Antibodies

1.            Introduction

a.            Antibodies are globulin proteins (immunoglobulins) that react specifically with the antigen that stimulated their production.

b.         There are three classes of globulins in the plasma: alpha, beta and gamma – antibodies are gamma globulins (20% of plasma proteins)

c.         There are five classes of antibodies: IgG, IgM, IgA, IgD, and IgE.

d.         Types of antibody clones:

i.            Polyclonal: antibodies that arise in an animal in response to typical antigens are heterogeneous, because they are formed by several different clones of plasma cells.

ii.            Monoclonal: antibodies that arise from a single clone of cells are homogenous.

2.            Antibody Structure

a.            Immunoglobulins are glycoproteins made of 2 light (Mr=25,000) and 2 heavy (Mr=50,000-70,000) polypeptide chains.

b.         The four chains are attached into a Y shape (linked by disulfide bonds) with a hinge region that allows flexible positioning of the arms of the molecule:

i.          Fab regions: the two arms contain antigen-binding sites that confer the antibody’s specificity.

ii.          Fc region: stem of the Y-shaped antibody.

c.            Domains of Immunoglobulins:

i.            domains are regions of the immunoglobuin chains composed of 3-dimensionally folded, repeating segments.

ii.          L chains have 2 domains each and H chains have 4 or 5 domains, separated by a short unfolded stretch.

iii.         each domain is designated by a letter that indicates whether it is on a L or H chain and a number that indicates its position.

iv.         L chains belong to one of two types, k (kappa) or l (lambda), on the basis of amino acid differences in their constant regions.

v.         both types occur in all classes of immunoglobulins but any one immunoglobulin molecule contains only one type of L chain.

d.            Variable and Constant domains:

i.          an L chain consists of one variable and one constant domain.

ii.          most H chains consist of one variable and 3 constant domains.

iii.         each domain is approximately 110 amino acids long.

iv.         the first domain on each L and H chain is highly variable and is designated as V_L or V_H; they have 3 hypervariable amino acid sequences (5-10 amino acids) at the amino-terminal end that form the antigen-binding site.

v.         the second and subsequent domains on both chains are much more constant in amino acid sequence and are designated C_L or C_H1, C_H2, and C_H3 _.

vi.         in addition to their interchain disulphide bonding, the globular domains bind to each other in homologous pairs, largely by hydrophobic interactions, as followed: V_H – V_L, C_H1 – C_L, C_H2 - C_H2, C_H3 - C_H3 .

e.         Domain structure:

i.          both the variable and constant domains are constructed from two sheets formed by adjacent strands of the polypeptide chain that pack together and which are linked by a disulfide bridge forming a cylindrical shape.

ii.          the sheets that form this structure are beta sheets and they form a beta barrel.

iii.         the beta barrel that is formed by immunoglobulin chains is called immunoglobulin fold.

f.          Regions of chains:

i.          variable regions are responsible for antigen binding.

ii.            constant regions are responsible for various biological functions such as complement activation and binding to cell surface receptors.

iii.         the structural variation of the H chains mediate a variety of biological functions and give rise to the different classifications of  the immunoglobulins.

g.         An individual antibody molecule consists of identical H and L chains due to:

i.          allelic exclusion

ii.            regulation within the B cell, which ensures the synthesis of either kappa or lambda L chains but not both.

3.            Antigen-binding site

a.            Hypervariable regions:

i.          the variable regions of both L and H chains have 3 hypervariable amino acid sequences at the amino-terminal end that form the antigen-binding site.

ii.          only 5-10 amino acids in the hypervariable region form the binding site.

b.            Framework regions:

i.          the rest of the V domain shows less variability and the regions and the regions between the hypervariable regions, which are invariant, are the framework regions.

ii.          the framework regions form the beta sheets that provide the structural framework of the domain, while the hypervariable sequences correspond to three loops at one edge of each sheet that are juxtaposed in the folded protein.

c.            Formation of antigen binding site:

i.          when the variable domains of light and heavy chains pair in the antibody molecule, the hypervariable loops from each domain are brought together.

ii.          this creates a single hypervariable site at the tip of the Fab fragment that forms the antigen binding site.

d.         These hypervariable loops determine specificity by forming a surface complementary to the antigen and are termed complementarity determining regions – CDR1, CDR2 and CDR3.

e.            Combinatorial diversity: generation of antibodies of different specificities by generating different combinations of heavy- and light chain variable regions.

f.            Antigenic determinants:

i.          regions of a molecule that are recognized specifically by antibodies are called antigenic determinants or epitopes.

ii.          such sites on protein surfaces are likely to be composed of amino acids from different parts of the sequence that have been brought together by protein folding.

iii.            epitopes of this kind are conformational or discontinuous epitopes since the site is composed of segments of the protein that are discontinuous in the primary sequence but are contiguous in the three-dimensional structure.

iv.         an epitope composed of a single segment of polypeptide chain is termed a continuous or linear epitope.

g.            Antigen-antibody binding:

i.          no covalent bonds are involved in the interaction between an antibody and an epitope.

ii.          the binding may involve electrostatic interactions, hydrophobic interactions, hydrogen bonds and van der Waals forces.

iii.         since these forces are relatively weak, the ‘fit’ between the antigen and its complementary binding site on the antigen receptor must occur over an area large enough to allow the summation of all the possible available interactions.

iv.         this requirement is the basis for the exquisite specificity observed in immunologic interactions.

v.         due to low levels of energy involved in the interaction between antigen and antibody, antigen-antibody complexes can be readily dissociated by low or high pH, high salt concentration or by chaotropic ions, e.g. cyanates.

h.         Affinity and Avidity:

i.          affinity denotes the intrinsic association constant between an antibody and a univalent ligand such as a hapten.

ii.          avidity is used to denote the overall binding energy between antibodies and a multivalent antigen.

iii.         this distinction is important as the overall binding of the antigen with antibody is affected by an increased number of sites on the antigen where the antibody can act.

4.            Antibody Functions

a.         Primary function:

i.          the primary function of antibodies is to bind antigens to B lymphocytes and initiate the production of additional antibodies.

ii.            because antibodies are found in the interstitial fluid and plasma, they are most effective against extracellular pathogens; bacteria, some parasites, antigenic macromolecules and viruses before they invade their host cells.

iii.         the surface of each B lymphocyte is covered with as many as 100,000 antibody molecules whose Fc ends are inserted into the lymphocyte membrane.

iv.         binding of antibody to Fab regions of the bound antibodies activate the B lymphocyte, converting it into a memory cell or antibody-secreting plasma cell.

b.            Opsonins:

i.          act as opsonins to facilitate recognition and phagocytosis of antigens.

ii.          label antigens so that other immune cells will attack them.

iii.         all antibody-bound substances are eventually ingested by phagocytes.

c.            Agglutination of antigens for easier phagocytosis.

d.         Fc binding:

i.            antibodies bound to antigens at their Fab regions will bind to other immune cells with the Fc end of the antibody.

ii.          through this mechanism, antibody-bound antigen can be recognized by a single Fc receptor, rather than requiring millions of different receptors.

e.            Activation of complement proteins.

f.            Activate mast cells, both directly and via complement, causing them to release chemicals that mediate the inflammatory response.

g.         Bind to viruses or to toxins produced by bacteria, preventing these substances from affecting host cells.

5.            Classes of Immunoglobulins

6.            Immunoglobulin Genes

a.         DNA rearrangement and RNA splicing are used to produce large number of different immunoglobulin molecules.

b.         Each immunoglobulin chain consist of a distinct variable (V) and constant (C) region.

c.         For each type of chain, there is a separate pool of gene segments located on different chromosomes –V, D (diversity, seen only in H chains), J (joining) and C gene segments.

d.            Synthesis of Light chain:

i.          light-chain variable region genes are constructed from two segments.

ii.          a variable(V) and a joining(J) gene segment in the genomic DNA are joined to form a complete light-chain variable-region gene.

iii.         the constant region is encoded in a separate exon and is joined to the variable-region gene by RNA splicing of the light-chain message to remove the L to V and the J to C introns.

iv.            immunoglobulin chains are extracellular proteins and the V gene segment is preceded by an exon encoding a leader peptide(L), which directs the protein into the cell’s secretory pathways and is then cleaved.

e.            Synthesis of Heavy chain:

i.          heavy-chain variable regions are constructed from three gene segments.

ii.          first the diversity(D) and J gene segments join, then the V gene segment joins to the combined DJ sequence, all at the genomic DNA level.

iii.         the heavy-chain constant-region sequences are encoded in several exons.

iv.         the constant-region exons, together with the leader(L) sequence, are spliced to the variable-domain sequence during processing of the heavy-chain gene RNA transcript.

v.         post-translational alterations remove the L sequence and attach carbohydrate moieties.

f.            Flanking sequences:

i.          flanking sequences of V, D, and J gene segments consist of a conserved block of 7 nucleotides (heptamer), followed by a spacer and a second conserved block of nine nucleotides (nonamer).

ii.          the heptamer-spacer-nonamer is called a recombination signal sequence (RSS).

iii.            recombination occurs only between gene segments located on the same chromosome and it can only link a gene segment flanked by a 12mer-spaced RSS to one with a 23mer-spaced RSS.

iv.         the 12mer-spaced and 23mer-spaced recombination signal sequences are brought together by interactions between proteins.

v.         the two DNA molecules are then broken and re-ligated.

7.            Generation of Antibody diversity

a.            Multiple gene segments:

i.          there are multiple copies of each of the gene segments that make up an immunoglobulin variable region.

ii.            different combinations of gene segments can be used in different rearrangement events.

b.            Rearrangement of gene segments:

i.            diversity arises through the pairing of different combinations of heavy- and light- chain variable regions to form the antigen-binding site.

ii.          a antibody molecule could be encoded by a DNA molecule composed of a set of recombined minigenes that made up a complete gene.

iii.         given small sets of these minigenes, which are randomly combined to make the complete gene, it is possible to produce a large repertoire of specificities from a limited number of gene fragments.

c.         VJ and VDJ Combinatorial association:

i.          the association of any V-gene segment with any J-gene segment can occur to form a ligh-chain variable region and similarly, any V can associate with any D- or J-gene segments in heavy-chain gene rearrangement.

ii.          all these distinct segments contribute to the structure of the variable region.

iii.         as there are about 200V_k and 5 J_k genes coding for the k-chain variable region, assuming random association, then 200 X 5 or 1000 k chains can be formed; with 100 V_l and 6 J_l genes, 600 l chains can be formed.

iv.            similarly, if there are about 200 V genes, 12 D genes and 6 J genes that can code for an H-chain variable region and these may also associate in any combination, then 200 X 12 X 6 or 14,400 different heavy chains can be formed.

d.            Random assortment of H and L chains:

i.          in addition to VJ and VDJ combinatorial association, any H chain may associate with any L chain.

ii.          thus, if any H chain can associate with any k or l chain, a total of 14.4x10⁶ different k-containing immunoglobulin molecules (1000 X 14,400), and 8.6x10⁶ (600 X 14,400) l-containing molecules can be generated from just 529 different genes by adding up all the H, k and l segments.

e.            Junctional and Insertional diversity:

i.          the precise positions at which the genes for the V and J, or the V, D and J segments are joined are not constant.

ii.            imprecise DNA recombination can lead to changes in the amino acids at these junction sites.

iii.         the absence of prevision in joining during DNA rearrangement leads to deletion or changes of amino acids (junctional diversity) that affect the antigen-binding site.

iv.         in addition, small sets of nucleotides (insertional diversity) may be inserted at the V-D and D-J junctions without the need for a template.

v.         the additional diversity provided is termed N regional diversity.

f.            Somatic mutations:

i.            generates diversity throughout the variable region and operates on B cells in secondary lymphoid organs after functional antibody genes have been assembled.

ii.          this process introduces point mutations into the variable regions of the rearranged heavy- and light-chain genes at a very high rate, giving rise to mutant immunoglobulin molecules on the surface of the B cells.

iii.         base changes that alter amino acid sequences are clustered in the CDR1 and CDR2 regions, while silent mutations which preserve amino acid sequence and do not alter structure are scattered throughout framework regions.

iv.         these mutations that occur in the V genes during the lifetime of an individual B cell increases the variety of antibodies produced by the B cell.

v.         the primary immune response commences with the formation of low-affinity antibodies, but as the immune response continues, somatic mutations occur that are then positively selected for, leading to production of antibody with greater affinity for antigen.

8.            Structural variation in constant regions

a.            Sequence differences between immunoglobulin heavy chains cause the various isotypes to differ in several characteristic aspects.

b.         These include the number and location of interchain disulfide bonds, the number of attached oligosaccharide moieties, the number of constant domains, and the length of the hinge region.

c.            Polymers:

i.          IgM molecules are found as pentamers while IgA in mucous secretions, but not in plasma, is mainly found as a dimer.

ii.          an additional separate polypeptide chain called the J chain promotes polymerization by linking carboxy-terminal cysteines, which are found only in the secreted forms of the M and A chains.

iii.         in the case of IgA, polymerization is required for transport through epithelia.

d.            Importance of Polymerization:

i.          the polymerization of immunoglobulin molecules is important in antibody binding to repetitive epitopes.

ii.          it increases the binding strength, or avidity.

iii.         IgM antibodies are able to recognize repetitive epitopes such as those expressed by bacterial cell wall polysaccharides.

9.            Functional specialization in Constant regions

a.         Binding to cell receptors:

i.          the Fc portions of IgG antibodies are recognized by Fc receptors expressed on the surface of phagocytic cells such as macrophages and neutrophils, which can thereby bind and engulf pathogens coated with antibodies of these isotypes.

ii.          the Fc portion of IgE is recognized by Fc receptors on mast cells in tissues, and basophils, which respond by releasing inflammatory mediators.

b.         Binding to complement:

i.          the Fc portions of antigen-antibody complexes can bind to complement and initiate the complement cascade, which helps to recruit and activate phagocytes and to destroy pathogens directly.

ii.            complement is activated by IgM and IgG, which initiate the cascade by binding to a protein called C1q through a region of charged residues on the constant domains of IgG and IgM.

c.            Delivery:

i.          the Fc portion function to deliver antibodies to places they would not reach without active transport.

ii.          these include the mucous secretions and also tears and milk and the fetal blood circulation by transfer from the pregnant mother.

iii.         this system uses Fc receptors, this time on the transporting cells of the appropriate epithelia or the placenta.

10.            Immunoglobulin class switching

a.         Initially, all B cells carry IgM specific for an antigen and produce IgM antibody in response to exposure to that antigen.

b.         Later, gene rearrangement and differential splicing permits the elaboration of antibodies of the same antigenic specificity but of different immunoglobulin classes.

c.         In class switching, the same assembled VH gene can sequentially associate with different CH genes so that the immunoglobulins produced later (IgG, IgA or IgE) are specific for the same antigen as the original IgM but have different biologic features.

d.         Isotype switch is stimulated in the course of an immune response by cytokines released by T cells.

e.         Control of class switching is dependent on:

i.            concentration of the various interleukins: IL-4 enhances IgE production whereas IL-5 increases IgA.

ii.            interaction of the CD40 protein on the B cell with CD40 ligand protein on helper T cell.

f.          Allelic exclusion: a single B cell expresses only one K-chain and one H-chain allele; i.e. either the paternal or maternal set is expressed but not both.

11.       B-cell antigen receptor

a.            Antibodies of all heavy-chain isotypes can be produced either in secreted form or as a membrane-bound receptor.

b.         The membrane forms of all isotypes are monomers: IgM and IgA only polymerize when secreted.

c.         In its membrane-bound form, the immunoglobulin molecule has a hydrophobic transmembrane domain which anchors it to the surface of the B lymphocyte; this domain is absent from the secreted form.

d.            Encoding:

i.          the last exon of the constant region gene contains the sequence encoding the transmembrane region of the heavy chain.

ii.          if the primary transcript includes all exons, the sequences encoding the carboxy-terminal of the secreted form is removed during RNA processing and the cell-surface form of immunoglobulin is produced.

iii.         if transcription is terminated before the last exon, only the secreted molecule can be produced.

iv.         both transcription termination and differential mRNA splicing are regulated to generate membrane or secreted forms.

12.            Activation of B cells

a.         Binding of membrane-bound immunoglobulins:

i.            transmembrane immunoglobulins are found in a complex with two other proteins, Iga and Igb, which are closely associated with immunoglobulin in the membranes of B cells.

ii.          Iga and Igb have extracellular single-domain immunoglobulin folds that bind nonconvalently to the immunoglobulin heavy chain, and cytoplasmic tails that contain specialized sequence motifs called immunoreceptor tyrosine activation motifs (ITAMs)

b.         Role of antigen:

i.          when antigen binds to the membrane-bound immunoglobulin at the surface of a B cell, it initiates a cascade of events in the cell interior that leads to its proliferation and differentiation of the progeny into antibody-secreting plasma cells.

ii.          tyrosine phosphorylation of the ITAMs is the first step in an intracellular signaling cascade triggered by antigen binding to the receptor.

iii.         the role of the antigen in triggering the signaling cascade is to crosslink the surface immunoglobulin molecules and therefore cause clustering of the signaling complex.

iv.         this brings receptor-associated tyrosine kinase molecules together with the ITAMs they phosphorylate.

c.         Role of helper T cells:

i.            crosslinking of surface immunoglobulin is insufficient on its own to activate B cells and most antigens do not have repeating epitopes and are not capable of crosslinking surface immunoglobulins.

ii.          the initiation of most B-cell responses depend on signals delivered by the helper T cells, which recognize antigen on the surface of B cells.

12.            Immune Memory

a.         The production of antibody after a priming contact with antigen may cease entirely within a few weeks but the immunized individual is left with a cellular memory (i.e. long-lasting memory cells) of this contact.

b.         When encountered with the same antigen again:

i.          the lag phase is considerably shorter.

ii.            antibody may appear in less than half the time required for the primary response.

iii.         the production of antibody is much greater.

iii.         higher concentrations of antibody are detectable in the serum.

iv.         the production of antibody may also continue for a longer period, with persistent levels remaining in serum months or even years later.

c.         There is also a marked change in the type and quality of antibody produced in the secondary response:

i.          there is a shift in class response, with IgG antibodies appearing at higher concentrations and with greater persistence than IgM, which may be greatly reduced or disappear altogether.

ii.          this may be also accompanied by the appearance of IgA and IgE.

d.            Maturation of the response occurs, such that the average affinity (binding constant) of the antibodies for the antigen increases as the secondary response develops.

e.         The driving force for this increase in affinity is a selection process during which B cells compete with free antibody to capture a decreasing amount of antigen.

f.          Thus, only those B cell clones with high-affinity Ig receptors on their surfaces will bind enough antigen to ensure that the B cells are triggered to differentiate into plasma cells.

g.         These plasma cells, which arise from a preferentially selected B cells, synthesize this antibody with high affinity for antigen.

h.         This capacity for making secondary or anamnestic (memory) response may persist for a long time (years in humans); this is the purpose of public health immunization programs.