Research Progress on the Molecular Mechanism of Immunoglobulin IgM

Immunoglobulin serves as a principal element of the human adaptive immune system. There are two forms of immunoglobulin: Ig serves as the main element of the B cell receptor system when located at the B cell surface through its transmembrane region but converts to an antibody when released externally by plasma cells. The human body contains five immunoglobulin categories based on heavy chain types which include IgM, IgD, IgG, IgE and IgA.

IgM represents both the most ancient immunoglobulin discovered through evolution and the initial antibody to emerge during human development and immune response. It plays an important role in the early stages of pathogen infection. IgM in serum mainly exists in the form of pentamers and a small amount of hexamers, and the structural characteristics of the polymer are crucial to its function: First, IgM produced before other antibodies usually has only low affinity for antigens, while the polymer structure increases the valence of IgM, First, it can bind antigens with strong total affinity (avidity); second, the polymer structure gives IgM a strong complement activation ability, allowing IgM to connect to natural immunity through the classical complement activation pathway; third, IgM pentamers can be secreted to the mucosal surface to participate in mucosal immunity. IgM pentamers are composed of 10 light chains (light chain, L), 10 heavy chains and 1 J chain (joining chain, J-chain), with a molecular weight of about 900 kD and 10 antigen binding sites. The light chain is a κ chain or a λ chain, both of which contain 1 variable region (VL) and 1 constant region (CL). The heavy chain contains 1 variable region (VH) and 4 constant regions (Cμ1–Cμ2–Cμ3–Cμ4). In addition, the carboxyl terminus of the IgM heavy chain contains a tail fragment (tp) consisting of 18 amino acids, which plays an important role in the assembly process of IgM polymers. The J chain is an important component of the IgM antibody, It plays an important role in the assembly and mucosal transport of IgM pentamers. In the absence of J chain, IgM can form hexamers to a certain extent.

Structural Biology Research on IgM

The research on IgM structure can be traced back to the 1960s. These early studies first used negative staining electron microscopy to observe IgM molecules isolated from serum and found that IgM was in the shape of a flat five-pointed star. With the development of structural biology technology, the structure of a single domain of IgM has been resolved, and the structure reveals that the Cμ2, Cμ3 and Cμ4 domains all adopt typical Ig folding, and it is found that Cμ4–tp is essential for the assembly of IgM polymers. In recent years, breakthroughs in sample preparation technology and electron microscopy technology have greatly promoted IgM-related structural biology research.

The Structure of IgM-BCR

Study IgM is a core component of IgM-BCR when it exists in the form of membrane-bound monomers, and plays a key role in the development of B cells in the bone marrow and spleen. BCR is composed of membrane-bound immunoglobulin (mIg) and Igα/Igβ (CD79a/CD79b) heterodimers. Among them, mIg is responsible for recognizing and binding antigens, while Igα/Igβ activates downstream signaling pathways through its immunoreceptor tyrosine activation motif (ITAM). BCR activates B cell activation and proliferation by specifically recognizing and binding antigens, prompting them to differentiate into plasma cells or memory cells, thereby producing antibodies to resist foreign pathogens and forming immune memory. Although the components of BCR were identified as early as 1990, its fine structure and assembly mechanism have not been revealed until recently. Researchers reported the cryo-electron microscopy structure of the human IgM-BCR complex. Structural analysis shows that membrane-bound IgM (membrane-bound IgM, mIgM) binds to Igα and Igβ in a stoichiometric ratio of 1:1:1. The assembly of the IgM-BCR complex is mediated by the extracellular IgM-Cμ4 and the Ig-like domain of Igα/Igβ, as well as the connecting peptide (CP) and transmembrane (TM) helix. In the extracellular region, the Cμ4 domain of the mIgM heavy chain is tightly stacked with the extracellular domain of Igα/Igβ; in the transmembrane region, the two heavy chains of mIgM and the two transmembrane helices of Igα/Igβ form a tight helical bundle, and the helices are stabilized by conservative hydrophobic and polar interactions; in the proximal membrane region, the CP between the mIgM-Fc terminus and the TM helix is inserted into the cavity formed by Igα/Igβ, further stabilizing the structure of the IgM-BCR complex. Free IgM exists in the form of polymers, In the resting IgM-BCR, only the monomeric state of IgM is observed. Structural analysis suggests that the Ig-like domain of Igα prevents IgM Cμ4 from forming multimers. Early studies have found that BCR activation is usually accompanied by the formation of BCR multimers. However, the potential mechanism of how antigen binding mediates the conversion of the monomeric form in the resting state to the multimeric form in the activated state remains to be further studied.

Structural Study of IgM Multimers

In 2020, researchers analyzed the 3.4 Å in vitro recombinant human SIgM cryo-electron microscopy structure, including the IgM-Fc (Fcμ) pentamer core region (Cμ3–Cμ4–tp), J chain and secretory component SC, allowing people to clearly observe the overall assembly of IgM pentamers at the atomic level for the first time. The structure shows that IgM forms pentamers in an asymmetric manner, and the five IgM monomers are arranged into a hexagon with a gap according to the principle of nearly perfect six-fold symmetry, and the gap angle is about 61°. The asymmetric structural feature is of great significance to the formation of IgM hexamer. It allows the sixth IgM monomer to bind to the gap relatively easily in the absence of J chain, thus forming IgM hexamer. Structural analysis revealed that Cμ3, Cμ4 and tp of IgM are all involved in the assembly of pentamer, among which tp at the carboxyl terminus of IgM is located in the center of the structure, providing the most important force to stabilize the IgM pentamer.

Structural Study of IgM and Receptors

Fc receptors are an important class of receptors in the human immune system. They play a key role in immune responses by binding to the Fc region of immunoglobulins. Different immunoglobulins bind to different Fc receptors, specifically triggering different downstream signaling pathways and immune responses. There are three types of IgM Fc receptors in the human body, namely pIgR, FcμR and FcαμR. The three are located in the same region of the chromosome and are structurally related. pIgR is a dual receptor for IgA and IgM, responsible for transporting IgA and IgM with J chains to the mucosal surface. Subsequently, the extracellular domain SC of pIgR is cleaved and secreted together with IgA and IgM to form SIgA and SIgM. SC is composed of 5 Ig-like domains (D1–D2–D3–D4–D5). The previous crystal structure revealed that SC presents a closed conformation in the unliganded state, and the overall shape is similar to an isosceles triangle. The D1 domain responsible for recognizing IgM is buried inside the triangle. In the SIgM complex structure, SC undergoes a dramatic conformational change, resembling a right triangle, and vertically binds to the plane formed by Fcμ–J. In this state, SC presents a stretched conformation, exposing the three complementary determining regions (CDR)-like loops of the D1 domain, thereby interacting with IgM. Structural study of IgM and CD5L

CD5-like antigen molecule (CD5L) is a protein expressed and secreted by macrophages, belonging to the scavenger receptor cysteine-rich superfamily (SRCR), also known as Spα or macrophage apoptosis inhibitor (AIM). CD5L has diverse functions. It can be used as a pattern recognition receptor to recognize a variety of pathogens and endogenous harmful substances, and can also inhibit apoptosis of thymocytes, T cells and other cells. In addition, CD5L is also involved in the pathological process of diseases such as obesity and atherosclerosis. Injection of recombinant CD5L protein can significantly reduce the mortality of septic mice, suggesting that CD5L may become a potential biological therapy for the treatment of sepsis. These studies show that CD5L plays an important role in immune homeostasis and disease development and treatment. In previous studies, researchers first found another protein in human serum IgM in addition to the J chain, which was later identified as CD5L.

Structural Study of IgM and Pathogen Proteins

IgM plays a vital role in the body’s resistance to infection and can promote the clearance of pathogens by efficiently activating the complement pathway. However, pathogens have evolved strategies to counter the immune function of IgM in the arms race of co-evolution with humans. Plasmodium falci parum infection causes the most severe malaria in humans. It can express a series of virulence proteins containing Duffy binding-like domains (DBL domains), such as VAR2CSA, TM284VAR1, DBLMSP and DBLMSP2. Early studies revealed that these virulence proteins may promote immune escape by recruiting IgM molecules. A study reported the cryo-electron microscopy structure of the complex of the above four virulence proteins and the IgM core region, revealing that there are many different interaction modes between Plasmodium falciparum proteins and IgM. Although the binding modes of these Plasmodium falciparum proteins and IgM are different, their binding sites on IgM are all located in the Cμ4 region, and the J chain is not involved in this interaction, suggesting that the IgM hexamer may also become a target of Plasmodium falciparum proteins. Another study found that VAR2CSA can bind to the IgM pentamer at a stoichiometric ratio of 2:1, with two VAR2CSA molecules binding to both sides of the IgM plane, and it is speculated that the Cμ1-Cμ2 region of IgM presents a bent conformation compared to the IgM plane, and is arranged alternately up and down around the two VAR2CSA molecules, thereby hindering the binding of VAR2CSA to chondroitin sulfate. Given that the VAR2CSA protein is expressed on the surface of infected red blood cells, if IgM can interact with VAR2CSA molecules from different red blood cell surfaces at the same time, It may bridge adjacent red blood cells, causing the rosetting phenomenon between red blood cells. This aggregation may interfere with the immune system’s recognition and clearance of infected red blood cells, thereby further providing a mechanism for malarial parasites to escape immunity.