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Immunoglobulin Structure and Function Lecture 8 Dr. Nabil MTIRAOUI, M.Sc, Ph.D Definition and Properties Outline of Lecture 1. Recognize the structure of immunoglobulin molecule 2. Know the different types of immunoglobulin. 3. Understand the biological activities of each type. 4. Differentiate between types of immunoglobulin. 5. Define isotype switching and antibody diversity. 6. Differentiate between types of FC receptors. Antibodies (or Immunoglubulins) The chemical information of immunoglobulin was provided by Tiselius and Kabat in the early 1940s. In 1950s, Porter and Edelman revealed the basic structure of immunoglobulin molecule. Antibodies are products of antigen- activated lymphocytes. They are the main effectors of humoral immunity. B- Definition Amount of protein + albumin globulins 1 2 Immune serum Ag adsorbed serum Mobility An antibody or immunoglobulin (Ig) is a glycoprotein that is made by plasma cells in response to an antigen and can recognize and bind to the antigen that caused its production. Antibodies are produced by B cell General Functions of Immunoglobulins Ag binding Can result in protection Valency Effector functions Fixation of complement Binding to various cells Usually requires Ag binding Basic Immunoglobulin Structure 1. 2. 3. 4. 5. 6. Fab region Fc region Heavy chain with one variable (VH) domain followed by a constant domain (CH1), a hinge region, and two more constant (CH2 and CH3) domains. Light chain with one variable (VL) and one constant (CL) domain Antigen binding site (paratope): It is the area of Ig molecules that interacts specifically with epitope of the Ag Hinge regions. Basic Immunoglobulin Structure Abs have more than one antigen combining site Some bivalent Ab molecules can combine to form multimeric Abs that have up to 10 combining sites. All immunoglobulin have a basic structure composed of 4 polypeptide chains connected to each other by disulfide bonds. Each light chain consist of 220 Aa and has a mass of approx. 25kDa. Each heavy chain consists of about 440 Aa and has a mass of 5070kDa. Basic Immunoglobulin Structure Both light and heavy chains contain 2 different regions constant and variable region The four chains are arranged in the form of a flexible “Y” with the hinge region and is termed as crystallizable fragment (Fc) and contains the site at which Ab binds. Top of the “Y” consist of two Ag binding fragments (Fab) that bind with antigenic determinant sites. The four chains are linked by disulfide bonds. Basic Immunoglobulin Structure Light chain: The light chain may be either of two distinct forms called “Kappa” and “Lambda” and can be distinguished by aa sequence of carboxyl portion of the chain. Heavy chain: In the heavy chain NH2 terminal has a pattern of variability similar to that of kappa and lambda of the light chain. The variable (V) regions. The first 100 or so amino acids at the N-terminal of both H and L chains vary greatly from antibody to antibody. These are the variable (V) regions. The amino acid sequence variability in the V regions is especially pronounced in 3 hypervariable regions. Together they construct the antigen binding site against which the epitope fits Only a few different amino acid sequences are found in the Cterminals of H and L chains. These are the constant (C) regions. The variable (V) regions. Variability Index HVR3 15 0 10 0 5 0 0 HVR2 HVR1 FR2 FR1 25 50 Amino acid residue FR3 7 5 FR4 10 0 The Hypervariable regions. Hypervariable regions: In the variable regions of both L&H chains, there are 3 extremely hypervariable amino acids sequences that form the Ag binding sites. The hypervariable regions form the region complementary in structure to the epitope. These regions are involved in the formation of paratope. The constant (C) regions. two different kinds of C regions for their L chains producing kappa (κ) L chains lambda (λ) L chains five different kinds of C regions for their H chains producing mu (µ) chains (the H chain of IgM antibodies) gamma (γ) chains (IgG) alpha (α) chains (IgA) delta (δ) chains (IgD) epsilon (ε) chains (IgE) Fc and Fab regions The proteolytic enzyme papain breaks each Ig molecule into 3 fragments at the hinge region. The single crystallizable fragment (Fc region) includes part of the constant domain that occupies the stem. There are 2 antigen-binding fragments (Fab region), which include the entire light chain and variable and constant portions of the heavy chain. Ig G Papain 2 Fab + Fc Fc and Fab regions Antigen-binding fragment (Fab) recognize Ag contain Ig idiotype Unique protein sequence that identifies each Ab Crystallizable fragment (Fc) define isotype of Ig bind FcR for all functional attributes of Ab Fab link to Fc by hinge region Fonction of Fc and Fab regions By binding to specific proteins the Fc region ensures that each antibody generates an appropriate immune response for a given antigen. The Fc region also binds to various cell receptors, such as Fc receptors, and other immune molecules, such as complement proteins. Thus, Ab mediates different physiological effects including opsonization, cell lysis, and degranulation of mast cells, basophils and eosinophils. Activation of complement Antibodies that bind to surface antigens on, for example a bacterium, attract the first component of the complement cascade with their Fc region and initiate activation of the "classical" complement system This results in the killing of bacteria in two ways; First, the binding of the antibody and complement molecules marks the microbe for ingestion by phagocytes in a process called opsonization; Secondly, some complement system components form a membrane attack complex to assist antibodies to kill the bacterium directly. Types of FC receptors FC receptor Affinity to Ig Cell distibution Function FcγRl (CD64) High binds IgG1a and IgG3 MQ. Neutrophils and eosinophils Phagocytosis, activation of phagocytes FcγRllA (CD32) Low MQ. Neutrophils and eosinophils,platelet Phagocytosis,cell activation (inefficient) FcγRllB (CD32) Low B lymphocytes Feed back inhibition of B cells FcγRlllA (CD16) Low NK ADCC FcεRl High bind monomeric IgE Mast cells, basophils, eosinophils Avtivation, degranulation of mast cell, basophils CLASSES (ISOTYPES) OF IMMUNOGLOBULINS Classes based on constant region of heavy chains Immunoglobulin A (IgA) Immunoglobulin D (IgD) Immunoglobulin E (IgE) Immunoglobulin G (IgG) Immunoglobulin M (IgM) Differentiation of heavy chains Length of C region, location of disulfide bonds, hinge region, distribution of carbohydrate Classes have different effector functions CLASSES (ISOTYPES) OF IMMUNOGLOBULINS Immunoglobulin G Structure Monomer (7S) IgG1, IgG2 and IgG4 IgG3 IgG It is the major Ig in normal serum, accounting for 75% of the total Ig pool. It is a monomeric unit (2 heavy chain &2 light chain), MW 160,000. It can bind 2 Ag molecules. 4 subclasses are known IgG1,2,3,4. Its biological activities include: Its half life time is 23 days and is the longest of all Igs. It is the major Ab in the secondary immune response. It is the only Ab that can cross placenta (IgG2 does not cross well) and provide immunity to the newborn during the first months after birth. Transfer is mediated by a receptor on placental cells for FC region of IgG . It diffuse into the extravascular neutralizing bacterial toxins (antitoxin). It enhance phagocytosis (opsonization) by coating bacteria and attaching by its FC portion to FC receptor on phagocytic cells. It can fix and activate complement (by IgG1 and IgG3) Immunoglobulin A Secretary Ab First line defense for microbes IgA : a doublet guards the entrance to the body. 170,000 MW in serum and 400,000 MW in external secretions, 15% of Ig in serum, found in the blood as a monomer, and in tears, saliva, colustrum, nasal, vaginal, prostatic and bronchial secretions as a dimer. • Blocks attachment of microbes to mucous membranes • It concentrates in body fluids such as tears, saliva, and secretions of the respiratory and gastrointestinal tracts. IgA Structure Serum - monomer Secretions (sIgA) Dimer (11S), sIgA molecule consists of two H2L2 units plus one molecule each of J chain and secretory component (SC or SP) Secretory Piece J Chain IgA Properties 2nd highest serum Ig Major secretory Ig (saliva, tears, respiratory, intestinal, and genital tract secretions.) Does not fix complement unless aggregated Binds to Fc receptors on some cells Immunoglobulin M Macroglobulin primary immune response Bacteriolytic •IgM usually combines in star-shaped clusters. pentamer, •It tends to remain in the bloodstream, 10% of blood Ig, • found on the surface of B lymphocytes. •Activates the complement system. IgM Structure Pentamer (19S) composed 5 H2L2 units plus one molecule of J chain Extra domain (CH4) J chain J Chain C4 Fixation of C1 by IgG and IgM Abs No activation Activation IgM Structure Properties 3rd highest serum Ig First Ig made by fetus and B cells Produced early in the primary response The most efficient Ig Fixes complement Agglutinating Ig Binds to Fc receptors B cell surface Ig Tail Piece Immunoglobulin D Membrane bound antibody Found on B-cell membrane Memory function 180,000 MW, activity is not well known. 0.2% of plasma Ig. 13% carbohydrate content. Also found on the surface of B lymphocytes, where it somehow regulates the cell's activation IgD Structure Monomer Tail piece Tail Piece IgD Structure Properties 4th highest serum Ig Expressed in B cell surface Ig Does not bind complement FUNCTIONS: B cell activation . Acts as receptor for Ag binding. Immunoglobulin E Activate mast cells Release vasoactive amines Respond to allergens Mediate hypersensitivity reactions 90 000 MW, largest immunoglobulin, present in extremely low levels in a healthy individual. IgE levels rise in response to parasitic infections and in allergic reactions. Bind and activate mast cells. Mast cells cause acute inflammatory response (e.g. swelling, redness, pain and itchiness). Hay fever is a condition caused by too much IgE activity. IgE Structure Monomer Extra domain (CH4) C4 IgE Structure Properties Least common serum Ig Allergic reactions Parasitic infections Does not fix complement FUNCTIONS: Responsible for immediate hypersensitivity or allergic reactions. o Binds to Fc receptors on basophils and mast cells. o Release of substances like histamine , bradykinin and other vasoactive ‘mediators’. o Properties of immunoglobulins: IgG IgA IgM IgD IgE 1. Serum conc. (%) 85 5-15 5-10 <1 <1 2. Mol. Wt. 160,000 170,000 & 385,000 960,000 184,000 188,105 3.Sed. coeff. 7S 7S 19S 7S 8S 4.Heavy chain class Gamma Alpha Mu Delta Epsilon 5.Light chain K&L K&L K&L K&L K&L 6. Valency 2 2 or multiple of 2 5 (10) 2 2 Dimer or Trimer Pentamer Monomer Monomer 7.No of basic 4Monomer polypeptide chains IgG IgA IgM IgD IgE 8.Placental transport + _ _ _ _ 9.Present in milk + + _ _ _ 10.Selectie secretion by seromucus gland _ + _ _ _ 11. Intravascular distribution(%) 45 42 80 75 50 12.Carbohydrate (%) 3 11 10 13 12 8.Subclasses IgG1-4 IgA1-2 _ _ _ IgG IgM IgA IgD IgE A.Classical ++ _ +++ _ _ B.Alternati ve _ + _ _ _ 15.Half life (days) 23 6 5 2-3 2-3 16.Princip-al site of action Serum Secretion Serum Receptor for B cells Mast cells 17.characteristic properties precipitins, antitoxins, compleme-nt fixation, late Ab Serum and secretory Abs Agglutinin, opsonin , lysin , early Ab Not known (B-cell activation) Reaginic Ab (anaphylaxis) 14.Complement fixation Thank You