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1、An enormously diverse collection of 108 related proteins found in the blood of vertebrates, specifically bind such foreign invaders (antigens) as bacteria, viruses or large molecules, and tag them for destruction (neutralization). Produced by plasma B cells.Biochemistry Lecture (Oct. 11, 2012)Extrem
2、e specificityGreat diversity2012 Nobel Prize in Chemistrylfor studies of G-proteincoupled receptorsRobert J. LefkowitzBrian K. KobilkaAn overview of theAdaptive Immune Response Complement systemOr PhagocytosisFound in salivatears and milkThe first made inprimary immuneresponse;Of high avidityIgG: do
3、minant in Secondary immune response; Most abundant in blood.von Behring(1854-1917) Nobel Prize in 1901“for his work on serum therapy, especially its application against diphtheria, by which he has opened a new road in the domain of medical science and thereby placed in the hands of the physician a v
4、ictorious weapon against illness and deaths.” lSide chains in the form of receptors existing in the “protoplasm”, which were able to bind distinct toxins with strict specificity, like “a key in a lock”.l Released into the blood, these receptors represented antitoxins (antibodies).Dr. Ehrlichs Magic
5、Bullet (an 1940 movie)Paul Ehrlich (18541915) Nobel Prize in 1908in recognition of their work on immunity lHeidelberger & Avery. (1923) The soluble specific substance of pneumococcus. J Exp Med. 38:73-9. lCulture fluids of pneumococci contained a substance that precipitates specifically with anti-pn
6、eumococci serum. lThe substance found not to be protein (contained no nitrogen) but a polysaccharide. lThis allowed the identification of the antibody present in an immunoprecipitate as protein. HeidelbergerAverylFelton LD. (1934) Pneumococcus antibodies what are they? Science 79:277-9. Pepsin and t
7、rypsin digest: loss of immunological characteristics.lTiselius A, Kabat EA. (1938) Electrophoresis of immune serum. Science. 87:416-7. lTiselius A, Kabat EA. (1939) An electrophoretic study of sera and purified antibody preparation. J Exp Med. 69:119-31.Electrophoresis of horse anti-pneumococcus ser
8、um before (A) and after (B) absorption of the antibody by antigen: only one protein band disappeared.ABAntigen absorption experimentKabatTiseliusg gg gRabbit anti-egg albumin serumunabsorbedabsorbedHorse anti pneumoccus serumlPauling L. (1940) A theory of the structure and process of formation of an
9、tibodies, J. Am. Chem. Soc. 62:2643-2657.Heterogeneity of sera;Complementariness (specificity);Weak bonding (H-bonds);Bivalent nature of antibodies (precipitin reaction; rule of parsimony)One single chain (of different configuration);MW of Ab: 160 kD;Antigen: be big enough.Pauling(1901-1994)B: stabl
10、eA &C: foldinto manyConfigurationsand Pro-richThe clonal selection theoryFrank M. Burnet(Nobel Prize, 1960) Antigen stimulates the amplificationof the type of B cell that produces one specific antibody.(Polyclonal antibodies)(monoclonal antibodies)Burnet FM (1957) A modification of Jerns theory of a
11、ntibody production using the concept of clonal selection, Austr. J. sci., 20:67-69lPorter R R. (1958) Separation and isolation of fractions of rabbit g g-globulin containing the antibody and antigenic combining sites. Nature. 182:670-1. lPorter, R. R., (1959) The hydrolysis of rabbit g g-globulin an
12、d antibodies with crystalline papain, Biochem. J.,78:119-127.AntigenBinding(Fab)Crystallizes (Fc)50kD 53kD80kDFractions I and II, nolonger form precipitatewith antigen, butinhibits theantibody-antigenprecipitate formationRodney Porter(Fred Sangers First Ph.D. student)“Rabbit y-globulin is formed of
13、two pieces with very similar structure joined to a third piece of quite different character.”lEdelman, G. M., (1959) Dissociation of g g-globulin, J. Am. Chem. Soc., 81:3155.lEdelman, GM, Poulik, MD. (1961) Studies on structural units of the g g-globulins. J Exp Med. 113:861-84. Treatment withreduci
14、ng agentmercaptoethanoland denaturanturea.ReducedNon-reducedBence Jones protein from urineLight chain from serumGerald M. EdelmanFrom the same patientlPorter RR. (1973) Structural studies of immunoglobulins. Science. 180:713-6. (review)lReduction without denaturant.lThe L chain does not react with a
15、ntiserum to Fc. The Nobel Prize in Physiology or Medicine 1972for their discoveries concerning the chemical structure of antibodiesGerald M. Edelman Rockefeller University USAb. 1929(studied consciousnessIn his later life)Rodney R. PorterUniversity of Oxford United KingdomB. 1917-1985 lFeinstein, A.
16、 and Rowe, A. J. (1965) Molecular mechanism of formation of an antigen-antibody complex. Nature. 205:147-9. A “click-open” theory proposed:A compact antibody opensupon binding to two antigens.(H chain of IgG)N-term.Hypervariable (HV) regions on the H chainCDR1CDR2CDR3Elvin Kabat1970sComparison of th
17、e amino acid sequences of the VH and VL regionsThe chemical structure of IgG as revealed by Porter and Edelman (1950S and 1960S).2lHuber et al. (1976) Crystallographic structure studies of an IgG molecule and an Fc fragment. Nature. 264:415-20. lHarris et al. (1992) The three-dimensional structure o
18、f an intact monoclonal antibody for canine lymphoma. Nature. 360:369-72. lOnly monoclonal antibodies and monospecific antibodies isolated from patients with multiple myeloma could be used for crystallization.lThe IgG molecule (H2L2) contains six domains, each has characteristic immunoglobulin folds.
19、 lEach hypervariable region (i.e., the CDRs) of the H and L chains exist as a loop, with the six loops close to each other in space. Structure of an intact IgG (of b b-sheets!)VLVHCH1CLCH2CH3CH2CH3VLCLVHCH1Harris LJ., 1992,Nature, 360:369-72.The “hinge” region(extended and flexible)An asymmetric con
20、formation!The three hypervariable Regions exist as three loops that are close to each other in space. lForming the immunoglobulin superfamily of hundreds of members, containing domains of a sandwich of two antiparallel b b-sheets stabilized by a disulfide bond.lInclude: cell surface antigen receptor
21、s, co-receptors and co-stimulatory molecules of the immune system, molecules involved in antigen presentation to lymphocytes, cell adhesion molecules, certain cytokine receptors, intracellular muscle proteins (titin), etc.lThe antigen binding sites are indeed made of the hypervariable regions (CDRs)
22、 of both the H and L chains (as was hypothesized).lConformational changes in the antibody and/or the antigen occur, allowing a complementary tight binding between a specific antigen and its specific antibody.lNoncovalent interactions are used for the antibody-antigen interaction.lThe Kd for antibody
23、-antigen interaction can be as low as 10-10 M. In this view, the HV regions of the Fab have been deleted: there is no contact between antibody and antigen. This ribbon structure shows the antibodys HV (purple) region of the Fab, and their interaction with an epitope of the antigen. FabAntigenHVAntig
24、enAn induced fit conformational change occurs in IgG upon binding to its antigenlAffinity chromatography: one-step protein purification using a specific antibody that is covalently attached to a resin.lImmunoblotting assay: including Western blotting and Enzyme-linked immunosorbent assay (ELISA); qu
25、alitative and quantitative detection of a specific antigen present in a mixture, which could be a solution, a gel, or a living cell, using its specific antibody that is labeled (e.g., being linked to an enzyme).(antibody, receptor etc) Specific antigen Nonspecific proteins Salt solution or other age
26、nt Only Specific antigens are retained on the columnThe principle and procedure of immunoblot assaysSecondaryantibody(labeled)Primary antibody(antigen specific)ColorlesssubstrateColored product(for easy detection)Protein mixtures are absorbed to aninert surfaceHorseradishperoxidaseColorlesssubstrate
27、Colored productWestern blotting to detect a specific antigen protein in a protein mixtureusing a specific antibody.Gel-separated antigen proteins are transferred onto a nitrocellulosemembrane before being probed withantibodies.lImmunoglobulins are vertebrate proteins evolved for removing potentially harmful foreign antigens of tremendous variation in structure.lThe diversity of Immunoglobulin structures explaining its extreme antigen specificity was demonstrated on both the amino acid sequence and tertiary structure levels.
生物化學：Chapter 5-3 Protein Function, modulation and evolution
