Immunoglobulin M

IgM is the oldest phylogenetic class of Ab which is expressed by all vertebrate species 154. IgM is the first Ab to be produced during humoral responses, do not undergo somatic hypermutation or Ab class switching and are therefore of low affinity 155. IgM exists as a membrane-bound form on B cells (BCR), in the circulation, and also on mucous membranes or in excretory gland secretions as secretory IgM 155. Circulatory IgM is present at ~1-2mg/ml in the blood of healthy adults, principally as a pentamer and occasionally as a hexamer 156. Unlike other immunoglobulin isotypes, circulatory IgM can be divided into non-immune and immune IgM 135. Non-immune IgM molecules are low affinity and broadly reactive Abs which are generated by the B-1 subset of B cells in the absence of exogenous antigens 157, 158. The multimeric structure of non-immune IgM compensates for the low antigen-binding affinity by creating 10 identical antigen-binding sites (Figure 1.6), which increase overall avidity when binding to multivalent antigens such as bacterial capsular polysaccharides 135, 156. In addition, the multimeric structure of IgM facilitates complement activation in a highly efficient manner. Immune IgM is produced by B-2 cells in response to pathogenic exposure and differs from natural IgM in its affinity, specificity repertoire, range of function, and antigen-binding site structure 155. The majority of circulating IgM molecules are considered to be non- immune IgM, as serum IgM levels are similar between animals grown in sterile conditions and normal animals 155. However, both non-immune and antigen-specific IgM are crucial for Ab mediated responses against pathogens.

IgM molecules share similar structure to other Ab isotypes, whereby two heavy chains are linked with two light chains to form an immunoglobulin (Ig) subunit of approximately 190kDa (Figure 1.6). The heavy chains (μ) defining the IgM isotype are composed of one variable (Vμ) and four constant Ig domains (Cμ1-Cμ4) 155. In

or tertiary structure 155. Pentameric IgM is composed of five subunits joined by a joining (J-chain), whereas hexameric IgM is composed of six subunits which lack a J-chain 159, 160. The J-chain is a 15kDa protein which covalently associates with pentameric IgM, through disulphide bond formation between cysteine residues in the J-chain and μ-tailpiece 161. The J-chain was shown to regulate intracellular polymerisation of IgM 162, as well as mediating transport of secretory IgM across epithelia by binding to the polymeric Ig receptor (pIgR) 161, 163. Hexameric IgM molecules lacking J-chains are unable to bind to pIgR, possibly owing to the fact that hexameric IgM activates complement 15-20 times more efficiently than pentameric IgM 164, 165. If transported to the gut mucosa which contains high antigen loads, hexameric IgM could cause robust complement activation and ensuing inflammation, resulting in damage the host 166. Therefore, it is possible that these sparse Abs have evolved without a J-chain to play a role in humoral rather than mucosal immunity 164,

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Attempts at obtaining a crystal structure of monomeric or polymeric IgM have been unsuccessful to date, most likely owing to the size and flexibility of IgM molecules

167

. As IgM shares basic domain architecture to IgE, a recent study utilised the similarities between the two immunoglobulins to build a homology-based structural model of pentameric IgM 135. This model proposed a non-planar, mushroom-shaped complex for pentameric IgM, in which the central C-terminal domain portion protrudes out of the plane created by the Fab and Cµ2 domains 135. Cryo-atomic force microscopy confirmed this unexpected conformation by imaging individual human IgM molecules. In this conformation, the protruding C-terminal domain could be targeted by Fc-binding proteins such as IgM receptors or IgM-binding malarial proteins 135. Under this assumption, the protrusion would be directed towards the cell surface upon formation of polyvalent attachments with antigen on the cell surface, resulting in a table-like conformation that would permit the exposure of its C1q binding site to the surrounding solution. 135.

Figure 1.6 Human IgM is a glycoprotein. Pentameric IgM is composed of variable and constant heavy chain and light chain domains (VH and CH or VL and CL, respectively), and features a J-chain.

A recent study determined the atomic details of mouse IgM Fc domains using X-ray crystallography, SAXS, and NMR spectroscopy 167. The structures of each domain (Cµ2, Cµ3, and Cµ4) were reconstructed to build a model for hexameric mouse IgM, which predicted a flexible star-shaped configuration around the inner Cµ4 core 167. In this model, the core structure (~180Å) projected out of the plane created by the Cµ2 and Cµ3 domains 167, which supports previous structural data of human IgM 135. Despite these recent advances, obtaining a crystal structure of human IgM will be pivotal to provide a better understanding of how the Fc region of the molecule mediates its effector functions.

There are five N-linked glycosylation sites present on each μ chain of circulatory IgM, located at Asn-171, Asn-332, Asn-395, Asn-402, and Asn-563. The light chains lack conserved N-linked glycosylation sites, and the J-chain contains a single site at Asn-48 (Figure 1.6). IgM is heavily glycosylated, as roughly 7-12% of the total mass of IgM is comprised of carbohydrates 155. Glycosylation of IgM is important for the secretion of IgM and its presentation on B cell surfaces. Complex glycans predominantly terminating in sialic acid or galactose occupy Asn-171, Asn-332, Asn-395, whereas Asn-402 and Asn-563 are 100% and 17% occupied with GlcNAc2Man5-9-containing oligomannose glycans, respectively 168. It is predicted

that complex glycans are confined to the non-antigen binding face of IgM due to the mushroom-shape adopted by pentameric IgM 135, 168. In this conformation, the glycans would be restricted from binding lectins once the Fab domain has bound antigens forcing IgM to adopt a crab-like conformation 135. This hypothesis is supported by the finding mannose-binding lectin (MBL) does not bind to Ag-bound IgM molecules 168. Therefore, these complex glycans may allow IgM to agglutinate lectin-containing microorganisms in the blood in the absence of specific antigen- binding 169. In the case of non-immune IgM binding as seen with malaria Fcμ- binding proteins, the orientation of IgM with respect to the parasite or erythrocyte plasma membrane is unknown. Intuitively, the orientation would be opposite to that seen with Fab bound IgM, with the C1q binding sites facing the plasma membrane and the IgM glycans therefore facing towards the solution. However, more research is required to clarify the orientation of IgM with respect to parasite IgM binding proteins, whether this be on the surface of merozoites or IEs.