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!
Transcript
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 incompatibilityOn 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 presentationThe 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 peptide-binding 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 space-filling 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-micro-globulin. 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 theorganism, can be „foreign” for the cell!Macrophages presenttheir 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 MHC-like protein.
• „Empty” and misfoldedMHC II are degraded, can not be transportedto 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.
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)
B and T cell receptors, immunglobulins
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.
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.
Where all this started?
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 transposaseHERMES, 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 transferaseRAG 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 hipermutationIn 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 ofimmunoglobulins, serving dif-ferent purposes (IgA protects mucosal surfaces, IgE fightseukaryotic 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.
IgM molecules (the oldest class) do not go through affinity maturation, they have lower affinity. However, they form pentameric structures with10 binding sites, increasing strenght of interaction by the number of binding sites.
