Membrane-bound Immunoglobulins and B-cell Receptor (Part 11- Antibody Basics)
Welcome to the 11th part of the 13-part series on Antibody basics.
Previous parts: Part 1, Part 2, Part 3, Part 4, Part 5, Part 6, Part 7, Part 8, Part 9, and Part 10
Immunoglobulins can exist in 2 forms: secreted immunoglobulin (sIg) and membrane-bound immunoglobulin (mIg). The five classes of antibodies IgG, IgA, IgD, IgE, and IgM can be expressed either as secreted immunoglobulin or membrane-bound immunoglobulin.
Structure of secreted and membrane-bound Ig
The basic structure of secreted immunoglobulin or membrane-bound immunoglobulin is the same. But their carboxyl-terminal domains differ in both structure and function. For instance, the carboxyl-terminal domain of each heavy chain of secreted immunoglobulin has a hydrophilic amino acid sequence of about 20 amino acids at the carboxyl-terminal end.
But in membrane-bound immunoglobulin (mIg), the carboxyl-terminal domain of each heavy chain contains three regions that anchor the mIg on the surface of B cell:
1. An extracellular hydrophilic “spacer” sequence composed of 26 amino acid residues
2. A hydrophobic transmembrane sequence
3. And a short cytoplasmic tail
The length of the transmembrane sequence is constant among all immunoglobulin isotypes, whereas the lengths of the extracellular spacer sequence and the cytoplasmic tail vary among the isotypes. For instance, the mIgM and mIgD cytoplasmic tails contain only 3 amino acids, mIgA has a cytoplasmic tail of 14 amino acids. On the other hand, the mIgE and mIgG contain a cytoplasmic tail of 28 amino acids.
The function of the membrane-bound Ig is to recognize and bind to a specific antigen. For B cell activation, the B cell should get the signal that the specific antigen has been recognized. But in each isotype of membrane-bound Ig, the cytoplasmic tail is too short to deliver the activation signal to B cell after binding to a specific antigen.
Therefore, the activation signal to the B cell is delivered by the two accessory proteins associated with the membrane-bound Ig. These accessory proteins are Ig-α and Ig-β. These proteins together form a heterodimer Ig-α/Ig-β held together by a disulfide bond. Thus, the membrane-bound Ig alone does not constitute the entire B cell receptor on B cells. Instead, the B cell receptor (BCR) is composed of 2 components. One is membrane-bound Ig, and the other component is Ig-α/Ig-β. The Ig-α chain has a longer cytoplasmic tail of 61 amino acids, and the cytoplasmic tail of Ig-β contains 48 amino acids.
The tails in both Ig-α/Ig-β are long enough to interact with the signaling molecules to induce the B cell activation.
Thus, to summarize the mechanism of B cell activation, upon the contact of mIg with specific antigen, the mIg delivers signals to Ig-α/Ig-β. Ig-α/Ig-β then interacts with signaling molecules to induce B cell activation. Once activated, the B cells proliferate and differentiate into effector B cells and memory B cells.
Effector B cells are known as plasma cells which secrete antibodies. The plasma cells produce and secrete antibodies specific to the antigen recognized by the B cell receptors. The secreted antibodies then bind to the pathogens or antigens and activate defense mechanisms that lead to the destruction of the pathogen.
The membrane-bound antibodies present on the B cells are components of B-cell receptors. And on the other hand, antibodies secreted by plasma cells are called secreted immunoglobulins.
Expression of membrane or secreted Immunoglobulin
The two forms of immunoglobulins: membrane-bound or secreted form, differ in the amino acid sequence of the heavy chain carboxyl-terminal domains. The secreted form of antibody has a hydrophilic sequence of about 20 amino acids in the carboxyl-terminal domain. In contrast, in the case of membrane-bound antibody, the carboxyl-terminal domain of each heavy chain contains an extracellular hydrophilic sequence, a hydrophobic transmembrane segment, and a short hydrophilic cytoplasmic tail at the carboxyl-terminal that extends into the cytoplasm.
Also, multiple constant gene segments encode the constant domain of the classes and subclasses of antibodies. CH gene segments are arranged in the order Cµ, Cδ, C𝛾3, C𝛾2, C𝛾1, C𝛾4, Cε, Cα1, and Cα2. Constant region gene segments are organized as a series of coding exons and non-coding introns. For instance, the Cµ gene segment that encodes the constant region of IgM contains four exons Cµ1, Cµ2, Cµ3, and Cµ4. These four exons correspond to the CH1, CH2, CH3, and CH4 domains of the heavy chain of an IgM antibody.
Cµ4 exon contains a nucleotide sequence represented as S at its 3’end that encodes the hydrophilic sequence in the CH4 domain of secreted IgM.
Apart from the S sequence, there are two additional exons M1 and M2, present downstream from the 3’ end of the Cµ4 exon. The M1 exon encodes for the transmembrane domain, and the M2 exon encodes for the hydrophilic cytoplasmic tail of the CH4 domain of membrane-bound IgM.
When a primary transcript of IgM molecule is formed by transcription of rearranged µ heavy chain gene, it contains two polyadenylation signal sequences or poly-A sites in the Cµ gene segment.
Site 1 is located at the 3’ end of S sequence, and site 2 is located at the 3’ end of the M2 exon. Therefore, if polyadenylation occurs at site 1, then the M1 and M2 exons are lost; thus, excision of introns and splicing of primary transcript produces mRNA that encodes for the secretory form of µ chain.
On the contrary, if the polyadenylation occurs on the second poly-A site, then, in this case, splicing removes S site at 3’end of Cµ4 exon, that encodes for hydrophilic carboxyl-terminal end of the secreted form and joins the remainder of Cµ4 exon with M1 and M2 exons, thus producing mRNA that encodes for the membrane-bound µ chain.
Therefore, differential processing of a primary transcript determines whether the secreted antibody or membrane-bound antibody will be produced. The mature naive B cells, which have never been exposed to an antigen, produce membrane-bound antibodies IgM and IgD. But upon recognizing and binding to a specific antigen, the naive B cells proliferate and get differentiated into plasma and memory B cells. The plasma cells then produce secreted forms of antibodies. On the other hand, the antibodies present on the surface of memory B cells are membrane-bound.
Simultaneous expression of IgM and IgD
In the case of heavy chain locus, Cµ and Cδ gene segments are present close together, and there is no switch sequence present between them. Therefore the entire VDJCµCδ rearranged gene will be transcribed into a single primary RNA transcript. IgM antibody has four constant domains because the hinge region is absent in it; therefore, there are four exons for the Cµ gene segment. On the other hand, the IgD antibody has three constant domains; therefore, there are three exons for the Cδ gene segment. Also, there are 2 poly-A sites for one C gene segment.
Therefore, the RNA transcript that contains Cµ and Cδ gene segments has 4 poly-A sites. Poly-A sites 1 and 2 are associated with Cµ, and poly-A sites 3 and 4 are associated with the Cδ gene segment. Therefore, if the polyadenylation occurs at site 2, present at 3’end of Cµ4 exon, then the mRNA formed after cleavage and splicing will encode the membrane-bound form of the IgM molecule.
Whereas if polyadenylation occurs at site 4, present at the 3’ end of Cδ3, the RNA cleavage and splicing remove the Cµ exons and produce mRNA encoding the membrane-bound form of IgD molecule.
In the case of mature B cells, both processes occur simultaneously. That is why mature B cells express both IgM and IgD on their membrane.
On the other hand, the polyadenylation of the VDJCµCδ primary RNA transcript at site 1 or site 3, followed by RNA cleavage and splicing in plasma cells, yields the secreted form of IgM or IgD antibodies.
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