Transcript Immunoglobulin Gene Rearrangement

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)
 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
Figure 33.2 Biochemistry (Berg) 5th ed.
IgG Cleavage by Papain
 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
(antigen-binding
Fragment)
(crystallizable)
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
 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
Constant Domain
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
Biochemistry, 5th edition
Any V gene can pair with any J gene
© 2002 W.H. Freeman & Company
 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
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
Biochemistry, 5th edition
 D genes encodes residues 95-97
© 2002 W.H. Freeman & Company
 Heavy chain: 51 V x 27 D x 6 J =8262
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: (200 + 120) x 8262 = 2.6 x 106
Variability in the exact points of segment
joining (VJ and VD) increases these values
by at least 100x.
Biochemistry, 5th edition
© 2002 W.H. Freeman & Company
The Basics of Ig Rearrangement
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 antigenrecognizing region of antibodies – crucial
for defense!
http://www.cas.vanderbilt.edu/bsci111b/immunology/supplemental.htm
Heavy and Light Ig Chains Undergo
Random Gene Rearrangement
J
V
Enhancer
5’
C
3’
*Light chain rearrangement
J
V
VL
V
D
5’
*Heavy chain rearrangement
J
3’
V
D J
VH
Adapted from Lodish, et al, 2008
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
Experimental Design
EcoRI
V
D
J
EcoRI
5’
3’
• All 3 genes located within EcoRI sites
rear• Each rearrangement gives a
specific signal
Do VHDJH rearrangements occur on
both chromosomes?
• If:
V
5’
D
J
3’
• 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”)
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
tp://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?