Antibody-Immunoglobulin Function Structure Antigen-antibody interaction Antibody isotypes Cellular and Molecular Immunology, Chapters 3 & 7

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1.Two heavy chains and two light chains 2.Disulfide bonds 3.Ig domains 4.Hinge region 5.Constant and variable regions 6.Two identical antigen-binding sites 7.Two function domains a. Bind to antigens b. Recruit effector cells and molecules 7. Heavy chains decide the class Structures of antibodies

Determining Ab Structure Work in 1950s and 60s using biochemical techniques Rodney Porter used partial proteolysis with papain - 2 identical antigen-binding fragments (Fab) and one “tail” fragment (Fc) Alfred Nisinoff used pepsin - one fragment with divalent antigen binding (F(ab2)’) Gary Edelman used  -ME to reduce Ig, resolving heavy (H) and light (L) chains Rodney Porter probed H and L chains with anti- Fab and anti-Fc antibodies: anti-Fab detected both H & L, but anti- Fc detected only H Combined work earned Porter and Edelman 1972 Nobel Prize

Fig. 3.3 The Y-shaped immunoglobulin molecule can be dissected by partial digestion with proteases. Fab-Fragment antigen binding Fc-Fragment crystallizable Fv-Fragment variable

Fine Structure of Ab “Variable” (V) and “Constant” (C) regions, defined by degree of sequence similarity between different antibodies Repeated structural motif (“immunoglobulin fold”): antiparallel beta strands form 2 beta sheets; intrachain disulfide leads to beta barrel structure V-type fold has 9 beta strands, C-type fold has 7 L chain has one V and one C domain; H chain has one V and 3-4 C domains Ag-binding site formed by V H + V L

Structure of Ig Light Chain Showing V- and C-type Immunoglobulin Folds

Variability of V-Region Variability isn’t evenly distributed: “hyper- variable” (HV) regions surrounded by less variable “framework” regions (FR) Defined by variability plots (number of different a.a./frequency of most common a.a.) HV regions correspond to loops between beta strands: B-C, F-G, and C’-C”, and can vary in both sequence and length HV regions also called “complementarity determining regions (CDR), because they make up the Ag-binding surface (H + L)

HV-Hypervariable region CDR-Complementarity-Determining Regions FR-Framework region FR1FR2FR3FR4

One geneOne protein antigens genes ??? The Immunological Conundrum: How is Variability Generated?

Generating Diversity: DNA Recombination In 1976, Hozumi and Tonegawa showed that Ab-producing cells have different DNA sequence than other somatic cells in Ig locus - evidence for DNA recombination (1987 Nobel Prize) Sequencing of Ig genes has identified gene segments that are brought together by recombination

Not in book

Ig Locus Rearrangement Sequence-specific: recombination signal is a conserved heptamer (CACAGTG), a spacer (12 or 23 non-conserved bases), and a conserved nonamer (ACAAAAACC) 12/23 Rule: Recombination machinery always joins a gene segment with a 12-bp spacer to another with a 23-bp spacer This ensures that the correct gene segments get joined (no V-V, H chain always has D between V and J, etc.)

V(D)J Recombination Machinery Uses both specific (RAG1/2) and ubiquitous factors RAG1/2 complex is the sequence-specific recombinase: recognizes recomb signal, brings a 12 and a 23 signal together, and cleaves DNA Cleaved DNA is repaired by general DNA repair factors

[Estimate]

Heavy – 65 (V); 27 (D); 6 (J) = 10,530 Kappa - 40 (V); 0 ; 5 (J)= Lambda - 30 (V) 0 ; 4 (J)= ,530 X 320 = 3 million (about 30 million in human, due to more V segments) Actually much much much more! (Junctional diversity, somatic mutations, nucleotide insertions, etc)

Great Diversity Allows for Exquisite Specificity Antibodies can distinguish between antigens differing by only small chemical substituents Precise part of an antigen recognized by the antibody is called an epitope, or determinant Small chemical epitope = hapten Linear epitope Conformational epitope (generated by folding)

Fig. 3.8 Antigens can bind in pockets or grooves, or on extended surfaces in the binding sites of antibodies. Antigenic determinant or epitope: The structure recognized by an antibody Haptens Continuous Discontinuous or or linear epitope conformational epitopes

1.Antibodies neutralize bacterial toxins and complete virus particles and bacterial cells. 2.Antibodies coating an antigen render it recognizable as foreign by phagocytes (macrophages and neutrophils), which then ingest and destroy it; this is called opsonization. 3.Antibodies activate of the complement system by coating a bacterial cell. Fig Antibodies can participate in host defense in three main ways. Functions of antibodies

Antibody Functions Ab’s have two “business” ends: antigen-binding arms and Fc tail. Effector function is mediated by Fc region Different classes (isotypes) use distinct C H gene segments, and have different effector functions Isotype switching occurs by another DNA recombination event (except IgM/IgD, which uses alternative RNA splicing)

Fig The structural organization of the main human immunoglobulin isotype monomers. Isotypes of Immunoglobulin

Fig The IgM and IgA molecules can form multimers.

Monoclonal Antibodies Monoclonal Antibodies - identical antibodies produced in large quantities by an immortalized hybridoma cell line Technique developed by Köhler and Milstein in 1975, earned Nobel Prize in 1984 Benefits: identical antibodies available in unlimited quantity; easily purified Drawbacks: may not be useful for all techniques; limited number of species (mouse, rat, hamster, rabbit)

Production of monoclonal antibodies. HGPRT-hypoxanthine:guanine phosphoribosyl transferase PEG-polyethylene glycol HAT-hypoxanthine- aminopterin-thymidine Cells lacking HGPRT are sensitive to the HAT medium Unfused B cells die within 1-2 days without stimulation