Immunoglobulin Gene Rearrangement

MCB720

January 20, 2011

Presented by: Alamzeb Khan & Maria Muccioli

http://www.theraclone-sciences.com/develop.php

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 108 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, 5th edition© 2002 W.H. Freeman & Company

Structure of Immunoglobulin G (IgG)

Figure 33.2 Biochemistry (Berg) 5th 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, 5th 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

VL can pair with any VH - a large number of different binding sites can be constructed by combinatorial association

Biochemistry, 5th 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 33.4. Biochemistry, 5th 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 98-110 encodes by J-genes) J genes are important to antibody diversity, b/c they form part of the hypervariable region

Biochemistry, 5th 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 98-113 D genes encodes residues 95-97 Heavy chain: 51 V x 27 D x 6 J =8262

Biochemistry, 5th edition© 2002 W.H. Freeman & Company

Different Antibody Possibilities

κ light-chain: 40 V x 5 J = 200λ light-chain: 30 V x 4 J x 4 C = 120

Heavy chain: 51 V x 27 D x 6 J =8262Total: (200 + 120) x 8262 = 2.6 x 106

Variability in the exact points of segmentjoining (VJ and VD) increases these values

by at least 100x.

Biochemistry, 5th 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

http://www.cas.vanderbilt.edu/bsci111b/immunology/supplemental.htm

5’ 3’

5’ 3’

V

VL

C

J

J

Adapted from Lodish, et al, 2008

Enhancer

J

VH

V

V

V D

D J

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 VH, D, & JH 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

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Experimental Design

• All 3 genes located within EcoRI sites

• Each rearrangement gives a specific signal

5’ 3’V JDEcoRI EcoRI

Do VHDJH rearrangements occur on both chromosomes?

• If:

• Then: No hybridization to 5’ D-specific probes should occur if there are VHDJH rearrangements on both chromosomes (this is predicted by the “deletional model”)

5’ 3’V JD

Interpreting the Results

• DNA digested with EcoRI andelectrophoresed 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 thedetermination 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

http://www.cartage.org.lb/en/themes/sciences/LifeScience/GeneralBiology/Immunology/Recognition/AntigenRecognition/Antibodydiversity/igdna.gif

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 (VH) represses further rearrangement

References

• Alt, et al (1984); “Ordered rearrangement of immunoglobulin heavy chain variable region segments”; EMBO 3(6), 1209-1219.

• Berg. J, et al (2002 & 2006); “Biochemistry”; W.H. Freeman & Co. (Chapter -Immunology)

• Lodish, et al (2008); “Molecular Cell Biology”; W.H. Freeman & Co. 1063-1073.

Questions?