Immunoglobulins Proteins


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 Immunoglobulins Proteins Background

Immunoglobulin, also known as antibody, is large, Y-shape glycoprotein molecules produced by plasma cells in identification and response to an immunogen such as bacteria and viruses. Immunoglobulin or antibodies play vital roles in immune defenses. Without medical intervention, individuals born with antibody deficiencies succumb to infections and die early in life. Even before the adaptive immune system has developed and produced antibodies of its own, fetuses and infants receive protection from maternal antibodies transferred across the placenta. At mucosal surfaces, antibodies are important for blocking the initial entry of bacteria and viruses into the body. If such a barrier is bypassed, then antibodies are employed in systemic defense. In addition to their role in killing pathogens and neutralizing viruses and toxins, antibodies coordinate the immune response by acting as adaptor molecules that bring antigens together with cells and proteins of the innate immune system. These interactions are made possible by the unique structure of antibodies.

Immunoglobulin (antibody) structure
Immunoglobulins are composed of four peptides, two identical light chains and two identical heavy chains. Each heavy chain is covalently linked to the other by one or more disulfide bonds, and a single light chain is linked to each heavy chain by an additional disulfide bond. At the quaternary level the immunoglobulin is Y-shaped. Each immunoglobulin chain is divided into a series of immunoglobulin domains that all share a common tertiary immunoglobulin fold held together by an intra-domain disulfide bond. Immunoglobulin domains are further subdivided into variable immunoglobulin domains and constant immunoglobulin domains. A single variable immunoglobulin domain is located at the amino-terminal end of each immunoglobulin chain, followed by one constant domain in the light chains (CL) and three to four constant domains in the heavy chains (CH). Together, one variable domain from a light chain and one from a heavy chain form the antigen-binding portion of the antibody. Since immunoglobulins have two pairs of heavy and light chains, each molecule has two antigen binding sites at the ends of the arms of the Y-shaped molecule, making it divalent. Variable domains vary extensively in their amino acid content as a result of the previously mentioned reorganization of the gene segments that encode them. It is this diversity of the variable domains that allows antibodies of each B cell and its clones to have unique antigen binding properties. By contrast, constant domains are highly conserved and form the structure of the immunoglobulin that allows it to interact with other components of the immune system to elicit immune effector functions that destroy and eliminate immunoglobulin-tagged antigens.
The original elucidation of the structure of immunoglobulin molecules was aided in the 1950’s and 1960’s by experiments carried out by Rodney Porter. In these experiments, immunoglobulins of the IgG class were proteolytically fragmented. Since the resulting fragments of the immunoglobulin are functionally different, it is still convenient to refer to the portions of an immunoglobulin by the names of these fragments. Digestion with pepsin cleaves immunoglobulins at the heavy chains between the first and second constant domains in a region known as the hinge. This releases two fragments, the F(ab’)2 , which is capable of binding antigen and the Fc, so named because the fragment is easily crystallized. Alternatively, papain cleaves also at the hinge, but at a position N-terminal to all the disulfide bonds that hold the heavy chains together. Therefore papain digestion releases a similar Fc fragment and two antigen binding Fab fragments. Primarily, it is through the Fc portion that other immune system components bind immunoglobulins to initiate immune effector functions. For this reason, immunoglobulin receptors that bind this portion the immunoglobulin are called Fc receptors (FcR).
The constant domains of each immunoglobulin peptide are encoded by constant region genes located downstream of the variable region genes, which recombine together to encode the variable domain of the same immunoglobulin chain. In humans, constant region genes are located at three loci. Immunoglobulin light chain constant regions are encoded by either the κ light-chain locus on chromosome 2 or by the λ light-chain locus on chromosome 22. Accordingly, the resulting light chains are referred to as κ and λ light. The heavy chain constant region locus on chromosome 14 has nine functional heavy chain constant region genes and three pseudogenes. The nine heavy chain regions that are encoded by the functional genes are grouped into five types: µ, γ, α, δ, and ε, which in turn define the five isotypes or classes of immunoglobulins: IgM, IgG, IgA, IgD and IgE respectively. IgG antibodies can be further subdivided into four subclasses (IgG1, IgG2, IgG3 and IgG4), whereas IgA antibodies are found as two subclasses (IgA1 and IgA2). The heavy chains of the antibody subclasses are designated γ1, γ2, γ3, γ4 (for IgG1, 2, 3 and 4) and α1, α2 (for IgA1 and IgA2). The constant region heavy chain genes are named according to the standardized ImmunoGeneTics nomenclature as IGH, followed by the appropriate letter and number to designate the specific subclass, i.e. IGHG3 for the γ3 gene, and IGHA1 for the α1 gene. Immunoglobulins of a common class have highly conserved peptide sequences, share common features and have similar functional properties. The immunoglobulin classes IgA, IgD and IgG have heavy chains containing three immunoglobulin constant domains with a hinge that separates the CH2 and CH3 domains. IgM and IgE by contrast have four CH domains and no hinge. Most of the differences between immunoglobulin subclasses are located in the hinge region.
The hinge region contributes to segmental flexibility and in IgA and IgG also to intermolecular covalent assembly. Variability of the length and amino acid content of the hinge region from each subclass results in a different degree of flexibility and to the overall shape of the immunoglobulin. The hinge of IgG consists of three segments, the upper, middle and lower hinge. The middle hinge is a rigid region that contains a variable number of cysteines, which form the inter-chain disulfide bonds by connecting two parallel polyproline double helices. It is connected to the Fc region by the lower hinge and to the Fab portion by the upper hinge. The lower hinge is involved in binding the low affinity Fc gamma receptors. In humans, the IGHG1, IGHG2 and IGHG4 genes include only one separate hinge exon, whereas the IGHG3 hinge is usually encoded by four exons and less frequently by two, three, or five distinct exons. Since the IgG3 hinge region is encoded by more than one exon, IgG3 molecules are characterized by hinge regions exhibiting several repetitions and are therefore longer than the hinge regions of the other IgG subclasses. Consequently, IgG3 molecules are the most flexible of all human IgG subclasses. IgA1 molecules include an elongated hinge region rich in proline residues. The IgA2 hinge region differs from the IgA1 hinge region by a 13- residue stretch of amino acids, presumably the result of an evolutionary response to bacterial IgA1 specific proteases. The human IgD hinge region includes 64 amino acid residues and is, therefore, longer than the hinge region of the other antibody classes (with the exception of IgG3). Hinge regions are characterized by evolutionary instability, as they are the most diverse at the interspecies and intraspecies level. Differences in the hinge of each molecule have been demonstrated to alter immunoglobulin affinity for antigen and immune complex formation. In some HIV in vitro studies IgG3 with its long hinge has been demonstrated to be more efficient than other IgG subclasses at neutralizing viruses in the absence of affinity differences. Hence the hinge region greatly contributes to antigen binding and antigen elimination.
Immunoglobulins are expressed as BCR or as secreted proteins based on the type of carboxyl-terminal tail they express. Immunoglobulins forming BCRs are anchored to the plasma membrane by a hydrophobic tail that makes up the transmembrane domain and a short cytoplasmic tail. The transmembrane domains of all antigen receptors, including TCRs, have residues that make up a conserved antigen receptor transmembrane motif (CART). This motif is believed to be necessary for proper association with the signaling molecules of the antigen receptor complex. For BCR, these signaling molecules are the invariant transmembrane proteins Ig-α and Ig-β, which contain the necessary cytoplasmic elements for signaling and antigen presentation. For all human immunoglobulin heavy chains, the immunoglobulin transmembrane tail is encoded by two exons (M1 and M2) found 3’ to the last CH exon of the gene, whereas secreted antibodies have a short hydrophilic tail that varies greatly between the different immunoglobulin isotypes. For all isotypes except IgD, the secretory tail is encoded at the 3’ end of the final CH exon. The IgD secretory tail has been found to be encoded by a separate exon (CH-S) located between the CH3 exon and the exons M1 and M2 of the Igδ gene (IGHD) in all species in which it has been characterized. Polyadenylation of polyA motifs found 3’ of the tail to be used and subsequent alternative splicing regulate production of the cytoplasmic tail.

Immunoglobulin subclasses and functions
Antibodies alone in some cases are sufficient to inactivate a pathogen to prevent infection or render toxins innocuous. These antibodies are called neutralizing antibodies. The mechanisms responsible for neutralization are not fully understood. Neutralizing antibodies may defend against viruses by occupying all possible virion sites of interaction with host cells, thus blocking infection. Evidence indicates that other antibody properties are usually required to prevent or stop infections. Fab fragments from neutralizing antibodies generally lose the ability to prevent disease. Indeed, many bacteria actively produce proteases that separate antigen binding Fab fragment from the Fc fragment of the antibody. It has been suggested that some gut bacteria may be able to use IgA Fab fragments created by their proteases to cloak antigen epitopes from other intact antibodies to escape targeting by the immune system. Other bacterial proteins like staphylococcal superantigen-like protein 7 bind the Fc region of IgA antibodies, thereby blocking interactions with complement and Fc receptors.
Each immunoglobulin subclass has different properties and consequently performs different functional roles in the immune system. Particularly, immunoglobulin functional differences include eliciting different immune effector mechanisms (complement-dependent cytotoxicity, antibody- dependent cellular cytotoxicity, and opsonization), physiological localization, ability to multimerize, ability to cross the placenta, and secretion at mucosal surfaces. Each subclass within an immunoglobulin class appears similar to the other (only a 5-10% gene difference for the IgG subclasses). However, there are important differences between them. Production of a specific subclass is controlled by genetic rearrangement at the heavy chain gene locus, a process called isotype switching. This process results in a heavy chain consisting of the same variable domain and with identical antigen specificity. The environmental milieu of a B cell dictates which subclass of antibody is produced and is a reflection of the assault with which the immune system is challenged. For example, a viral infection stimulates T helper type I cytokines that will result in isotype switching to produce antibody classes best able to fight intracellular infections.
IgM and IgD with the same antigen specificity are coexpressed on the surface of naïve B cells and act as the initial BCR to bind antigen. No other combination of two immunoglobulins can be expressed. Uniquely, Igµ and Igδ genes are transcribed in a single RNA molecule which is then polyadenylated 3’ of either the Igµ exons or the Igδ exons and then spliced. Of the two, IgD is the least understood. Results from a few recent studies show that IgD and IgM may modulate B cell activation differently. Secreted IgD makes up only about 0.5% of serum immunoglobulins and very little research has been performed to determine its role. Both membrane and secreted IgD can bind to receptors expressed on T cells, hence modulating immune responses. IgM constitutes 5-10% of the total serum immunoglobulin. Low affinity IgM is the first immunoglobulin secreted in a primary immune response and is thought to represent the major isotype of natural antibodies. Natural antibodies are secreted in the absence of prior exposure to antigen, and often have broad antigen specificity. Natural antibodies may play an important role by priming the immune response through an initial capture of pathogens for processing by APC. Multimeric IgM is efficient at activating the classical complement pathway. IgD does not form multimers and does not activate the classical complement pathway.
Both IgA and IgM multimerize and are transported across epithelial cells to mucosal surfaces. IgM molecules form pentamers and IgA predomintly forms dimers, although IgA polymers can exist also in trimeric and tetrameric forms. Multimerization is facilitated by the attachment of a polypeptide chain called the J chain. The J chain in turn can associate with the poly-immunoglobulin receptor (pIgR) expressed on the basolateral surface of epithelial cells. The pIgR then endocytoses and transports the immunoglobulin to the apical surface. The pIgR is then cleaved to release the immunoglobulin, leaving a portion of the pIgR (referred to as the secretory component) still attached to the molecule. IgA is the most abundant immunoglobulin class, accounting for 10-15% of serum immunoglobulin and for almost all of secreted immunoglobulin (76-90% in the gut mucosae, 69-86% in nasal, lacrimal and parotid glands). In the mucosae, IgA acts via immune exclusion by coating pathogens and preventing them from binding to the mucosal linings. IgA also helps with clustering of pathogens and their subsequent expulsion from the mucosae. IgA is considered an anti-inflammatory immunoglobulin. Through binding Fc receptors (FcαRI or CD89), IgA can down regulate the effector functions of leukocytes from the myeloid lineage, such as inflammatory cytokine release induced by IgG immune complexes. Somewhat paradoxically, IgA immune complexes can interact with the same receptor to induce leukocyte effector functions.
IgG makes up 80% of serum immunoglobulins with the relative abundance of the human subclasses being IgG1>IgG2>IgG3>IgG4. IgG are usually high affinity antibodies and are produced late in a primary immune response. IgG modes of action differ between subclasses. IgG3 followed by IgG1 then IgG2 can activate complement, whereas IgG4 cannot. IgG1, IgG3 and IgG4 are transported across the placenta to protect the fetus. IgG1 and IgG3 are most efficient at binding Fc receptors.
IgE has a very low serum concentration and is mostly bound to mast cells and eosinophils through the high affinity IgE Fc receptor. Antigen cross-linking of Fc receptor bound IgE on these cells results in the release of type I hypersensitivity reactions mediators. Type I hypersensitivity reactions range from common allergies to anaphylaxis. In addition, IgE plays an important role in defense against parasites.


Immunoglobulin reference
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