- The Immunoglobulins
- Structure and Function of Immunoglobulins
- Immunoglobulins structure and function pdf reader
- Antibody structure and function
- Structure of immunoglobulins
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- Immunoglobulin Structure and Classes
Immunoglobulins are heterodimeric proteins composed of two heavy H and two light L chains. They can be separated functionally into variable V domains that binds antigens and constant C domains that specify effector functions such as activation of complement or binding to Fc receptors.
The variable domains are created by means of a complex series of gene rearrangement events, and can then be subjected to somatic hypermutation after exposure to antigen to allow affinity maturation.
Each V domain can be split into three regions of sequence variability, termed the complementarity determining regions, or CDRs, and four regions of relatively constant sequence termed the framework regions, or FRs. There are five main classes of heavy chain C domains.
The constant domains of the H chain can be switched to allow altered effector function while maintaining antigen specificity. In , von Behring and Kitasato reported the existence of an agent in the blood that could neutralize diphtheria toxin. The term antigen is a contraction of this term.
Structure and Function of Immunoglobulins
Thus, an antibody and its antigen represent a classic tautology. In , Tiselius and Kabat used electrophoresis to separate immunized serum into albumin, alpha-goblulin, beta-globulin, and gamma-globulin fractions.
Absorption of the serum against the antigen depleted the gamma-globulin fraction, yielding the terms gamma globulin, immunoglobulin Ig , and IgG. More than years of investigation into the structure and function of immunoglobulin has only served to emphasize the complex nature of this protein. Typically, receptors bind to a limited and defined set of ligands.
However, while individual immunoglobulin also bind a limited and defined set of ligands, immunoglobulins as a population can bind to a virtually unlimited array of antigens sharing little or no similarity.
This property of adjustable binding depends on a complex array of mechanisms that alter the DNA of individual B cells.
Immunoglobulins also serve two purposes: that of cell-surface receptors for antigen which permit cell signaling and cell activation and that of soluble effector molecules which can individually bind and neutralize antigens at a distance. The molecular mechanisms that permit these many and varied functions are the focus of this chapter. Immunoglobulins Igs belong to the eponymous immunoglobulin super-family IgSF.
Both Ig L chains contain only one C domain, whereas Ig H chains contain either three or four such domains.
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H chains with three C domains tend to include a spacer hinge region between the first C H 1 and second C H 2 domains. These solvent exposed surfaces offer multiple targets for docking with other molecules.
The H and L chains at the top deconstruct the antibody at a nucleotide level. The chains at the bottom deconstruct the protein sequence. See text for further details.
Antibody structure and function
Early studies of Ig structure were facilitated by the use of enzymes to fragment IgG molecules. Papain digests IgG into two Fab fragments, each of which can bind antigen, and a single Fc fragment. Pepsin splits IgG into an Fc fragment and a single dimeric F ab 2 that can cross-link as well as bind antigens. Single Fv fragments can be genetically engineered to recapitulate the monovalent antigen binding characteristics of the original, parent antibody.
Intriguingly, a subset of antibodies in a minority of species [camelids 5 , nurse shark 6 ] lack light chains entirely and use only the heavy chain for antigen binding. While these unusual variants are not found in human, there are a number of ongoing attempts to humanize these types of antibodies for therapeutic and diagnostic purposes e.
Immunoglobulin-antigen interactions typically take place between the paratope , the site on the Ig at which the antigen binds, and the epitope , which is the site on the antigen that is bound.
Structure of immunoglobulins
This ability to identify component parts of the antigen independently of the rest makes it possible for the B cell to discriminate between two closely related antigens, each of which can be viewed as a collection of epitopes. It also permits the same antibody to bind divergent antigens that share equivalent or similar epitopes, a phenomenon referred to as cross-reactivity.
Immunization of heterologous species with monoclonal antibodies or a restricted set of immunoglobulins allowed the identification of both common and individual immunoglobulin antigenic determinants. Individual determinant s , termed idiotype s , are contained within V domains.
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Common determinants, termed isotypes , are specific for the constant portion of the antibody and allow grouping of immunoglobulins into recognized classes, with each class defining an individual type of C domain. Determinants common to subsets of individuals within a species, yet differing between other members of that species, are termed allotypes and define inherited polymorphisms that result from gene alleles.
Ig heavy and light chains are each encoded by a separate multigene family, 9 , 10 and the individual V and C domains are each encoded by independent elements: V D J gene segments for the V domain and individual exons for the C domains.
The primary sequence of the V domain is functionally divided into three hypervariable intervals, termed complementarity determining regions CDRs that are situated between four regions of stable sequence termed frameworks FRs Figure 1.
Each V gene segment typically contains its own promoter, a leader exon, an intervening intron, an exon that encodes the first three framework regions FR 1, 2, and 3 , CDRs 1 and 2 in their entirety, the amino terminal portion of CDR 3, and a recombination signal sequence RSS. Each J for joining gene segment begins with its own recombination signal, the carboxy terminal portion of CDR 3, and the complete FR 4 Figure 1 , Figure 2.
The creation of a V domain is directed by the recombination signal sequences RSS that flank the rearranging gene segments. Each RSS contains a strongly conserved seven base pair, or heptamer, sequence e. These spacers place the heptamer and nonamer sequences on the same side of the DNA molecule, separated by either one or two turns of the DNA helix. A one turn recombination signal sequence 12 base pair spacer will preferentially recognize a two turn signal sequence 23 base pair spacer , thereby avoiding wasteful V-V or J-J rearrangements.
Initiation of the V D J recombination reaction requires recombination activating genes 1 and 2 RAG-1 and RAG-2 , which are almost exclusively expressed in developing lymphocytes. These breaks are then repaired by ubiquitously expressed components of a DNA repair process, known as nonhomologous end-joining NHEJ , that are common to all cells of the body. Terminal deoxynucleotidyl transferse TdT , which is expressed only in lymphocytes, can variably add non-germline encoded nucleotides N nucleotides to the coding ends of the recombination product.
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The clean cut ends of the signal sequences enable formation of precise signal joints. However, the hairpin junction created at the coding ends must be resolved by re-nicking the DNA, usually within four to five nucleotides from the end of the hairpin. The cut ends of the coding sequence are then repaired by the non-homologous end joining proteins. Ku70 and Ku80 form a heterodimer Ku that directly associates with DNA double-strand breaks to protect the DNA ends from degradation, permit juxtaposition of the ends to facilitate coding end ligation, and help recruit other members of the repair complex.
DNA-PKcs phosphorylates Artemis, inducing an endonuclease activity that plays a role in the opening of the coding joint hairpin. V gene segments can be grouped into families on the basis of sequence and structural similarity. The typical numbers of functional gene segments are shown.
The H chain locus, on chromosome 14q A pair of one turn recombination signal sequences flanks each D H. Recombination begins with the joining of a D H to a J H gene segment, followed by the joining of a V H element to the amino terminal end of the DJ intermediate.
A H chain rearrangement can yield as many as 38, different VDJ combinations. The addition of nine N nucleotides on either side of the D gene segment yields can yield up to 64,, different CDR-H3 junctional sequences.
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B The view is looking into the binding site as an antigen would see the antigen binding site. The V H domain is on the right side. The central location of CDR-H3, which due to N addition is the focus for repertoire diversity, is readily apparent. While combinatorial joining of individual V, D, and J gene segments maximizes germline-encoded diversity, the junctional diversity created by VDJ joining is the major source of variation in the pre-immune repertoire Figure 4.
First, D H gene segments can rearrange by either inversion or deletion, and each D H can be spliced and translated in each of the three potential reading frames. This gives each D H gene segment the potential to encode six different peptide fragments. Third, the terminus of each rearranging gene segment can undergo a loss of one to several nucleotides during the recombination process. Fourth, TdT can add numerous N nucleotides to replace or add to the original germline sequence.
N nucleotides can be inserted between the V and the D, as well as between the D and the J. The imprecision of the joining process and variation in the extent of N addition permits generation of CDR-H3s of varying length and structure.
Taken as a whole, somatic variation in CDR3, combinatorial rearrangement of individual gene segments and combinatorial association between different L and H chains can yield a potential pre-immune antibody repertoire of greater than 10 16 different immunoglobulins. All CH genes can undergo alternative splicing to generate two different types of carboxy termini: either a membrane terminus that anchors immunoglobulin on the B lymphocyte surface or a secreted terminus that occurs in the soluble form of the immunoglobulin.
Cocktails of cytokine signals transmitted by T cells or other extracellular influences variably activate the I exon, initiating transcription and thus activating the gene. A final mechanism of immunoglobulin diversity is engaged only after exposure to antigen. SHM allows affinity maturation of the antibody repertoire in response to repeated immunization or exposure to antigen. Creation of immunoglobulin diversity is hierarchical. Production of a properly functioning B cell receptor is essential for development beyond the pre-B cell stage.
This is followed by four to six cycles of cell division Alternative splicing permits co-production of IgM and IgD. The IgM and IgD on each of these cells share the same variable domains. The life span of mature B cells expressing surface IgM and IgD appears entirely dependent on antigen selection. After leaving the bone marrow, unstimulated cells live only days or a few weeks.
Immunoglobulin Structure and Classes
The reaction to antigen leads to activation, which may then be followed by diversification. The nature of the activation process is critical. T cell independent stimulation of B cells induces differentiation into short-lived plasma cells with limited class switching. T-dependent stimulation adds additional layers of diversification, including somatic hypermutation of the variable domains, which permits affinity maturation, class switching to the entire array of classes available, and differentiation into the long-lived memory B cell pool or into the long-lived plasma cell population.
In general, the C domain of the H chain defines effector function, while the paired V domains of the antibody confer antigenic specificity.
This Fc fragment defines the isotype and subclass of the immunoglobulin. Despite amino acid differences between the isotypes and subclasses, each CH region folds into a fairly constant structure consisting of a three strand-four strand beta sheet pinned together by an intrachain disulfide bond. The Fc fragment mediates effector function by binding to the Fc receptor on effector cells or activating other immune mediators such as complement. The Fc region can also affect the affinity or kinetics of binding of the antibody by the Fv region, and thus influence antigen recognition or binding.
Immunoglobulins are glycoproteins and the glycans associated especially with the Fc domain of immunoglobulins have been shown to affect antibody function. The extent of glycosylation varies by isotype Figure 6. The core of this complex biantennary type of sugar is a heptasaccharide consisting of N-acetylglucosamine and mannose.
Variation in glycosylation is seen between IgG molecules as well as within the two sites on the same molecule due to differences in terminal sialic acid, galactose, N-acetylglucosamine and fucosylation of the core.
These differences can lead to as many as 32 possible glycosylation patterns. The glycans at this site interact with a hydrophobic pocket on the Fc domain that stabilizes the immunoglobulin structure. At a similar site in CH2 of IgD, Asn, mutations that prevent glycosylation are associated with the loss of IgD production, suggesting that glycans in CH2 can be essential for immunoglobulin stability.