p art i immnoglobulins are proteins proteins are specified by genes there are too few genes to...

IMMUNOLOGY

Bios 328a textbook-based study of immunologySpring 2003

http://www.lehigh.edu/~sk08/Courses/Bios328/mainpage.htm

PART I

• Immnoglobulins are proteins• Proteins are specified by genes• There are too few genes to specify all the

antibodies.– i. e., ~32,000 genes < 10,000,000,000 Ab’s

• How is Ig diversity specified genetically?

Ig proteins are specified by genetic “cassettes”

• Light chains are specified by “variable” (V), “joining (J), and “constant” (C) gene segments (aka “cassettes”).

DNA rearrangementand

alternative RNA spicing

Ig proteins are specified by genetic “cassettes”

• Heavy chains are specified by “variable” (V), “diversity” (D), “joining (J), and “constant” (C) gene segments (aka “cassettes”).

DNA rearrangementand

alternative RNA splicing

Another view….

PART II

• Cassettes rearrange… • How does this happen?

• How do you get one “V” fusing to one “J” (in a light

chain)?• In a heavy chain, a “D” fuses with a “J”; then the fused DJ

cassette fuses with a “V” cassette…

• The orderliness of this process implies that there are genetic instructions. What are they?

Cassettes rearrange…

• The heptamer is a palindrome– (i.e., it exhibits two-fold rotational symmetry.)

• The nonamer is AT-rich

– “Turns” refer to the DNA helix…

Cassettes rearrange…

One turn – two turn rule…

• one turn and two turn are “recombination signal sequences”

• one turn only reacts with a two turn

• Recombination signal sequences are the substrates of enzymes RAG-1 and RAG-2

(“RAG” = recombination-activating gene)

So… cassettes are marked by RSS (i.e., they are substrates for recombination.)

Thus, cassettes can be fused.

What is the consequence?

Look at mouse:

A mouse has:

134 VH, 13 DH, 4 JH segments

85 V, 4 Jsegmentsand 2 V, 3 J segments

Thus, a mouse has:

134 13 4 = 6968 heavy chains 85 4 = 340 kappa chain

and 2 3 = 6 lambda chains

6968 (340 + 6) = 2,410,928 antibodies

PART III (the HARD part…)

2.4 106 < 1010

So, there must be additional mechanisms of diversity other than “fusing” “cassettes”

How does a RAG enzyme work?

Junctional flexibility

The “hairpin loop”

Junctional flexibility, “P” nucleotides, and “N” nucleotides are added to CDR3

Somatic hypermutation

One turn – two turn rule…• one turn only

reacts with a two turn

• crossover between direct repeats (same transcriptional orientation) leads to deletion

• crossover between indirect repeats leads to inversion

PART IV• How do immunoglobulins assemble?

• Some immunoglobulins are in the surface membrane of immature B-cells while other immunoglobulins of the same idiotype are secreted by mature B-cells. What’s the difference?

• Similarly, identical variable regions can be shared among different isotypes. How?

• B-cells are diploids with two sets of genetic instructions. How does just one set get expressed?