Docstoc

Antibodies

Document Sample
Antibodies Powered By Docstoc
					                                  Antibodies
It is estimated that each individual has an incredible 1011 different
antibodies. Each of these antibodies is produced by a different B-
lymphocyte. If this lymphocyte is activated it will divide and differentiate
into memory B cells and plasma cells. The memory cells are long lived cells
that enable a faster immune response to the same antigen in the future,
thereby providing immunity to that particular infection. Concurrently,
plasma cells produce large numbers of antibodies to fight the current
infection.

Antibodies and Immunoglobulins
These two terms are often used synonymously and in most cases they do
mean the same thing. However they are not strictly identical terms.
Antibodies are immunoglobulins and the B-lymphocyte receptor is also an
immunoglobulin. Antibodies are free in plasma whilst the B-lymphocyte
receptor is membrane bound. Another important term is the
Immunoglobulin superfamily. Immunoglobulins are glycoproteins; that is,
they are proteins that have sugar groups bound to them. Within
immunology there are several other glycoproteins that have a great deal of
homology with immunoglobulins (such as the T-lymphocyte receptor) and
hence they are referred to as members of the immunoglobulin superfamily.

Structure of Immunoglobulins
Immunoglobulins are made up of two identical heavy chains and two
identical light chains. The heavy chains are coded for by one gene on
chromosome 14. There are two types of light chain – known as  and ; the
genes are on chromosomes 22 and 2 respectively. There is no functional
difference between  and  chains. Each immunoglobulin has two antigen
binding sites. These sites are formed from the variable domains of the light
and heavy chains together. The light chains have one variable region and
one constant region. The heavy chains have one variable region and three
or four constant regions. There are five different heavy chains which are
known by the Greek letters  and  The picture shows the basic
common structure of immunglobulin molecules. (Move mouse over image)
Error! Hyperlink reference not valid.


One of the earlier studies done on immunoglobulins was treatment with
proteases. When treated with papain, immunoglobulins are cleaved just
below the hinge region producing two different fragments.. These are know
as Fab (Fragment Antigen Binding) and Fc (Fragment crystallisable). This is
important as the crystallisable fragment contains the tail of the constant
regions of the immunoglobulin. Many cells, such as macrophages, have
receptors that bind to the constant region of antibodies. These receptors are
known as Fc receptors (FcR). (Move mouse over image)

Error! Hyperlink reference not valid.

Classes of Immunoglobulin
There are five classes of immunoglobulins: IgM, IgG, IgA, IgE and IgD. The
class of immunoglobulin is defined by the heavy chain; i.e. IgM has heavy
chains, IgG, heavy chains etc. Each of these classes of immunoglobulin
have different properties.

Immunoglobulin   Heavy   Molecular         Serum       Complement    Placental   Phagocyte    Mast
                 Chain    weight     Level     Half     Activation   Transfer     Binding     Cell/
                          (kDa)      (mg       live                                          Basophil
                                     ml-1)    (days)                                         binding
IgG     IgG1      1       146         9       21         ++           +++          +
        IgG2      2       146         3       20          +            +
        IgG3      3       165         1       7          +++           ++          +
        IgG4      4       146        0.5      21                       +
IgM                       970        1.5      10         +++
IgA     IgA1      1       160        3.0      6                                    +
        IgA2      2       160        0.5      6                                    +
IgE                       188       5x10-     2                                    +          +++
                                       5

IgD                       184       0.03       3

IgM
In plasma IgM is a pentamer (held together by a protein known as a ‘J-
chain’(J for joining)) and thus it can cross link several antigens. This cross-
linking causes precipitation of pathogens and toxins in plasma. IgM (in a
monomer form) is also the B-lymphocyte receptor. The difference between
secreted IgM and the B-lymphocyte receptor is one domain on the protein;
either a secretory component that enables it to be released into plasma or a
membrane-bound component that binds the immunoglobulin to the cell
membrane. This is achieved by alternate splicing of the mRNA to either
contain a secretory region or a trans-membrane region on the tail of the
molecule. Secreted IgM is the first antibody produced by B-lymphocytes.
IgG
IgG is the most abundant in plasma. It is able to activate complement and is
a powerful opsonin. It also crosses the placenta and thus provides passive
immunity to the neonate.




IgA
IgA is primarily involved in mucosal immunity. Both IgA1 and IgA2 are
dimers and in addition they contain a J chain similar to IgM and an S chain
(S for secretory) that prevents the antibody from being broken down by the
host proteases found on epithelial surfaces. IgA is the type of antibody
found in breast milk and thus breast feeding introduces these antibodies
directly to the epithelial surface of the gastrointestinal tract of the infant.
IgE
The precise function of IgE is not well understood. IgE is thought to be
important in allergic reactions because it is very potent in activating mast
cells and basophils and hence induces histamine release which is known to
be a major mechanism of allergy. IgE also appears to play an important role
in fighting parasitic infections along with eosinophils. Large worms cannot
be ingested by phagocytes and so they are killed by a process known as
exocytosis. The invading worm becomes coated with IgE which then binds
to the Fcreceptor on the eosinophils that release toxic granules onto the
parasite.




IgD
IgD is found at low levels in plasma but its function is unclear. Mice
deficient in IgD show no immunocompromise and thus it is not thought to
be important. In naïve B cells it is also co-expressed as a membrane-bound
immunoglobulin with IgM.
Immunoglobulin Production.
The ability to produce an incredible array of immunoglobulins that bind to
foreign antigens is key to the ability of the immune system to mount an
adaptive response to infection. This diversity is produced by a number of
mechanisms.

Lymphocytes break what is known as somatic theory. Somatic theory states
that every cell within a complex organism has the same genetic material
within it and the differences between the cells are determined by which
genes are expressed. Lymphocytes, uniquely, are able to rearrange their
genome. B-lymphocytes do this with the immunoglobulin genes and T-
lymphocytes with the T-lymphocyte receptor genes. The mechanisms used
are essentially the same for both with a few important differences. Genetic
rearrangement is very well controlled within the cell. This is vital as
otherwise it would be extremely carcinogenic. The genes responsible for
this are know as RAG-1 and RAG-2. (Recombination Activating Genes).

This genetic rearrangement takes place in several steps. Firstly the heavy
chain. The variable region of an immunoglobulin molecule is made up of
the variable regions of the heavy and light chain. The heavy chain variable
region is made from three segments. These three are know as the Variable
segment (VH), the Diversity segment (DH) and the Joining segment (JH).
The native genome has multiple copies of each of these exons.
There are 65 different variable segments, 27 different diversity segments
and 6 different joining segment. Downstream from these segments is the
coding for the conserved region of the heavy chain.

The first step in gene rearrangement is for one of the D segments to be
joined to one of the J segments. This all happens at the DNA level under the
control of the RAG1 and RAG2 gene products. (Move mouse over image).

Error! Hyperlink reference not valid.Error! Hyperlink reference not valid.
Any J segment can join to any D segment.

In the same way the DJ segment then joins to one of the 65 V segments.
(Move mouse over image).
Error! Hyperlink reference not valid.
Again, any V segment can join to any D segment.

The gene is then transcribed into RNA. The gap between the J segment and
the beginning of the conserved region is removed by splicing (as are the
introns within the conserved region). This produces an IgM heavy chain.
(The L segment codes for a leader part of the mRNA which is not translated
and thus does not code for part of the peptide.)

The process is essentially the same for the light chains with two differences.
Firstly there are two genes for the light chain –  and  (either one can be
used). Secondly there are no D segments in the light chain, only J segments.

The overall process is summarised below.
The  light chain gene contains 40 variable segments and 5 J segments. The
 gene 30 V and 4 joining segments. Hence there are 40 x 5 = 200 different
combinations for the  light chain and 30 x 4 = 120 for the  light chain.
Thus by the random arrangement of J with D segments alone, there are 320
different light chains that are possible. Similarly with the heavy chain: 65 V
segments x 27 D segments x 6 J segments = 10530 different heavy chains. If
any heavy chain can combine with any light chain then that would result in
10530 x 320 = 3369600 (3.4 million) different antibodies that B-
lymphocytes could produce.

3.4 million (3x106) is quite a large number but it is a long way from 1011.
The answer as to how this much diversity is produced lies in the means by
which these different segments are joined together. It is not simply a means
of excising the intervening sequences and joining together the segments but
two processes occur within the joints to vastly increase the diversity of
these peptides. This is referred to as joint diversity.

Joint Diversity: N- and P- nucleotides
N-nucleotides are so-named because they are not coded for by the gene and
P-nucleotides are palindromic sequences that are added at the joints
between segments.

P-Nucleotides
When Rag-1 and Rag-2 cleave the ends of a D segment and a J segment
they form a hairpin loop in the DNA. A hairpin loop is formed when the
two anti-parallel strands of DNA are joined together:
Then, single stranded cleavage of one of the strands of DNA two or three
bases from the loop takes place: (Move mouse over image)

Error! Hyperlink reference not valid.
This will, by definition, result in a palindromic sequence.

The unwound hairpins thus make 'sticky-ends' of the DNA which can be
brought together:




N-Nucleotides
In heavy chain rearrangement and sometimes in light chain rearrangement,
another enzyme TdT (Terminal deoxynucleotidyl Transferase) is active. It
acts to add random nucleotides to the long single stranded end. Anything
up to 20 nucleotides may be added.
The 'sticky ends' are then brought together and the gaps filled in by DNA
repair enzymes: (Move mouse over image)

Error! Hyperlink reference not valid.

By these mechanisms at D-J and V-D joints in heavy chains and at V-J
joints in light chains, massive diversity is produced. The number of
nucleotides added is random. If this is not a multiple of three it will
produce a frame shift. Frame shifts produce non-functioning peptides and
therefore only 1 in every 3 rearrangement results if a functioning peptude
and so this is a very wasteful process.

Somatic Hypermutation
All of these processes occur in the immature B cell as they are necessary to
produce the B-cell receptor molecule. One final process that occurs in
mature B lymphocytes is somatic hypermutation. Somatic hypermutation
is the mutation of the immunoglobulin gene in a mature B-lymphocyte.
This occurs in the V regions of the immunoglobulin gene where point
mutations are made.

The major signal for B-cell proliferation is binding of antigen. If
hypermutation produces an immunoglobulin molecule that has a higher
affinity for the antigen than the original molecule, this B cell is then
selected in a kind of 'micro-evolution' because the new immunoglobulin
molecule will bind the antigen more tightly. This process produces more
effective antibodies that have better affinity for the antigens.

Isotype Switching
The different classes of antibody are referred to as isotypes. Naïve B-
lymphocytes express IgM (and IgD). In the event of activation they divide
and differentiate. Some of the daughter cells become plasma cells, secreting
large amounts of IgM. Other daughter cells become memory cells and
undergo isotype switching, producing either IgG or IgE or IgA. Isotype
switching is also done at the DNA level and thus is irreversible.

The heavy chain genes are laid out as follows: the 65 V segments are
followed by the 27 D segments and then by the 6 J segments. Downstream
from the J segments are all the conserved segments. First the  and then
the  segments. An immature B cell undergoes the gene rearrangements
described above to form the genetic code for the variable part of the
immunoglobulin molecule. This is then fixed for the life of the B cell.
Whatever antigen specificity it has is maintained, regardless of which
isotype of immunoglobulin it produces. This is because the variable part is
transcribed into mRNA along with whichever conserved segment it is
expressing.

In the naïve cell the  and  segments are both transcribed into mRNA. One
of these is removed by alternate splicing. Thus translating the mRNA
produces either an IgM molecule or an IgD molecule. Since it is a random
process that determines which conserved segment is removed, IgM and IgD
are co-expressed by the same cell. Further downstream are the coding
regions for, then  and finally . In order for these to be
expressed the intervening sequences of DNA are removed. For example, to
form an IgA1 antibody, the cell would excise all of the segments between
the VDJ segment and the  segment. Hence, the , andsegments
are all lost from the genome.

				
DOCUMENT INFO