IMMUNOGLOBULINS STRUCTURE AND FUNCTION Arpad Lanyi BSc Public Health 5th week, 2015

IMMUNOGLOBULINS Definition Glycoprotein molecules that are present on B cells (BCR) or produced by plasma cells (usually referred to as antibodies) in response to an immunogen (antigen that provokes immune response)

B CELL ACTIVATION Gene rearrangement in an immature B cell leads to the expression of membrane-bound IgM and IgD on the mature B cell surface. After encounter with an antigen this isotype is produced in a secreted form by the plasma cell (IgM largely while IgD only is small amounts). All isotypes (IgA, IgD, IgE, IgG and IgM) can be made in two forms: the bounded immunoglobulins serving as BCRs, and soluble secreted antibodies by the plasma cells. (The difference b/w membrane bound and secreted is a hydrophobic part and hydrophilic parts at the carboxy terminus, respectively).

Immunoglobulin STRUCTURE 2x Heavy chain (light blue) 2x light chain (dark blue) Variable regions  antigen binding Constant regions disulfide bond carbohydrate CL VL CH2 CH3 CH1 hinge region VH

FLEXIBILITY OF ANTIBODIES

mIg sIg BCR (B cell receptor) Antibody Transmembrane domain Associated chains for signaling Cytoplasmic domain SOLUBLE (freely circulating) MEMBRANE BOUND! Antigen binding  effector functions Produced by plasma cells Antigen recognition  B cell activation

ANTIBODY DOMAINS AND THEIR FUNCTIONS Antigen recognition Variable domain Ag Ag Constant domains Effector functions

B CELL ACTIVATION B cell BCR oligomerization results in B cell activation, proliferation and differentiation

ANTIGEN BINDING Antigen Binding Fragment (Fab) Complement binding site Placental transfer Constant fragment (Fc) Binding to Fc receptors on phagocytic cells

HYPERVARIABLE REGIONS B cell development in the red bone marrow  DNA recombination (somatic gene rearrangement) of gene segments encoding variable domains of heavy and light polypeptide chains is responsible for generation of B cells with highly variable specificity Epitope CDR1 CDR2 CDR3 Light chain Heavy chain CDR = complementarity determining region = hypervariable region

The three-dimensional structure of immunoglobulin C and V domains Shown is the structure of one light chain with its constituent C domain (left panel) and V domain (right panel). The inset shows the location of the light chain in a Fab of IgG. The folding of the polypeptide is depicted as a ribbon diagram in which the thick colored arrows correspond to the parts of the chain that form the β strands of the β sheet. The arrows point from the amino terminus to the carboxy terminus. Adjacent strands in each β sheet run in opposite directions (antiparallel), as shown by the arrows, and are connected by loops. The C domain has four β strands in the upper β sheet (yellow) and three in the lower (green). The V domain has five strands in the upper sheet (blue) and four in the lower (red).

DIFFERENT VARIABLE REGIONS  DIFFERENT ANTIGEN-BINDING SITES  DIFFERENT SPECIFICITIES the same applies to TCRs!

Sequence variability of H/L-chain constant regions ISOTYPE (CLASS) Sequence variability of H/L-chain constant regions IgG - gamma (γ) heavy chains IgM - mu (μ) heavy chains IgA - alpha (α) heavy chains IgD - delta (δ) heavy chains IgE - epsilon (ε) heavy chains Each isotype has a distinct constant region and the isotype of the antibody determines the effector functions….

PHASES OF B CELL RESPONSE Gene rearrangement in an immature B cell leads to the expression of membrane-bound IgM and IgD on the mature B cell surface. After encounter with an antigen this isotype is produced in a secreted form by the plasma cell (IgM largely while IgD only is small amounts). All isotypes (IgA, IgD, IgE, IgG and IgM) can be made in two forms: the bounded immunoglobulins serving as BCRs, and soluble secreted antibodies by the plasma cells. (The difference b/w membrane bound and secreted is a hydrophobic part and hydrophilic parts at the carboxy terminus, respectively).

YIsotype Switching during B Cell Development PE SWITCHING Isotype Switching during B Cell Development During the initial stages of a B cell’s primary response to antigen, it produces and secretes IgM. Later in the primary response or during subsequent responses, different heavy chain isotypes may be expressed by the progeny of the original IgM-producing clone. Such “switching” occurs at the DNA level, resulting in the production of an Ig protein with the same V region but a different C region. Thus, over the lifetime of a B cell clone, it may produce antibodies of the same specificity but different isotypes.

MAIN CHARACTERISTICS OF ANTIBODY ISOTYPES IgG1-IgG4 IgA1-IgA2

ANTIBODY PRODUCTION DURING THE PRIMARY AND THE SECONDARY IMMUNE RESPONSES Level of antibodies secondary response against antigen A Primary response against antigen A primary response against antigen B Days napok Antigen A Antigen A and B

EFFECTOR FUNCTIONS OF ANTIBODIES Antibody-mediated immune responses Fab part: NEUTRALIZATION Fc part: OPSONIZATION followed by opsonized phagocytosis (macrophage; IgG) ADCC (NK cell; IgG) mast cell degranulation (parasite, allergy; IgE) COMPLEMENT ACTIVATION

NEUTRALIZATION

Complement binding site Binding to Fc receptors Antigen binding Complement binding site Placental transfer Binding to Fc receptors

OPSONIZED PHAGOCYTOSIS Flagging a pathogen Antigen binding fragment (Fab) binds the pathogen  the Fc region is accessible for Fc-receptors of phagocytic cells, facilitating (speeding up) the process of phagocytosis

Opsonization facilitate and accelerate the recognition of the pathogens by phagocytes Main opsonins: antibodies Complement molecules Acute-phase proteins (CRP, SAP) Phagocytes must express receptors for the opsonins: IgG  FcγRI C3b  CR1

Antibody Dependent Cellular Cytotoxicity (ADCC)

MAST CELL DEGRANULATION FcεRI + IgE Mast cells, basophils and activated eosinophils in mucosal surfaces play a role in the defense against parasites and express the FcεRI. FcεRI has such high affinity that the IgEs, specific for many different antigens- cannot dissociate. Upon antigen binding and FcεRI cross-linking the mast cell is activated (degranulation) inflammatory mediators released, acting on vessel permeability- swelling, pain etc, and acting on smooth muscle cells  Sneezing, coughing, vomiting, diarrhea. Directly killing the parasite by toxic granule content or Indirectly flushing it. An unnecessary response to an innocuous substance (pollens etc.) are an unfortunate side effect of the highly specialized and powerful antibodies. (A) High-affinity FcRs on the surface of the cell bind antibodies before it binds to antigen. (mast cell) (B) Low-affinity FcRs bind multiple Igs that have already bound to a multivalent antigen. (macrophage, NK cell)

The complement system The complement system is a set of plasma proteins that act in a cascade to attack and kill extracellular pathogens. Approximately 30 components: activating molecules complement receptors regulator factors membrane proteins wich inhibit the lysis of host cells Most of the complement proteins and glycoproteins are produced in the liver in an inactive form (zymogen). Activation is induced by proteolitic cleavage.

Amplification of the complement cascade limited proteolysis inactive precursors Complement activation involves proteolytic cascades, in which an inactive precursor enzyme, called a zymogen, is altered to become an active protease that cleaves and thereby induces the proteolytic activity of the next complement protein in the cascade. As the cascade proceeds, the enzymatic activities result in tremendous amplification of the amount of proteolytic products that are generated. These products perform the effector functions of the complement system. enzyme activating surface Activating surface needed!

Pathways of complement activation The activation of the complement system may be initiated by three distinct pathways, all of which lead to the production of C3b (the early steps). C3b initiates the late steps of complement activation, culminating in the production of peptides that stimulate inflammation (C5a) and polymerized C9, which forms the membrane attack complex, so called because it creates holes in plasma membranes. The principal functions of major proteins produced at different steps are shown. Cellular and Molecular Immunology, 7th ed., 2014 Elservier

Complement binding site Binding to Fc receptors Antigen binding Complement binding site Placental transfer Binding to Fc receptors

SUMMARY The alternative pathway of complement activation is triggered by changes in the local physicochemical environment that are caused by the constituents of some bacterial surfaces. The alternative pathway acts at the earliest times during infection. The lectin-mediated pathway is initiated by the mannose-binding lectin of plasma, which binds to carbohydrates found on bacterial cells and other pathogens. The lectin-mediated pathway is induced by infection and contributes to innate immunity. The classical pathway is initiated in the innate immune response by the binding of C-reactive protein to bacterial surfaces, and in the adaptive immune response by the binding of antibodies to pathogen surfaces.

Complement binding site Binding to Fc receptors Antigen binding Complement binding site Placental transfer Binding to Fc receptors FcRn on the placenta facilitate the transfer of maternal IgG to the body of the fetus

PRODUCTION OF IMMUNOGLOBULINS IgG transport is so efficient that at birth babies have as high a level of IgG in their plasma as their mothers These transfers are a form of passive immunization. The babies protection by IgG and IgA is against those pathogen that the mother has mounted The children are most vulnerable during the first year of life (esp.3-12m) when maternal IgGs have disappeared but the de novo synthesis is at low level

Maternal IgG is transported by the neonatal Fc receptor (FcRn) across the placenta to the fetus The bulk of materno-fetal IgG transfer in humans occurs antenatally across the syncytiotrophoblast of the placenta. Syncytiotrophoblasts are bathed in maternal blood and internalize serum containing maternal IgG. FcRn is expressed in the internal vesicles of the syncytiotrophoblast. On acidification in the endosome, FcRn binds to maternal IgG and transcytoses it to the fetal circulation where it is released at physiological pH.

The receptor FcRn transports IgG from the bloodstream into the extracellular spaces of tissues At the apical (luminal) side of the endothelial cell, IgG and other serum proteins are actively taken up by fluid-phase pinocytosis. In the endocytic vesicle, the pH becomes acidic and each IgG molecule associates with two molecules of FcRn. The IgG is carried by FcRn to the basolateral face of the cell and away from the degradative activity of the lysosomes. At the basal side of the cell, the more basic pH dissociates the complex of IgG and FcRn, and the IgG is released into the extracellular space.

IgG half-life FcRn is also present in the adult and involved in protecting IgG from degradation Accounts for the long (3 week) half-life of IgG compared to other Ig isotypes Therapeutic agents that are fused to IgG Fc regions take advantage of this property e.g. Enbrel (TNFR-Fc) Also explains why B cell deficient patients need IVIG injections only every 3-4 weeks

Pathological consequences of placental transport of IgG (hemolytic disease of the newborn) Passive anti-D IgG anti-Rh IgM Rhesus incompatibility: In case a fetus is Rh+ (meaning he expresses the D antigen on his RBCs surface) and the mother is RH- (no D antigens and no anti-D antibodies) after the first delivery when some fetal RBCs mix with maternal circulation, the mother will initiate a primary immune response towards the D antigen. These antibodies as it is the first immune response will be of IgM isotype and therefore not able to pass the placenta. However, with time, isotype switching might take place that will result in the production of IgG antibodies against the D antigen. These are now able to pass through the placenta, thus, in the second pregnancy if the fetus is Rh+ his RBCs will be attacked by maternal anti-D IgG antibodies, causing the mild to severe ‘hemolytic disease of the newborn’ (destruction of red RBCs anemia) To avoid that, i.m. anti-D IgGs are administered to the mother. These will bind any D antigens on fetal RBCs that entered the mothers circulation and prevent her from developing anti-D antibodies. The IgGs eliminate the RBCs coated with D antigens through opsonisation and elimination by macrophages and granulocytes. … One might say, well IgGs pass the placenta, then these anti D antibodies can attack the fetus RBCs- true, but the clinical course of such an event is benign and requires no treatment.

is mediated by the poly-Ig receptor (pIgR) Transcytosis of dimeric IgA antibody across epithelia is mediated by the poly-Ig receptor (pIgR) Dimeric IgA is made by plasma cells lying just beneath the epithelial basement membranes of mucosal tissues, such as the gut. The IgA dimer bound to the J chain diffuses across the basement membrane and is bound by the poly-Ig receptor on the basolateral surface of an epithelial cell. Receptor binding is mediated by the CH3 constant domains of the IgA. The bound complex crosses the cell (transcytosis) in a membrane vesicle and is delivered to the apical surface. There the receptor undergoes cleavage, which releases a complex of dimeric IgA bound to a fragment of the receptor called the secretory component or secretory piece. Importantly, the carbohydrate (blue hexagon) of the secretory piece tethers the IgA to the mucus that coats the apical surface, thus preventing the antibody from being washed away into the gut lumen and beyond. The residual membrane-bound fragment of the poly-Ig receptor is nonfunctional and is degraded.