Chapter 2

1. Chapter 2 Antibody Structure and the Generation of B-Cell Diversity

2. The Structural Basis of Antibody Diversity

3. Antibodies and Antigens • Antibodies…. • Five chemically and physically distinct classes of antibodies (IgG, IgA, IgM, IgD, IgE). • Bind with high specificity and affinity. • Produced by the B lymphocytes in response to infection. • Circulate as a major component of the plasma in blood and lymph. • Bind to pathogenic microorganism and their toxins in the extracellular spaces of the body. • Antibody repertoire might be as high as 1016. • Antigens…. • Are bound by antibodies. • Are biological macromolecules. • proteins and carbohydrates most common antigens

4. Antibody (Ab) - Immunglobulins • Antibodies are the secreted form of proteins known more generally as immunoglobulins (Igs) • Mature B cells express Igs (B cell receptor) • When Ag binds Igs, the B cell proliferates and then differentiates into a plasma cell • Plasma cell secretes Ab with same specificity as the membrane-bound Igs

5. Antibody Functions Antibody functions in host defense: • Antibody recognizes and binds its corresponding antigen • Antibody targets the bound antigen to other components of the immune system • each of these functions is performed by different parts of the antibody molecule

6. Antibody Structure • Antibodies are glycoproteins • Basic unit of four polypeptide chains (2 pairs L & H chains) • covalent disulfide bridges • noncovalent interactions • Total MW~150kDa • Two identical heavy chains (H chains) (50 kDa each) • Two identical, smaller, light chains (L chains) (25 kDa each) • Arms of Y • L chains paired with N-terminal pair of H chain - Covalently linked by a disulfide bond. • Stem of the Y • consists of the paired C-terminal portions of the two-H chains

7. Antibody Variability • H and L chains are divided into V and C regions • The V region • variability = great diversity • concentrated in the N-terminal region • paired V regions of a H and L chain • form the antigen binding site • two identical antigen binding sites • The C region • constant region • determines the fate of the antigen • less variation in amino acid sequence between antibodies N-terminal C-terminal

8. Antibody Affinity Affinity is the tightness of binding of an antibody binding site to an antigen. • The tighter the binding, the less likely the antibody is to dissociate from antigen. • Different antibodies to the same antigen vary considerably in their affinity for an epitope. • Antibodies produced by a memory response have higher affinity than those in a primary response.

9. Antibody Valence and Avidity • Valence of an antibody is the maximum number of antigenic determinants with which an antibody can react. • Multiple binding sites on an antibody dramatically increases its binding (avidity) to antigens on particles such as bacteria or virus. • Example: two binding sites on IgG are 100 times more effective at neutralizing virus than two unlinked binding sites. • Avidity is the firmness of association between a multideterminant antigen and the antibodies produced against it. • This combined effect (avidity) results from synergy of the binding strengths of each binding site.

10. Antibody Valence and Avidity

11. Proteases Cleave Antibody into Fab and Fc Fragments • Papain cleaves the Ig molecule into three pieces: • Two Fab (Fragment Antigen Binding) fragments • Antigen binding part • One Fc fragment (Fragment Crystallizable) • Part responsible for effector functions. • Pepsin cleaves IgG to yield 1 F(ab’)2 fragment and the Fc fragment • Fc fragment is broken into a number of smaller pieces

12. Antibody Classes • Different antibody classes with different biological activities have evolved to deal with diverse antigens. • Heavy-chain constant regions define five main isotypes. • Heavy chains , , , ,  • IgA, IgD, IgE, IgG and IgM • Each isotype has a different effector functions – with some overlap. • Example: IgA is the most common antibody in mucosal secretions while IgM is mainly found in the plasma, and both are most effective at those locations

13. Isotypes of Heavy and Light Chains • Heavy-chain constant regions define five main isotypes of antibodies: IgA, IgD, IgE, IgG, and IgM • Differences in length and sugars of heavy chain • Only two isotypes of light chain • kappa () and lambda () • Each antibody has either  or  chains • The antigen binding site is composed of two variable domains (VH and VL) • Constant domain of light chain is CL • Constant domain of the heavy chain is CH • The  heavy chain of IgG has three domains - CH1, CH2, CH3 • Some other isotypes have four C domains

14. IgG (MW~150 kD) • Four different subclasses (IgG1, IgG2, IgG3 and IgG4) • Each subclass has slightly different H-chains and corresponding differences in their function. • Found both in vascular and extravascular spaces as well as in secretions. • Most abundant Ig in the blood. • Provide the bulk of immunity to most blood borne infectious agents. • The only antibody class to cross the placenta to provide passive humoral (innate) immunity to the developing and newborn infant.

15. IgA (In Serum~170 kD, External Secretions ~420 kD Dimer) • Major Ig present in external secretions - colostrum, milk and saliva. • In addition to the L-chains and the IgA heavy chain () secreted IgA also contains two other polypeptide chains- • secretory component (SC) and J-chain (joining chain) • SC is part of the molecule (poly-Ig receptor) involved in the transepithelial transport of exocrine IgA and stabilizes IgA against proteolytic degradation. • The two four-chain units composing secretory IgA are held together by the J-chain through disulfide bridges. • A first line of defense against microbes entering through mucosal surfaces. • Respiratory, gastrointestinal and genitourinary tracts • Secretory (dimeric) IgA is synthesized locally by plasma cells in mammary and salivary glands and along the respiratory, gastrointestinal and genitourinary tracts. • IgA prevents colonization of mucosal surfaces by pathogens and mediates their phagocytosis.

16. (1) Protected from proteases (5) (2) (3) (4) Receptor-mediated endocytosis

17. IgM (~900 kD) • IgM is the first antibody produced by B cells. • Is the antigen receptor (B cell receptor). • Expressed as a four-chain unit - two  H-chains and two L-chains. • Also present as a soluble molecule in the blood. • J-chain is associated with IgM in the blood and initiates the polymerization of its subunits. • In the circulation, IgM is composed of five four-chain units with 10 combining sites (recall avidity slide). • Has high avidity for antigens and is very efficient per molecule in dealing with pathogens especially early in the immune response before sufficient quantities of IgG have been produced.

18. IgD • Present in low quantities in the circulation • (0.3 mg/ml in adult serum) • IgD functions primarily as an antigen receptor on B cells. • B cells express both IgM and IgD that are specific for the same antigen. • When IgM and IgD on a B cell interact with an antigen, the antigen is internalized and processed and presented to helper T cells. • This triggers B cells to proliferate and differentiate into plasma cells, thus initiating the development of a humoral immune response • Humoral-pertains to extracellular fluid including the plasma and lymph.

19. IgE • Present in serum at very low levels • nanograms per milliliter • Plays significant roles in • enhancing acute inflammation • protection from infection by worms • Allergic reactions are predominantly associated with IgE • After antigen stimulation induces plasma cells to produce IgE • IgE binds to receptors on mast cells which are specific for the Fc regions of IgE. • Antigen reintroduced into a previously sensitized individual binds to IgE on ‘armed’ mast cells and triggers release of the pharmacologically active agents –histamine (involved in immediate hypersensitivity such as hay fever and asthma).

21. Structural Organization of Human Immunoglobulin Isotypes • Differences in length of the heavy-chain C regions and locations of the disulfide bonds. • The hinge region present in IgG, IgA, IgD but not in IgM, IgE. • Isotypes differ in the distribution of N-linked carbohydrate groups • All occur as monomers in their membrane-bound form. • Soluble, secreted form of IgD, IgE and IgG are always monomers. • IgA forms monomers and dimers. • IgM forms pentamers.

22. Immunoglobulins are flexible • The hinge region is a flexible tether, allowing independent movement of the two Fab arms. • The angle between the arms can vary from 0° in dimers, to 60° or 90° degrees in triangles or squares.

23. Immunoglobulin Chains are Folded into Compact and Stable protein Domains • The V region at the N-terminal end of each H or L chain is composed of a single variable domain (V domain, VH or VL). • VH and VL domains together form an antigen-binding site. • The other domains have little or no sequence diversity within a particular isotype and are termed the constant domains (C domains) . • Constant region of a light chain is composed of a single CL domain • Constant region of a H chain is composed of three or four C domains (isotype dependent) • the  heavy chains of IgG have 3 domains-Ch1, CH2, Ch3 • other isotypes have four C domains (previous slide) • In the complete IgG molecule the pairing of the four polypeptide chains produces three globular regions, corresponding to the two Fab arms and the Fc stem (refer to fig 2.5)

25. Immunoglobulin Chains are Folded into Compact and Stable protein Domains Immunoglobulin-like domain = Immunoglobulin superfamily

26. Ag-binding Site is Formed from the Hypervariable Regions of a Heavy Chain and a Light Chain V Domain • V domains of light and heavy chains have hypervariable regions (HV), flanked by framework regions. • Three hypervariable regions in each domain.

27. The Hypervariable Regions of Antibody V Domains Lie in Discrete Loops at One End of the Domain Structure • The hypervariable regions of antibody V domains lie in discrete loops at the end of the domain. • The pairing of the heavy and light chains bring together the hypervariable loops and create a composite hypervariable surface that forms the antigen-binding site. • This surface determines specificity and diversity. • The hypervariable loops are also called the complementarity-determining regions(CDRs).

28. Epitopes for Antibody Binding are Exposed • Antibodies that are most effective are those that bind the exposed surface of a pathogen. • Epitope is the part of the antigen to which an antibody binds. • An antigen that contains more than one epitope is called multivalent. Poliovirus

29. Physical Properties of Antibody Binding Sites • The antibody binds to an antigenic determinant or epitope, commonly: glycoproteins, polysaccharides, glycolipids, and proteoglycans. • Epitopes can be linear, or discontinuous (when different parts of the antigen are folded together). • The binding forces are non-covalent: electrostatic, hydrogen bonds, van der Waals forces and hydrophobic forces. • The binding strength is known as the affinity of the antibody for the antigen.

30. Epitopes Can Bind to Pockets, Grooves or Extended Surfaces in the Antibody Binding Sites • The type of antigen bound by an antibody depends on the shape of the antigen-binding site. • Antigens that are small molecules can be bound within deep pocket. • Linear epitopes from proteins or carbohydrates can be bound within clefts or grooves. • Binding of conformation epitopes of folded proteins takes place over an extended surface area.

31. Antibodies Binding to Three different Types of Epitope Peptide epitope (red) bound to pocket in CDR loops Peptide epitope in groove between two V domains Fab binding to surface epitope of lysozyme

32. Monoclonal Antibodies • 1975 Kohler and Milstein developed a procedure to create cell lines producing predetermined, monospecific and monoclonal antibodies (mAb). • Standardized procedures involving fusion of an immortal cell (i.e. myeloma tumor cell) with a specific predetermined antibody-producing B cell from immunized animals or humans to create hybridoma cells producing monospecific and monoclonal antibodies (mAb). • Monoclonal antibodies important in a variety of therapeutic, diagnostic and research settings.

33. Production of Monoclonal Antibodies • Produced from a single clone from antibody-producing cells and thus have identical Ag-binding sites and identical isotypes. • Antibodies can be humanized (mouse CDRs into human antibody) for therapeutic purposes.

34. The flow cytometer allows individual cells to be identified by surface molecules.

35. Generation of Immunoglobulin Diversity

36. In all cells (except B cells) Ig genes are in a fragmented form that cannot be expressed. • The Ig H-chain and L-chain loci consists of families of gene segments containing alternative versions of parts of the Ig variable region. • Germ-line DNA contains multiple gene segments encoding portions of a single Ig heavy or light chain. Germ-line DNA Rearranged Functional DNA

37. In B cell germ-line DNA gene segments are rearranged into functional genes. • Mature B cells contain chromosomal DNA that is no longer identical to germ-line DNA. • Genomic rearrangement is an essential feature of lymphocyte differentiation. Germ-line DNA Rearrangement Rearranged Functional DNA

38. Generation of Diversity: Antibody Genes • DNA encoding Igs is found in three unlinked gene groups. • One group encodes kappa () L-chains • One lambda () L-chains • One H-chains • Each L-chain gene groups has multiple different copies of V gene segments and J segments. • In addition, in the kappa () chain group there is one gene segment encoding the constant regions of kappa () chains. • In the lambda () groups there are four lambda () chain constant regions gene segments. • The H-chain gene groups has multiple different copies of V, D, and J gene segments and one gene segment for each of the constant regions for the different antibody classes and subclasses.

40. Generation of Diversity: Gene Rearrangement • During development, a single B cell randomly selects from its H-chain gene group, one V, one D and one J gene segment for rearrangement. • The B cell then selects a L-chain from the kappa () or lambda () gene group, selecting one V and one gene J gene segment for rearrangement. • These gene segments then recombine to create a gene (VJ) encoding the L chain variable region and a gene (VDJ) encoding the H chain variable region.

41. Generation of Ig Diversity in B Cells Prior to Contact with Antigen • In the germ line configuration, the Ig genes are fragmented into gene segments along the chromosome. • Individual gene segments must be rearranged. • This only occurs in B cells. • It occurs during their development from precursors in the bone marrow. • When gene rearrangements complete, heavy and light chains are produced and membrane bound Ig is expressed on the cell surface. • The B cell can now recognize and respond to Ag.

44. Chromosomal Arrangement of Ig Genes • Heavy-chain locus on chromosome 14;  light-chain on chromosome 2;  light-chain on chromosome 22. • Different segments encode leader peptide (L), variable region (V) and constant regions (C). • Heavy-chain locus contains C genes for all isotypes. • The C genes are ready to be transcribed. • The V genes first undergo rearrangement.

45. Immunoglobulin Heavy- and Light–Chain Loci • The V regions of light-chain comprised of two segments: variable (V) and joining (J). • Heavy chain also contains D segments between V and J. • First and second hypervariable region of light-chain (CDR1, 2) are encoded by different V segments, the third CDR is determined by the V/J junction.

46. The Germline Organization of the Human Immunoglobulin Heavy-Chain and Light-Chain Loci

47. Somatic Recombination of Ig • V-region sequences are constructed from gene segments • L-chain, single recombination VJ • H-chain, 2 recombinations: first DJ, then VDJ. • C-regions at not rearranged at this point. All cells B cells

48. The V, J (and D) segments are combined at random, thus many variations possible. Human  light chain: 40 V segments and 5 J segments - therefore 200 combinations (40 x 5). Human  light-chain: 30 x 4 = 120. Human heavy-chain: 65 V, 27 D, 6 J (65 x 27 x 6 = 10,530 combinations). #’s of Functional Gene Segments Used to Construct the Variable Regions of Human Ig H and L Chains

50. Mechanism of V-region DNA Rearrangements • There are unique recombination signal sequences (RSSs) flanking each germ-line V, D, and J gene segment. • One RSS is located 3’ to each V gene segment and 5’ to each J gene segment, and on both sides of each D gene segment. • These sequence are signals for the recombination process that rearranges the gene.