutely) on J chain. In contrast, polymeric IgM forms efficiently in the absence of J chain.
The predominant form of human and mouse IgM is pentamer. By way of comparison, IgM from frog (Xenopus) is predominantly hexamer, IgM from bony fish is predominantly tetramer, and IgM from cartilaginous fish (shark) is predominantly pentamer. The predominance of pentamer in mouse and human IgM notwithstanding, it was evident that these IgM’s could also exist as hexamer. Subsequent studies using recombinant DNA expression systems indicated that hexamer is a major form of mouse IgM, when the IgM is produced under conditions where the incorporation of J chain is prevented, either by producing IgM in cells that lack J chain or by producing IgM with a µ heavy chain that lacks the cysteine in the tailpiece. In summary, hexameric IgM never contains J chain; pentameric IgM can be formed so as to include or not include 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. In fact 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 J chain-containing pentamer that reflects evidence that 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.
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
To gain insight into the detailed three-dimensional structure of the µ chain, the individual Cµ2, Cµ3 and Cµ4tp domains were produced separately in E. coli and then analyzed by a variety of methods, including sedimentation rate, X-ray crystallography, and NMR spectroscopy. As in the case of other immunoglobulins, the domains of the µ heavy chain have the characteristic overlying β-sheets comprising seven strands, stabilized by the intra-domain disulfide bonds. Overall, the IgM constant region has a “mushroom-like” structure, where the Cµ2-Cµ3 domains are a disk analogous to the head of the mushroom and the Cµ4tp domains protrude like a short stem.
IgM interacts with several other physiological molecules:
- IgM can bind complement component C1 and activate the classical pathway, leading to opsonization of antigens and cytolysis.
- IgM binds to the polyimmunoglobulin receptor (pIgR) in a process that brings IgM to mucosal surfaces, such as the gut lumen and into breast milk. This binding depends on J chain.
- Two other Fc receptors that bind IgM—Fcα/µ-R and Fcµ-R -- have been detected. Fcα/µ-R, like pIgR, binds polymeric IgM and IgA. Fcα/µ-R can mediate endocytosis, and its expression in the gut suggests a role in mucosal immunity. Fcµ-R (formerly known as Toso/Faim3) binds IgM exclusively and can mediate cellular uptake of IgM-conjugated antigen. Inactivation of the corresponding genes in knock-out mice produces a phenotype, but the physiological functions of these receptors are still uncertain
Regulation of the immune response
Specific immunoglobulins that are injected into animals together with their antigen can influence the antibody response to this same antigen. Endogenous antibodies produced after a primary immunization can also affect the antibody response to a booster immunization, suggesting that similar effects occur during physiological conditions. The ”regulatory” effects can be either positive or negative. That is, depending on the type of antigen and the isotype of the antibody, the effect can be suppression or enhancement of the antibody response. Such effects are well illustrated by experiments involving immunization with xenogenic (foreign) erythrocytes (red cells). For example, when IgG is administered together with xenogeneic erythrocytes, this combination causes almost complete suppression of the erythrocyte-specific antibody response. This effect is used clinically to prevent Rh-negative mothers from becoming immunized against fetal Rh-positive erythrocytes, and its use has dramatically decreased the incidence of hemolytic disease of the newborn. In contrast to the effect of IgG, antigen-specific IgM can greatly enhance the antibody response, especially in the case of large antigens. Thus, when IgM specific for erythrocytes is injected into animals (including humans) together with erythrocytes, a much stronger antibody response to the erythrocytes is induced than when erythrocytes are administered alone. Several lines of evidence indicate that the ability of IgM to activate complement is required for its enhancing effect. That is, IgM-mediated enhancement does not occur in animals that have been depleted for complement component C3, nor in mutant animals lacking complement receptors 1 and 2. Similarly, mutant IgM that cannot activate complement does not enhance the immune response. A possible explanation for IgM-mediated enhancement is that B lymphocytes capture IgM-antigen-complement complexes and transport the complexes into areas in the spleen where efficient immune responses are generated. Because IgM is produced early in an immune response, this might be important in the initiation of antibody responses.
In germ-line cells (sperm and ova) the genes that will eventually encode immunoglobulins are not in a functional form (see V(D)J recombination). In the case of the heavy chain, three germ-line segments, denoted V, D and J are ligated together and adjoined to the DNA encoding the µ heavy chain constant region. Early in ontogeny, B cells express both the µ and the δ heavy chains; co-expression of these two heavy chains, each bearing the same V domain depends on alternative splicing and alternative poly-A addition sites. The expression of the other isotypes (γ, ε and α) is effected by another type of DNA rearrangement, a process called Immunoglobulin class switching.
IgM antibodies appear early in the course of an infection and usually reappear, to a lesser extent, after further exposure. IgM antibodies do not pass across the human placenta (only isotype IgG).
These two biological properties of IgM make it useful in the diagnosis of infectious diseases. Demonstrating IgM antibodies in a patient's serum indicates recent infection, or in a neonate's serum indicates intrauterine infection (e.g. congenital rubella syndrome).
The development of anti-donor IgM after organ transplantation is not associated with graft rejection but it may have a protective effect.
IgM in normal serum is often found to bind to specific antigens, even in the absence of prior immunization. For this reason IgM has sometimes been called a "natural antibody". This phenomenon is probably due to the high avidity of IgM that allow it to bind detectably even to weakly cross-reacting antigens that are naturally occurring. For example, the IgM antibodies that bind to the red blood cell A and B antigens might be formed in early life as a result of exposure to A- and B-like substances that are present on bacteria or perhaps also on plant materials.
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