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B cell effector functions II Overview of antibody response and changes in antibodies Antibody class switching -brief review of effector functions Class switching mechanism- part 1 overview and targeting -distinction from V(D)J recombination
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The purpose of the antibody response is to tag specifically the microbe, marking it for appropriate disposal.
Antibody protein sequence and bioactivity changes during the immune response
Note: there are exons encoding the membrane and secreted forms of each of the antibody heavy chains.
Class switch is a DNA recombination, distinct from
VDJ recombination, that occurs in immune responses
RNA splicing controls expression of
membrane vs secreted IgM
1) expression of a recombination machine
2) targeted “accessibility” mediated by nearby enhancers and promoters
From Gearhart and Bogenhagen PNAS 80:3439, 1983
Strand bias, transitions>transversions,
bias vs pyrimidines (as assessed on coding strand).
From Betz et al, PNAS 90:2385.
1. Occurs at high rates: 10 -4 -10 -3 /bp/generation.
2. Occurs by untemplated single base substitutions.
3. Restricted to a brief period of B cell differentiation.
4. Restricted to the rearranged V region and its immediate flanking sequences.
5. Occurs in germinal centers with T cell help.
6. Occurs throughout the V region but more frequently in RGYW (A/GGC/TA/T) motifs.
7. Mutations in kappa light chain transgenes require intronic and 3’ enhancers
but not in the V region promoter or V coding region.
BioEssays 20:227–234, 1998
-If mutations are routinely removed from replicating DNA, a process that prevents repair locally would target mutation. If so, knockouts of DNA repair genes would have little or no effect.
-If mutations are introduced by massive local DNA damage, possibly needed to overwhelm the normal repair mechanisms, then repair mutants would have increased mutation rates in the targeted regions (near assembled VDJs).
-Alternatively, DNA repair enzymes may be needed to generate mutations.
Mismatch repair in eukaryotes
Mut mutants in bacteria have “mutator” phenotype
Msh2,6 +/- linked to hereditary non-polyposis colon cancer (HNPCC)
Martin and Scharff Nat. Rev. 2:605 (2002)
Marti et al. J. Cell. Physiol. 191:28 (2002)
The authors suggested that the
mutator might target the “wrong” strand for repair,
in effect co-opting the mismatch
repair process to introduce
Cascalho et al, Science (1998) 279:1207
Table 2. Distribution of Mutations in Hot Spots vs Background Mutations
Position Msh2+/- Msh-/-
39 TGT 2.1% (11) 4.6% (7)
56 AGC 2.7% (14) 9.3% (14)
62 GCA 1.8% (9) 4.6% (7)
253 GCT 1.6% (8) 6.6% (10)
8.2% (42) 25.1% (38)
Rada et al. (1998) Immunity, 9:135.
… but what would cause the initial mismatch?
A surprising finding: these three processes are dependent on a single gene, activation induced deaminase (AID). AID-deficient mice do not switch to IgG, IgA, IgE or show much antibody hypermutation.
is APOBEC-1, an RNA editase involved in lipid metabolism.
Figure 2. Occurrence of somatic mutation in one DNA strand in the G1 phase of the cell cycle. Somatic mutation was induced in BL2 cells in the G1 phase of the cell cycle. Single cells were either analyzed for mutations in the V4-39 gene after 90 min of stimulation or isolated in single wells and left for 24 or 48 h (one or two divisions) before analysis. (a) Three representative mutations in the V4-39 gene, which show a mixed sequence. (b ) Visualization of one, two and four cells. Note the streptavidin beads that cross-link the biotinylated anti-IgM bound at the cell surface. (c ) Three patterns were observed when two BL2 cells that differed at a single position in their V gene (nucleotide 57) were amplified. In addition to the expected configuration of amplification of a mixed sequence (left), cases of biased amplification were observed, which resulted in amplification of either of the two "alleles" (middle and right). (d) Schematic representation of mutation occurrence on a single strand of DNA, segregated in the same 50:50 proportion after cell division.
Faili, A. et al. Nature Immunology 3, 815 - 821 (2002)
in the Sµ region can occur under switching culture conditions, but prior to switching, consistent with a common mechanism for the two
types of DNA modification.
are associated with switch recombination