Immunoglobulin Gene Rearrangement MCB720 January 20, 2011 Presented by: Alamzeb Khan & Maria Muccioli

Immunoglobulin (Ig)  A defense system against foreign bodies (antigens)  Immunoglobulin are antibodies that protect cells from foreign bodies (antigens)  B- lymphocytes cells secrete more that 10 8 different types of immunoglobulin  Contain different binding surfaces for binding to antigens  Immunoglobulin bind to a specific site on the antigens called “epitope” or “antigenic determinant” Biochemistry, 5 th edition © 2002 W.H. Freeman & Company

Structure of Immunoglobulin G (IgG) Figure 33.2 Biochemistry (Berg) 5 th ed.  25 kd Light chain  50 kd Heavy chain  Chains linked by disulfide bonds  Lights chains contain two immunoglobulin domains  Heavy chain consists of four Immunoglobulin domains

IgG Cleavage by Papain (antigen-binding Fragment) (crystallizable)  Cleaved into three fragments by papain  Fab (antigen-binding fragment  Fc (Crystal fragment, b/c it can be crystallized)  Fc doesn’t bind antigen, but helps in other biological activities (e.g lysis of target cells) Biochemistry, 5 th edition © 2002 W.H. Freeman & Company

Five Classes of Immunoglobulin  IgG bind only two antigens  IgM has 10 binding sites, it bind antigens having multiple identical epitomes  IgA- antibody in external secretion, such as tears, saliva, and intestinal mucus.  IgA- the first line of defense against bacterial and viral antigens  Function of IgD is unknown  IgE protect against parasites

Concentration of Different Igs in Serum

Sequence Diversity of Antibodies Variable Domain Constant Domain  Hyper-variable loops are made of variable amino acids  V L can pair with any V H - a large number of different binding sites can be constructed by combinatorial association Biochemistry, 5 th edition © 2002 W.H. Freeman & Co.

Antigen Cross-linking Antigen Cross-Linking. Because IgG molecules include two antigen-binding sites, antibodies can crosslink multivalent antigens such as viral surfaces Figure Biochemistry, 5 th edition © 2002 W.H. Freeman & Company.

κ Light Chain Hypervariability  Made of only V and J gene combination  Any V gene can pair with any J gene  200 different combinations are possible  κ light-chain: 40 V x 5 J = 200  Hypervariable κ Light-chain consists of 110 residues (1-97 encoded by V genes and encodes by J-genes)  J genes are important to antibody diversity, b/c they form part of the hypervariable region Biochemistry, 5 th edition © 2002 W.H. Freeman & Company

Gene Rearrangement for Heavy Chain  The variable domain of heavy chain is made of three segments (V,D, & J)  VH genes encode residues, 1-94  JH encodes residues  D genes encodes residues  Heavy chain: 51 V x 27 D x 6 J =8262 Biochemistry, 5 th edition © 2002 W.H. Freeman & Company

Different Antibody Possibilities κ light-chain: 40 V x 5 J = 200 λ light-chain: 30 V x 4 J x 4 C = 120 Heavy chain: 51 V x 27 D x 6 J =8262 Total: ( ) x 8262 = 2.6 x 10 6 Variability in the exact points of segment joining (VJ and VD) increases these values by at least 100x. Biochemistry, 5 th edition © 2002 W.H. Freeman & Company

Variable (V), Diversity (D), and Joining (J) gene segments – Recombine to form antigen-recognizing (variable) regions of immunoglobulin chains (heavy chain rearranges first) – Somatic recombination catalyzed by RAG1 & RAG2 recombinases – Recognition Signal Sequences (RSS) – nonamer & heptamer repeats separated by 12 or 23 base pair spacer regions – Segments w/ different spacer regions can recombine successfully – Allow for diversity of the antigen- recognizing region of antibodies – crucial for defense! The Basics of Ig Rearrangement

5’ 3’ 5’ 3’ V VLVL C J J Adapted from Lodish, et al, 2008 Enhancer J VHVH V V VD DJ Heavy and Light Ig Chains Undergo Random Gene Rearrangement *Light chain rearrangement *Heavy chain rearrangement

Ordered rearrangement of immunoglobulin heavy chain variable region segments F W Alt, G D Yancopoulos, T K Blackwell, C Wood, E Thomas, M Boss, R Coffman, N Rosenberg, S Tonegawa, and D Baltimore GOALS: - To determine the joining order of V H, D, & J H segment rearrangement in the heavy Ig chain - To find out if a complete rearrangement on one chromosome suppresses further rearrangement on the other chromosome METHODS: Cultured B-lymphoid cells Designed hybridization assay with specific probes for each potential recombination event

rear Experimental Design All 3 genes located within EcoRI sites Each rearrangement gives a specific signal 5’ 3’ V JD EcoRI

Do V H DJ H rearrangements occur on both chromosomes? If: Then: No hybridization to 5’ D-specific probes should occur if there are V H DJ H rearrangements on both chromosomes (this is predicted by the “deletional model”) 5’ 3’ V JD

Interpreting the Results DNA digested with EcoRI and electrophoresed on an agarose gel Bands indicate hybridization to a probe The position of each segment with respect to 5’ or 3’ is indicated in parenthesis (previously determined by Kurosawa & Tonegawa in 1981) This analysis allowed for the determination of the order of V, D, & J rearrangements

Conclusions D -> J rearrangement occurs first (on both chromosomes), followed by DJ -> V Recombination occurs by “deletional joining” No V -> D or D -> D rearrangements found If a functional gene is constructed on one chromosome via the DJ -> V rearrangement, the other chromosome does not undergo further recombination

Model of Ig Heavy Chain Rearrangement

Summary Immunoglobulins (antibodies) are produced in B-cells & bind to specific antigens Diversity of the antigen recognition region is maintained by “random” rearrangements in the variable region of the V, D (heavy chain only), and J segments Heavy chain Ig gene rearrangement occurs via an ordered mechanism: D -> J, followed by DJ -> V via deletional joining Formation of a functional heavy chain gene (V H ) represses further rearrangement

References Alt, et al (1984); “Ordered rearrangement of immunoglobulin heavy chain variable region segments”; EMBO 3(6), Berg. J, et al (2002 & 2006); “Biochemistry”; W.H. Freeman & Co. (Chapter -Immunology) Lodish, et al (2008); “Molecular Cell Biology”; W.H. Freeman & Co

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