414/514 Immunology Principles 415/515 Immunology Principles and Practice

Immunoglobulin Structure and Function

Antibodies are immunoglobulins ... glycoproteins that are formed in response to an immunogenic antigen (immunogen) and which react specifically with that antigen

• Antibodies are formed in response to antigens, as first demonstrated by von Behring and Kitasato, who discovered in 1890 that immunity against diphtheria and tetanus is due to toxin-neutralizing antibodies
• Antibodies are important for destruction of transfused blood cells as shown by Landsteiner, who first classified human blood groups as A, B, AB and O ... and subsequently discovered Rh factor.
• Antibodies are globulins - Tiselius and Kabat (1939) demonstrated that most of the antibody in a hyperimmune rabbit migrates as gamma globulin when subjected to electrophoresis
• Overall structure of IgG antibody molecules - Porter, Edelman and Nisonoff were the primary researchers involved in deciphering the overall structure of IgG antibody molecules
  • Porter (1950s) started these studies by showing that partial digestion with papain reduced the sedimentation coefficient of IgG antibody from 7S to 3.5S with loss of only a small amount of protein
    • electrophoresis revealed that there were actually three fragments generated from the antibody molecules
    • ion exchange chromatography allowed isolation of the three fragments into two fragments that bound antigen but wouldn't precipitate it (these univalent fragments were called Fab for "fragment antigen-binding") and one fragment that did not bind antigen but crystallized when purified (called Fc for "fragment crystallizable")
  • Edelman (1959) showed that IgG antibodies were composed of two types of polypeptides (heavy and light chains) held together by disulfide bonds and noncovalent bonds when he discovered that
    • reduction (with 2-mercaptoethanol) and dissociation (with urea), followed by
    • electrophoresis in polyacrylamide gels containing urea yielded two bands, both of which migrated faster than IgG (Edelman's Nobel Lecture)
  • Porter (1959-1963)
    • isolated heavy (H) and light (L) chains by partial reduction of IgG followed by gel filtration in the presence of propionic acid (low pH), then showed that there were two H and two L chains in each IgG molecule by taking into account their molecular weights (~50kD for H and ~25kD for L), the relative amounts of each he isolated, and the molecular weight of intact IgG (~150kD)
    • identified heavy and light chain components in papain digestion fragments of IgG using antibodies specific for Fab and antibodies specific for Fc (anti-Fab reacted with both heavy and light chain, whereas anti-Fc reacted only with heavy chain) (Porter's Nobel Lecture)
  • Nisonoff (1960) showed that pepsin digestion of IgG antibodies yielded one bivalent fragment composed of two Fab fragments, named F(ab')₂
• Stick model of IgG antibody structure - bilaterally symmetrical molecule with two heavy chains that interact throughout their C-terminal halves, and two light chains aligned with the heavy chains at their N-termini and held together with a combination of noncovalent and covalent (disulfide) bonds; these molecules are "decorated" with carbohydrate moieties ... thus antibodies are glycoproteins
• Isotypes of antibody - characteristics and functions:

• Relationship of antibody heterogeneity to specificity for antigen
  • Hilschmann and Craig (1965) compared light chains from a number of antibodies with different specificities to show that the N-terminal half of light chain contains virtually all the heterogeneity (amino acid variability) and proposed that this heterogeneity is directly related to (responsible for) antibodfy specificity
  • Dryer and Bennett (1965) proposed their two-gene, one polypeptide model ... the idea that the light and heavy chain polypeptides comprising antibody molecules are each encoded in more than one gene ... many for the variable region and one for the constant region of each chain
  • Edelman (1969) sequenced human IgG (using the naturally homogeneous immunoglobulin (myeloma protein) from a patient with multiple myeloma (a disease characterized by a plasma cell tumor called a plasmacytoma), thus confirming the arrangement of heavy and light chains in the "stick" model and paving the way for others, who used both myeloma proteins and purified antigenically induced antibodies to compare antibody sequences more thoroughly
  • Wu and Kabat (1970)
    • compared sequences of light and heavy chains from a number of antibodies with different specificities to show that the heterogeneity in the N-terminal half of light chain and the N-terminal quarter of heavy chain is virtually all due to three areas of very high amino acid varibility that they called hypervariable regions
    • affinity labeled antibodies to show that amino acids in the hypervariable regions (HVR) contain the the amino acid residues that interact with antigenic determinants by making direct contact with their constituents (amino acids, sugars, etc.), thus showing that HVR amino acids make up the complementarity determining regions (CDR) of antibody molecules ... these regions contain the amino acids that actually interact with the antigenic determinant for which the antibody is specific
• Three-Dimensional Structure of Antibodies
  • Poljak and others (1970s ... 1990s) developed the methods leading to elucidation of the three-dimensional structure of antibodies, which required a combination of sequence studies and x-ray crystallography ... and now uses nuclear magnetic resonance and electron spin resonance to do this
  • antibodies are quaternary aggregates of globular protein domains arranged in such a manner that the Fabs are accessible to antigenic determinants and the Fc is available to bind to complement and/or Fc receptors (FcR) on phagocytic cells
    • domains are comprised of three-dimensionally folded polypeptide structures, which are universal in antibodies (and many other immunologically-relevant structures)
      • Fv (fragment variable)
        • comprised of the N-terminal domains of Fab
        • antibody combining sites are located near the N-terminal regions of the heavy and light chains that make up the Fv
        • certain amino acids in these sites make up the floor and sides of a shallow groove (or a slight projection) on the surface of the Fv that actually interacts with antigenic determinants
      • immunoglobulin fold (Ig fold) - responsible for determining the structure of Fv, which helps establish its ability to bind antigen
        • the Ig fold is comprised of ~110 amino acids and are arranged in antiparallel sets of beta-pleated sheets
        • loops that connect the beta-pleated sheets contain the amino acids that comprise the hypervariable regions (HVRs), or complementarity-determining regions (CDRs), of the antibodies
          • since these loops are on the surface of the domain, the side chains of these amino acids can come into contact with antigenic determinant constituents
          • interaction of CDR amino acids with antigenic determinant constituents occurs via multiple noncovalent interactions involving:
            • hydrogen bonding
            • electrostatic interactions
            • van der Waal's forces
            • hydrophobic interactions
          • thus, it should be clear that CDRs determine the specificity and affinity (strength of binding) of antibody combining sites as a result of the numbers and kinds of noncovalent bonds they can form with antigenic constituents
        • idiotypic determinants of antibodies are the result of the unique arrangements of amino acids that make up antibody combining sites
    • biological activities (IgG and IgM complement binding, IgG and IgE FcR binding, IgG transplacental transport, IgA transport through mucous membrane epithelial cells) are associated with structures in the Fc portion of antibody molecules
    • allotypic differences are due to mutation-induced changes in a restricted number of amino acids, mostly in the constant portions of heavy and light chains (but some are within the framework regions of the variable portions of heavy and light chains)
• Genetic basis of antibody combining site diversity
  • Tonegawa showed that the Dryer and Bennett hypothesis was essentially correct ... many gene segments or gene fragments (exons) in the germ-line DNA encode the information that is ultimately used to denerate light chain combining site diversity (Tonegawa's Nobel Lecture)
  • Hood showed that a very similar type of genetic arrangement also occurred in heavy chain genes
  • Gene segments are rearranged during B cell maturation to generate the active gene that codes for the antibody combining site a B cell can synthesize
  • Antibody is produced using the information encoded in the rearranged (activated) genes in B cells