Immunoglobulins Harper s biochemistry Chapter 49
Immune system Detects and inactivates foreign molecules, viruses, bacteria and microorganisms Two components with 2 strategies B Lymphocytes (humoral immune response): Soluble antibodies (immunoglobulins) Secreted by plasma cells Recognize and bind to foreign molecules. T Lymphocytes (cellular immune response): Killer cells that display foreign substances on their surface Use specific receptors on their surface
Immunoglobulins The binding to the foreign molecules is: Specific High affinity Effector functions: biological effects that result as consequence of antigen binding like Inactivation, degradation, lysis Can recognize and interact with foreign molecules even if not encountered before Huge number of different kinds (~10 8 ) Synthesis is stimulated by invasion of the body
Formation of immunoglobulins (Ig) Antigen - Foreign molecules to which Ig binds - Can elicit antibody formation ( immounogen) - Macromolecule; Protein, polysaccharide, nucleic acid Epitope Particular site on antigen to which antibody binds Antigenic determinant Hapten small molecule, not an antigen Antigen if attached to macromolecule
Formation of antibodies against small molecules OH Attach DNP to Bovine Serum Albumin Dinitrophenol small molecule a
Formation of antibodies against DNP after injection of DNP-BSA to rabbit
Isolation of antibodies (AB) against DNP from rabbit Antisera: The serum obtained which contains the antibodies (AB) AB formed can bind free and attached hapten AB can be purified by affinity chromatography AB obtained are NOT one kind of molecules but a mixture of antibodies with the same specificity
Monoclonal antibodies One kind of antibodies Produced from clone of one single cell
Principles of preparation of monoclonal antibodies Multiple Myeloma: Malignant growth of a single kind of B lymphocytes in human. Large amounts of one kind of antibodies are produced ( but unknown specificity). Mice strains with multiple myeloma are available Can be transplanted from one mouse to another. Same kind of antibody is produced. Cells can be cultured (cell line)
Antibodies produced Monoclonal Large amounts Desired specificity
Structure of antibodies Immunoglobulin G ( IgG ) is the major class of antibodies in the plasma. 150 kda Tetramer of Two heavy chains 50 kda Two light chains 25 kda Connected by disulfide bonds
light chain Heavy chain
IgG can bind and cross link antigens with multi binding sites
IgG is not a rigid molecule It has segmental flexibility
Classes of immunoglobulins IgG IgA IgM IgD IgE
Properties of immunoglobulin classes Class Concent. mg/dl Mass kda Heavy chain Chain structure IgG 1000 150 γ Monomer IgA 200 170-500 α Dimer or trimer IgM 120 900 μ Pentamer IgD 3 180 δ Monomer IgE 0.05 190 ϵ Monomer
Classes of immunoglobulins IgG IgA IgM??? IgD IgE
Properties of immunoglobulin classes Class IgG IgA The highest in concentration in the serum The major class in external secretions IgM The first class to appear in serum after exposure to antigen IgD Unknown role IgE A role in conferring protection against parasites Allergic reactions
Can t bind antigen Cleavage of IgG by papain (Limited proteolysis) F ab 50 kda Binds antigen Can t cross-link F c 50 kda Readily crystalyzes
Amino acid sequence of antibodies 2 L chains 25 kda 214 AA 2 H chains 50 kda 446 AA AA sequence L chain (multiple myeloma patients) 1-110 vary from one patient to another 111 214 same in many patients
Amino acid sequence of antibodies AA sequence H chain (multiple myeloma patients) 1-113 vary from one patient to another 114 446 same in many patients
Variable and constant regions of light and heavy chains 3 stretches (7-12 amino acids) hypervariable V L V H C L V L C H 3 V H
Immunoglobulin fold Pair of β sheets Each built of antiparallel β strands Connected by disulfide Three loops at one end of the structure Hypervariable stretches The binding surface complementarity determining regions (CDR)
The molecular basis of Antibodies diversity The Variable and constant regions Multiple V variable genes are separate from single C gene in embryonic DNA embryonic cell V 1, V 2, V 3..V n + C differentiated cell VC
Dreyer - Bennett hypothesis V V V V V Single germline C gene separate from multiple V genes V V V V C V V V C Rearranging V and C genes V V
The molecular basis of Antibodies diversity Immune system can generate > 10 8 antibodies proteins Human genome contains ~ 40,000 genes!
V genes in embryonic cells do not encode the entire variable gene V genes encode the first 97 amino acids Array of 40 segments J genes encode the last 13 amino acids Array of 5 segments near the C gene V 1, V 2,. V 40 J 1, J 2, J 5 C VJ VJC
Genes encoding heavy chains are present on chromosome 14 The variable gene is assembled from 3 segments 51 ~27 6 D join to J H then V is joined to DJ H
Number of possible combinations For κ 40 * 5 = 200 For λ 30 * 4 = 120 For H 51 * 6 *27 = 8262 Combination of 320 L and 8262 2.6 * 10 6 Somatic mutations increases the diversity
Class switching Proper immune response: Secreted antibodies must have appropriate effector function IgM IgG or IgA... Specificity unchanged Light chain unchanged V H unchanged C H is changed Gene rearrangement: movement of VDG from a site near one C gene to a site near another C gene Shifting of the recognition domain different effector function unchanged binding specificity
Immune system can distinguish between self and nonself Protection from invasion by foreign organisms is the primary function of immune system. Attacking host cells is avoided. How can immune system distinguish Cells that react strongly with self- antigens are killed early in the development of immune system