Dr. Nabil MTIRAOUI, M.Sc, Ph.D

1. Immunoglobulin Structure and Function Dr. Nabil MTIRAOUI, M.Sc, Ph.D Lecture 8

2. Definition and Properties

3. Outline of Lecture Recognize the structure of immunoglobulin molecule Know the different types of immunoglobulin. Understand the biological activities of each type. Differentiate between types of immunoglobulin. Define isotype switching and antibody diversity. Differentiate between types of FC receptors.

4. 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 B- lymphocytes. • They are the main effectors of humoral immunity.

5. - + albumin Amount of protein globulins   1 2 Immune serum Ag adsorbed serum Mobility Definition • 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

6. General Functions of Immunoglobulins • Ag binding • Can result in protection • Valency • Effector functions • Fixation of complement • Binding to various cells • Usually requires Ag binding

7. Basic Immunoglobulin Structure • 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.

8. 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 50-70kDa.

9. 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.

10. 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.

11. 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 C-terminals of H and L chains. • These are the constant (C) regions.

12. HVR3 150 Variability Index 100 HVR2 HVR1 50 FR2 FR1 FR4 FR3 0 25 75 100 50 Amino acid residue The variable (V) regions.

13. 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.

14. 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)

15. 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

16. Antigen-binding fragment (Fab) recognize Ag contain Igidiotype 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 Fc and Fab regions

17. 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.

18. 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.

19. Types of FC receptors

20. 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

21. CLASSES (ISOTYPES) OF IMMUNOGLOBULINS

22. IgG1, IgG2 and IgG4 IgG3 Immunoglobulin G • Structure • Monomer (7S)

23. 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)

24. 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.

25. Secretory Piece J Chain 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)

26. IgA • Properties • 2nd highest serum Ig • Major secretoryIg (saliva, tears, respiratory, intestinal, and genital tract secretions.) • Does not fix complement unless aggregated • Binds to Fc receptors on some cells

27. Immunoglobulin M Macroglobulin primary immune response Bacteriolytic • IgMusually 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.

28. J Chain C4 IgM • Structure • Pentamer (19S) composed 5 H2L2 units plus one molecule of J chain • Extra domain (CH4) • J chain

29. C1r C1r C1s C1s C1q C1q Fixation of C1 by IgG and IgM Abs No activation Activation

30. Tail Piece 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

31. Immunoglobulin D • 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 Membrane bound antibody Found on B-cell membrane Memory function

32. Tail Piece IgD • Structure • Monomer • Tail piece

33. 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.

34. Immunoglobulin E • 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. Activate mast cells Release vasoactive amines Respond to allergens Mediate hypersensitivity reactions

35. C4 IgE • Structure • Monomer • Extra domain (CH4)

36. IgE • Structure • Properties • Least common serum Ig • Allergic reactions • Parasitic infections • Does not fix complement • FUNCTIONS: • Responsible for immediate hypersensitivity or allergic reactions. • Binds to Fc receptors on basophils and mast cells. • Release of substances like histamine , bradykinin and other vasoactive ‘mediators’.

37. Properties of immunoglobulins:

40. Thank You