The first isotype of immunoglobulin secreted during the primary humoral response to an antigen is IgM. In order for genes of the other isotypes of Ig to be expressed, there is a second DNA rearrangement which occurs in B lymphocytes following antigenic stimulation. This rearrangement is termed heavy class switching (or isotype switching). In this DNA rearrangement a previously rearranged VHDHJH recombines with a different CH gene segment. The precise mechanism is not yet clear, but it is known that there are specific switch sites located 2-3 kb upstream from each CH gene segment with the exception of Cdelta.
The switch site is composed of multiple copies of a short highly repetitive sequence. It has been hypothesized that there are switch site specific recombinases which recombine the DNA between two switch sites leading to the looping out and excision of the sequences between the two switch sites. These circular excision products have been identified in B lymphocytes undergoing heavy class switching.
Rearranged heavy chain genes in the mouse:
5'--L-V-D-J--s--Cmu----Cdelta--s-Cgamma3--s--Cgamma 1--s--Cgamma 2b--s--Cgamma2a--s--Cepsilon--s--Calpha--3'
Certain cytokines have been shown to regulate heavy class switching. For instance, IL-4 (an important Th lymphocyte cytokine) has been shown to trigger the switch from Cmu to Cgamma1 and the switch from Cgamma1 to Cepsilon. In other words, when the Ig isotype switches from IgM to IgE there is a two-step rearrangement. Once functional rearrangements have occurred, Ig expression is regulated in large part by post-transcriptional processing of Ig primary transcripts.
*Poly A polymerase adds a poly A tail at a poly A adenylation site
*introns are excised
*exons are connected by RNA splicing
Now you have a mature mRNA which is ready to leave the nucleus and enter the cytoplasm so that it may be engaged by a ribosome for translation.
Heavy chain C gene segments are organized as a series of coding exons and non-coding introns. Each exon corresponds to a C H domain.
For example:
Cmu contains:
CH1 CH2 CH3 CH4
Expression of membrane-bound or secreted Immunoglobulin.
The secreted Ig and membrane-bound Ig differ in the amino acid sequences (in the carboxyl terminal domains) of the heavy chain.
Secreted form - has a terminal hydrophilic sequence of about 20 amino acids.
An S exon codes for this hydrophilic piece.
Membrane form - has an extracellular hydrophilic sequence followed by a hydrophobic transmembrane sequence, followed by a short hydrophilic cytoplasmic segment at the terminus.
M1 exon codes for transmembrane segment.
M2 exon codes for cytoplasmic segment.
The primary transcript of a rearranged mu (IgM) heavy chain gene contains 2 polyadenylation sites [indicated by*]
5'--L-VDJ--CH1---CH2---CH3---CH4-S *---M1---M2*-3'
If cleavage of primary transcript and addition of poly A tail occurs at site 1, M1 and M2 are lost, leaving S, producing an mRNA encoding the secreted form of Ig,
If cleavage and polyadenylation occurs at site 2, mRNA encoding the membrane form of Ig is produced.
In mature [but naive] B cells only the membrane form is synthesized.
In plasma cells you have predominantly the secreted form.
5'--L-VDJ--Cm--Cd--3'
in one long primary transcript. Therefore, there are actually four possible poly A sites.
If sites 3 or 4 are used RNA splicing will remove the Cm exons and produce mRNA for membrane or secreted form of the IgD heavy chain.
A mature B cell expresses both IgM and IgD on its membrane so processing by both pathways must occur simultaneously.
H and L chains are encoded on different chromosomes and the mRNAs are translated on separate polyribosomes of RER (rough endoplasmic reticulum). Leader sequence guides the chains into the lumen of the RER where it (leader sequence) is removed. Assembly of 2L and 2H chains occurs as the chains pass through the RER.
The assembled immunoglobulin now leaves the RER in membrane-bound vesicles which transport it to the golgi apparatus. In the golgi, further post-translationaly modifications (such as glycosylation) occur and then the "completed" immunoglobulin is packaged into membrane-bound vesicles which transport the Ig to the plasma membrane.
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