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Antigens, MHC proteins, antigen presentations
During the evolution receptors specialized for the recognition of molecular
patterns not found in host organisms. Toll-like receptors, mannose-binding
lectin, etc. (pattern recognition receptors) bind only „foreign” molecules.
Plants have very many PRPa, mammals and birds only a few: we have versatile
variable, recombinant receptors, recognizing antigens.
Using these receptors we have to distinguish between „self” and „non-self”
antigens, tolerable and danger molecules.
Different strategies are needed to combat extracellular and intracellular
enemies, to recognize mutant or abnormal „self” antigens (eg. tumor cells).
The smallest recognizable sequence, the unit of immune recognition is the
epitop, a 8-25 aa long peptide. (Without post-translational modifications
there are 500 000 000 000 different nona-peptides!)
Proteins coded by our own genome („self”), proteins in our food and in the
protective micro-flora should not be attacked. Unimportant proteins should
be tolerized. How to distinguish? Learning!
Antigen presentation on the surface of the cells:
discovery
Historical description of tissue and organ „transplants”
– sheep blood and monkey testicles
Tissue incompatibility, genetic basis of incompatibility
– blood groups
HLA: human leukocyte antigens, MHC: major histocompatibility genes/proteins
The task of the MHC I class proteins is personal identification of the cell: no
mutations, no parasites should exist within the cells.
MHC II belongs to immune cells: antigens of identified enemies are posted as an
MHC II complex
Polymorphism of MHC proteins within the
population: protection
Non-conventional antigen-presenting molecules
are responsible for the presentation of lipids, NAs
Tissue incompatibility
Acut rejection of foreign tissue (HVG: host versus
graft disease) / grafted immune cells fight the
host: GVH: graft versus host disease)
The immune system recognizes tissues from other
individuals (allogenic) or species (xenogenic)
Tissue incompatibility
On this figure the parents are inbred animals (homozygotes for all genes, A/A, B/B)!
In human populations individuals, who are homozygous for all MHC genes are
extremely rare, parental tissues can not be used as grafts, only in some cases.
Genetics of tissue compatibility
Genes of the human MHC proteins. MHC I class is coded by A, B
and C genes, MHC II are coded by D (DP, DQ and DR) genes
Antigen presentation
The purpose of antigen presentation is the preservation of the unity of the
organism, limitation of mutations and detection of intracellular parasites.
Peptides of all proteins are presented on the cell surface in a complex with MHC
proteins
The structure of MHC I proteins
• Schematic model and structure based on X ray crytallography: the red line
represent the presented peptide
Generation and presentation of antigenic peptides
• Not the intact antigen is
presented, only some of
its peptides (epitopes).
• Peptides of self antigens
are generated by
proteasomes.
• Peptides of all cellular
proteins, including
nuclear, mitochondrial,
etc. are presented on the
surface of the cell.
Identity of the cells
• Proteasomes degrade proteins in
the cytoplasm.
• TAP transporters use the energy
of ATP to pump peptides into the
lumen of the ER
• Actually, TAP proteins insert
these peptides into the peptidebinding pockets of MHC I
proteins
Specificity and peptide-binding of MHC I
• Two alpha helices of the MHC I bind the
peptide, their composition determines the
affinity of the MHC protein.
• Due to gene duplication and co-dominant
inheritance we have different MHC
proteins.
Peptide binding of the MHC I
• The space-filling model shows the intimate link between tha MHC I protein
and the presented peptide. The peptide is inserted into the binding groove
during the development of the MHC I conformation.
• Ribbon and spacefilling models
MHC I polymorphism
• In the human population there are extremely large numbers of MHC alleles.
While the overall structure of the protein is tricktly preserved, sequences of the
peptide-binding pockeds are very diverse.
Antigen presentation of MHC I: step by step
Calnexin (a chaperone) helps the assembly of the MHC I protein and beta-microglobulin. If a peptide (generated by proteasomes and) pumped by the TAP proteins
fits into the pocket of the MHC I, the protein gains it proper conformation for
transport to the cell memrane. „Empty” or misfolded MHC I can not be transported,
they are degraded..
Function of MHC II
• MHC II molecules are present on immune cells
• Peptides presented by MHC II are generated by lysosomes: these are
peptides of foreign proteins endocytosed by the cells.
• Proteins „foreign”for
the cell can be „self”
proteins: macrophages
engulf debris of dead
cells.
• What is „self” for the
organism, can be
„foreign” for the cell!
Macrophages present
their own peptides on
MHC I, endocytosed
antigen on MHC II.
Structure of the two classes of MHC proteins
• One chain of MHC I and both chains of MHC II are encoded by MHC genes.
MHC II molecules have extremely large variability, with many alpha and
beta alleles. Overall structures of the two classes are very similar.
The peptide-binding grove is encoded
by one gene in MHC I, by two genes in
MHC II proteins.
Specificity of the two classes of MHC
• Peptides boound by MHC II are much longer, up to 20-22 amino acids – their
variabity is also much higher. Specificity of binding is determined by the
alpha helices: in certain position the presented peptide should contain certain
amino acid residues.
How MHC II works?
• MHC II is also synthesized in the ER. However, it can not bind peptides there,
because trimers of chaperone li (invariable chain) is bound by the pockets (forming
a complex of 9 polypeptides ( 3 li, 3 α and 3 β subunits). This complex is transported
into the lysosomes.
• Endocytosed antigens
are digested in the
lysosomes. li is also
digested, its last peptide,
„clip” is exchanged to a
foreign epitope with the
help of DM, an MHClike protein.
• „Empty” and misfolded
MHC II are degraded,
can not be transported
to the surface.
Polymorphism of MHC genes
• Unlike any other genes, MHC genes have very high number of alleles, which
have different epitope specificity.
B and T cell receptors,
immunglobulins
During the evolution receptors evolved which bind molecules (or molecular
patterns) not present in the host organism (only in pathogenic organisms). These
pattern recognition receptors (PRR) signal danger, if the host is infected.
Detection of very many pathogens requires very many receptors. This strategy is
genetically expensive and always leaky: , „new” pathogens are not detected.
Plants, mushrooms, worms and insects have many PRRs, birds and mammals
developed a more economical and more flexible strategy: recombinant
receptors.
Two classes of immune cells (B and T cells) have cell surface receptors, which are
encoded by recombinant genes. The recombination is cell specific: each cell has
an individual receptor, different from all the others.
Useless or auto-reactive cells are destroyed (clonal deletion). Protective cells
proliferate very extensively (clonal expansion).
B cells (plasma cells) produce the soluble form of their receptor (immuneglobulins)
in large quantities.
B and T cell receptors,
immunglobulins
Only a section of the receptor is recombinant, the
variable domain, which interacts with the antigen. The
rest of the molecule is made of constant domains.
B cell receptors (BCR) have heavy and light chains,
each with one variable and 3-4 or 1 constant domains,
respectively, T cell receptors have two chains, with one
constant and one variable domains.
BCR (and immunoglobulins, Ig) bind antigens with the
variable regions of their heavy and light chains
(specificity is determined by the two chains together)
Antigen-binding sites
Both heavy and the light chains
contribute to antigen recognition.
Different fingers of the two chains are
labelled with different colors.
The lower picture shows, how the
binding sites surround the antigenic
molecule
Structure of constant and variable domains
Beta sheets of the domains form
layers, with loops protruding from
the domains.
Each domain is stabilized by one
disulphide bridge.
The Ig domain is one of the most
frequent domains found in proteins
which interact with other proteins.
Antigen-binding loops of the variable domain
Variable domains have hiper-variable regions:
these are the loops that interact with the antigen
3 loops are located on
the light chain, 3 loops
on the heavy chain.
These „fingers” hold
the antigenic molecule
Where all this started?
In 2002 they foound a chitin-recognizing PRR in a primitive fish, with
sequence homology to immunoglobulins. This gene is never recombined in the
fish, it is only one of the many PRRs, detecting insect or fungal pathogens.
Where all this started?
Approx. 450 million years ago a mobile genetic element got inserted into the
ancient Ig gene.
Later on the recombinational signal sequences (RSS) and the two, genetically
linked, but not homologous recombinase genes (RAG1 and RAG2) got separated
from each other.
Unlike other transposons, these were not silenced, but used by the host, but their
effect became limited to several regions of several genes.
Arrangement of immunoglobulin genes
B cell and T cell receptor genes are made up of gene fragments (bordered by
recombinational signal sequences (RSS). During the maturation of B and T cells, the
Rag enzymes rearrange the genes. Two or three type of fragments are found in the
genes: variability (v), diversity (d) and joining (j). The rearranged genes contain only
one of each of these fragments. High number of fragments in each classes allows very
high number of possible variations. Due to further mechanisms the number of possible
combinations is almost unlimited.
Recombination leads to loss of genomic sequences
RAG recombinases recognize 7 and 9
bp long sequences separated by 12 or
23 bp spacers.
Recombination is directed by the
signal sequences,
depending on the direction of these
sequences recombination results in
deletion or inversion.
The closest relative of our RAG
recombinase is the transposase
HERMES, which is active in flies
Rearrangement of the BCR/Ig heavy chain
First the recombination removes
sequences between the selected D
and J segments, then between the
selected V and the joined DJ.
Within V, D and J segments one
can find the codes for the 3
antigen-binding loops of the
variable domain.
When the variable domain is ready,
a pre-mRNA is transcribed,
resulting in the mature mRNA after
splicing out the introns.
Rearrangement of the BCR/Ig light chain
RAG recombinase removes sequences
between the selected V and J segments.
There are no D segments, the
recombination takes only one step.
Within V and J segments one can find
the codes for the 3 antigen-binding
loops of the variable domain.
When the variable domain is ready, a
pre-mRNA is transcribed, resulting in
the mature mRNA after splicing out the
introns.
Any heavy chain can be combined to
any light chain to increase the number
of possible combinations.
Terminal transferase
RAG recombinase produces hairpin DNA, which is
cleaved to produce ovehanging ends.
Terminal transferase (terminal deoxynucleotide
transferase, TdT) is a unique DNA polymerase: it works
without a template, producing nonsense sequences.
The activity of TdT before joining the ends of the
recombined gene fragments produces further variability.
Successful rearrangement of one allele inhibits the
rearrangement of the second allele. Otherwise the second
allele undergoes a similar process.
Cells with frameshift mutations leading to truncated
polypeptide chains or cells with non-functional Ig
molecules are eliminated during maturation.
Affinity maturation: somatic hipermutation
In the germinal center of the lymph nodes B cells undergo a unique somatic
hipermutation. The enzyme adenosine deaminase (ADA) generates a number of
mutations, affecting the region of the Ig gene coding for the variable domain.
This process, called affinity maturation generates different clones of the
antigen-recognizing B cell. The ones, with the highest affinity to antigen will
survive and produce immunoglobulins, the others will apoptose.
Isotype switch
There are different classes of
immunoglobulins, serving different purposes (IgA protects
mucosal surfaces, IgE fights
eukaryotic parasites, IgG and IgM neutralizes extracellular pathogens and
activates the complement system).
Different classes have different constant domains, coded by different Ig genes.
Depending on the pathogens, B cells produce different types of Ig classes to
optimalize defence. Isotype switch is another genetic recombination: the variable
domain is combined with the constant domains of the selected isotype.
Isotype switch and soluble immunoglobulins
In immature B cells only cell surface BCR is produced. After affinity maturation and
isotype switch mature B cells convert to plasma cells (or memory B cells) and start to
produce soluble immunoglobulins.
Soluble immunoglobulins are produced from the same pre-mRNA as BCR, but
alternative splicing removes the trans-membrane domain.
Un-switched Ig molecules: the IgM class
IgM molecules (the oldest class) do not go through
affinity maturation, they have lower affinity.
However, they form pentameric structures with
10 binding sites, increasing strenght of interaction
by the number of binding sites.