Antibody Structure Antibody Structure and Functionand Function
Keri C. Smith, Ph.D.Keri C. Smith, Ph.D.
January 22, 2008January 22, 2008(or)
Anatomy and Physiology of Antibodies
OverviewOverview
Physical properties of antibodiesPhysical properties of antibodiesStructural and molecular featuresStructural and molecular features
Differences between types of antibodiesDifferences between types of antibodiesFunctional properties of antibodiesFunctional properties of antibodies
Just a reviewJust a review
Antibodies are gammaAntibodies are gamma--globulinsglobulins
Multiple MyelomaMultiple Myeloma
Case Study #19
γ κ
Anatomy of Ig Light and Heavy Anatomy of Ig Light and Heavy ChainsChains
Porter and Porter and NisonoffNisonoff used enzymes to used enzymes to cleave Ig, studied function of isolated cleave Ig, studied function of isolated fragmentsfragmentsEdelman treated Ig with reducing agents to Edelman treated Ig with reducing agents to cleave Scleave S--S bonds, studied the resulting S bonds, studied the resulting polypeptide chainspolypeptide chains
IgIg
Fragmentation and Domain Fragmentation and Domain StructuresStructures
Fab
portion can bind antigenFc
portion can be crystallized
Ig Light ChainsIg Light Chains
Two types of Light chains are found in Ig of Two types of Light chains are found in Ig of all animals, all animals, aaaa sequence differssequence differsKappa chainsKappa chains--Human 60%, mice 95%Human 60%, mice 95%Lambda chainsLambda chains--Human 40%, mice 5%Human 40%, mice 5%
IgIg
Heavy ChainsHeavy Chains
5 classes of H chains in humans5 classes of H chains in humansSimilarities in Similarities in aaaa sequence, but each class has sequence, but each class has a unique sequencea unique sequenceH chains named with Greek letters H chains named with Greek letters corresponding with the class name, IgG, IgA, corresponding with the class name, IgG, IgA, IgM, IgE, IgM, IgE, IgDIgD ((γγ, , αα, , μμ, , εε, , δδ))IgG has 4 subclasses, IgA has 2 subclassesIgG has 4 subclasses, IgA has 2 subclasses
DomainsDomains
Early studies showed regularity of Early studies showed regularity of structure of all the Ig classesstructure of all the Ig classesEach 100Each 100--110 110 aaaa has a 60 has a 60 aaaa SS--S loopS loopV domains code the paratope, binds V domains code the paratope, binds AgAgC domains code regions important C domains code regions important for mediating secondary biological for mediating secondary biological functions, ie binding Complement, functions, ie binding Complement, crossing the placenta.crossing the placenta.
Human Ig Domain FunctionsHuman Ig Domain Functions
Ig Hinge RegionsIg Hinge Regions
Hinge regions on IgG, Hinge regions on IgG, IgA and IgA and IgDIgD are coded are coded by distinct by distinct exonsexonsShort span of Short span of aaaabetween 1between 1stst and 2and 2ndnd C C domainsdomainsRich in Cys and ProRich in Cys and ProProvides for flexibility Provides for flexibility of the moleculeof the moleculeIs readily accessible to Is readily accessible to solvent and enzymessolvent and enzymes
Ig Variable and Hypervariable Ig Variable and Hypervariable RegionsRegions
Amino terminal Amino terminal aaaa sequence was shown to sequence was shown to vary from one L chain to anothervary from one L chain to anotherKabatKabat and Wu developed the Variability Plot and Wu developed the Variability Plot to measure degree of variationto measure degree of variationFound 3 Hypervariable regions in both L and Found 3 Hypervariable regions in both L and H chain V regions.H chain V regions.These are epitope contact These are epitope contact aaaa regions, CDR regions, CDR ((complementaritycomplementarity determining regions)determining regions)
Definition of VariabilityDefinition of Variability
The ratio of the number of different amino The ratio of the number of different amino acids at a given position to the frequency of acids at a given position to the frequency of the most common amino acid at that position the most common amino acid at that position is defined as VARIABILITY.is defined as VARIABILITY.Example: 63 L chains were sequenced and Example: 63 L chains were sequenced and four amino acids were found at position 7. four amino acids were found at position 7. Serine occurred 41 times, ie frequency Serine occurred 41 times, ie frequency 41/63=0.65. Thus 4/0.65=6.15 Variability41/63=0.65. Thus 4/0.65=6.15 Variability
Variability PlotVariability Plot
CDR in 3DCDR in 3D
Ig Classes or IsotypesIg Classes or Isotypes
Result from structural variation in the heavy Result from structural variation in the heavy chainschainsAre 5 major classes or isotypes of Ig in Are 5 major classes or isotypes of Ig in HumansHumansIgG, IgA, IgM, IgE, IgG, IgA, IgM, IgE, IgDIgDSubclasses: Subclasses: IgGIgG (4) and (4) and IgAIgA (2)(2)
Features of Features of IgIg
IsotypesIsotypes
Ig AllotypesIg Allotypes
Allelic variants of Ig Constant regionsAllelic variants of Ig Constant regionsCoCo--dominant autosomal Mendelian genesdominant autosomal Mendelian genesAre allotypic variants of Are allotypic variants of γγ, , αα, and , and κκ chainschainsAllotypes used forensicallyAllotypes used forensicallyAllotype may confer some biological Allotype may confer some biological advantage for some infectious agentsadvantage for some infectious agents
Human Kappa Light Chain Human Kappa Light Chain AllotypesAllotypes
ALLOTYPEALLOTYPE AMINO ACID AMINO ACID DIFFERENCESDIFFERENCES
Km(1)Km(1) Val @ 153, Val @ 153, LeuLeu
@ 191@ 191
Km(1,2)Km(1,2) Ala @ 153, Ala @ 153, LeuLeu
@ 191@ 191
Km(3)Km(3) Ala @ 153, Val @ 191Ala @ 153, Val @ 191
Allotype effects in infectionAllotype effects in infection
Children with G2m(23) allotype had higher Children with G2m(23) allotype had higher levels of immunity to H. levels of immunity to H. influenzaeinfluenzae type B type B than G2m(23)than G2m(23)--negative children and the negative children and the Km(1) allotype was found more effective Km(1) allotype was found more effective than the other kappa chain allotypes.than the other kappa chain allotypes.AmbrosinoAmbrosino, et al. 1985, J. , et al. 1985, J. ClinClin. Invest. 75:1935; . Invest. 75:1935; GranoffGranoff, et al. 1986, J. Inf. Dis. 154:257., et al. 1986, J. Inf. Dis. 154:257.
Ig IdiotypesIg Idiotypes
IdiosIdios: Greek, : Greek, unique for Ab of one specificity.unique for Ab of one specificity.Are epitopes in Ig V regionsAre epitopes in Ig V regionsProvide a natural autoimmune mechanism for Provide a natural autoimmune mechanism for regulating Ab levelsregulating Ab levelsAntiAnti--id antibodies are routinely found and id antibodies are routinely found and downdown--regulate production of Ab.regulate production of Ab.
Two Examples of IdiotypesTwo Examples of Idiotypes
Clinical Relevance of Clinical Relevance of IdiotypesIdiotypes
Active immunotherapy with 1E10 antiActive immunotherapy with 1E10 anti--idiotypeidiotype vaccine in patients with small cell vaccine in patients with small cell lung cancer: report of a phase I trial.lung cancer: report of a phase I trial.
NeningerNeninger et al. Cancer Biol. et al. Cancer Biol. TherTher. 2007. 6:145. 2007. 6:145
IsoIso, , AlloAllo, or , or IdioIdio??????
Ig Structural FeaturesIg Structural Features
IgG Structural FeaturesIgG Structural Features
IgG Structural Features IIIgG Structural Features II
IgGIgG
SubclassesSubclasses
IgG1, IgG2, IgG3, IgG4IgG1, IgG2, IgG3, IgG4Have about 90% homology in their amino Have about 90% homology in their amino acid sequencesacid sequencesDiffer in the numbers and arrangement of the Differ in the numbers and arrangement of the interchaininterchain disulfide bondsdisulfide bondsThis results in differences in their functional This results in differences in their functional propertiesproperties
IgGIgG
Subclasses (2)Subclasses (2)
Important Differences Between Human IgG Subclasses
IgG1 IgG2 IgG3 IgG4
Occurrence (% of total IgG) 70 20 7 3
Half-life 23 23 7 23
Complement binding + + +++ —
Placental passage ++ ± ++ ++
Binding of monocytes +++ + +++ ±
IgGIgG
Subclasses (3)Subclasses (3)
IgGIgG
Biological Properties (1)Biological Properties (1)
Most common Most common IgIg in serumin serumDistributed approximately equally between Distributed approximately equally between intravascular and intravascular and extravascularextravascular spacesspacesLong halfLong half--lifelife
IgGIgG
RecylcingRecylcing
IgG Biological Properties (2)IgG Biological Properties (2)
Agglutination and PrecipitationAgglutination and PrecipitationPlacental passage (G1, G3, G4)Placental passage (G1, G3, G4)OpsonizationOpsonizationAntibody dependent cell mediated Antibody dependent cell mediated cytotoxicity (G1, G3)cytotoxicity (G1, G3)Complement Activation (G1, G2, G3+)Complement Activation (G1, G2, G3+)Toxin/Viral NeutralizationToxin/Viral Neutralization----Passive Passive AbAbinfusioninfusionBacterial immobilizationBacterial immobilization
Three Roles for Three Roles for IgGIgG
Clinical CorrelationClinical Correlation
Hemolytic disease of the fetus/newborn. Hemolytic disease of the fetus/newborn. Maternal IgG antibodies specific for RhD are Maternal IgG antibodies specific for RhD are actively transported across the placenta, actively transported across the placenta, opsonizeopsonize fetal RhD+ RBC for fetal RhD+ RBC for phagocytosisphagocytosisby liver cells, fetal by liver cells, fetal hematocrithematocrit drops to drops to dangerous or fatal levels. Case 45.dangerous or fatal levels. Case 45.
Antibody Dependent CellAntibody Dependent Cell-- Mediated CytotoxicityMediated Cytotoxicity
IgGIgG
ReviewReview
Gamma Globulin, Enzymatic Fragmentation
L & H Chains, V & C Domains, Hinge Regions, Variability and Hypervariable
regions
4 Isotypes
(Classes)
Allotypes, Idiotypes
IgG
Anatomy: γ
H Chains, κ/λ
L Chains
IgG
Physiology: Agglutination, Placental Passage, Opsonization, ADCC, Complement Binding, Toxin/Viral Neutralization, Bacterial Immobilization, Recycling
IgMIgM
Structural FeaturesStructural Features
IgM Biological PropertiesIgM Biological Properties
Efficient bacterial/viral Efficient bacterial/viral agglutinatoragglutinator, , potentially 10 potentially 10 paratopesparatopes/molecule/moleculeIsohemagglutininIsohemagglutinin--naturally present Ab naturally present Ab reactive with A/B blood groups, barrier to reactive with A/B blood groups, barrier to random transfusion & transplantationrandom transfusion & transplantationMost efficient Ig for mediating Complement Most efficient Ig for mediating Complement fixationfixationPrimary antibody response to challengePrimary antibody response to challenge
Affinity and AvidityAffinity and Avidity
Due to itDue to it’’s structure, s structure, IgMIgM has can have the has can have the same same affinityaffinity, but a higher , but a higher avidityavidity for antigenfor antigen
Blood Groups and Blood Groups and IsohemagglutininsIsohemagglutinins
Jessica Jessica SantillanSantillan
Heart transplant patient
Duke University Hospital, Feb. ‘03
Patient was type O, Donor was type A
Kinetics of a Normal Antibody Kinetics of a Normal Antibody ResponseResponse
IgAIgA
Structural FeaturesStructural Features
IgA SubclassesIgA Subclasses
IgA Secretion MechanismIgA Secretion Mechanism
Biological Properties of IgA Biological Properties of IgA AntibodiesAntibodies
IgA is abundant on mucosal surfaces as a IgA is abundant on mucosal surfaces as a ““First Line DefenseFirst Line Defense””..IgA is bactericidal for Gram negative IgA is bactericidal for Gram negative organisms in the presence of Lysozyme.organisms in the presence of Lysozyme.IgA is an efficient viral IgA is an efficient viral agglutinatoragglutinator, , preventing viral attachment to epithelial cell preventing viral attachment to epithelial cell viral receptors.viral receptors.Plays a role in passive immunotherapy Plays a role in passive immunotherapy –– can can be transferred from mother to child in breast be transferred from mother to child in breast milk. milk.
IgDIgD
Structural FeaturesStructural Features
Biological Properties of Biological Properties of IgDIgD AntibodiesAntibodies
IgDIgD is not found in significant amounts in is not found in significant amounts in serum.serum.Principal function involves initial Ag Principal function involves initial Ag triggering of B cells while bound to the triggering of B cells while bound to the membrane on the surface of B cells.membrane on the surface of B cells.
IgEIgE
Structural FeaturesStructural Features
Biological Properties of IgE Biological Properties of IgE AntibodiesAntibodies
IgE mediates Type I hypersensitivity IgE mediates Type I hypersensitivity reactions.reactions.IgE antibodies bind to IgE antibodies bind to FcFcεε receptors on Mast receptors on Mast cells.cells.Ag binding with the IgE antibody induces Ag binding with the IgE antibody induces degranulationdegranulation, secretion of histamine, , secretion of histamine, heparin, and other pharmacologic agents.heparin, and other pharmacologic agents.Are high IgE levels in parasitic infections.Are high IgE levels in parasitic infections.
IgEIgE--FcFcεε
StructureStructure
IgE CrossIgE Cross--linking Leads to Mast linking Leads to Mast Cell TriggeringCell Triggering
Clinical VignettesClinical Vignettes--AllergiesAllergies
Case #32 Case #32 –– Acute Systemic Anaphylaxis: 22 monthAcute Systemic Anaphylaxis: 22 month--old, ate a cookie with peanut butter in it, describes old, ate a cookie with peanut butter in it, describes the symptoms (BP 40/0, norm is 80/60, pulse 185, the symptoms (BP 40/0, norm is 80/60, pulse 185, norm 80norm 80--90, respiration 76, norm 20. Gave 90, respiration 76, norm 20. Gave epinephrine, saline, antiepinephrine, saline, anti--histamine, corticosteroids. histamine, corticosteroids. Case #33 Case #33 –– Allergic Asthma: 14 year old with Allergic Asthma: 14 year old with persistent wheezing, reduced peak flow rate in lungs persistent wheezing, reduced peak flow rate in lungs of 180 liter minof 180 liter min--11 (normal> 350(normal> 350--400 liter min 400 liter min --11) and ) and reduced expiratory volume. Elevated serum reduced expiratory volume. Elevated serum IgEIgEand and eosinophilseosinophils. Treated with inhaled . Treated with inhaled steriodssteriods, oral , oral antihistamine, and antihistamine, and immunotherpeuticimmunotherpeutic antigen antigen injectionsinjections
KISS OF DEATHKISS OF DEATH
Kinetics of a Normal Antibody Kinetics of a Normal Antibody ResponseResponse
Features of Features of IgIg
IsotypesIsotypes
Ig Ig SuperfamilySuperfamily
There are structural similarities to Ig of the There are structural similarities to Ig of the molecules of numerous membrane bound molecules of numerous membrane bound glycoprotein molecules such as the MHC glycoprotein molecules such as the MHC molecules and the T cell receptor molecules. molecules and the T cell receptor molecules. T cell receptors and triggering will be T cell receptors and triggering will be covered in another lecture.covered in another lecture.
Ig Ig SuperfamilySuperfamily——Some MembersSome Members
Antibody EngineeringAntibody Engineering
Single Chain Fv (Single Chain Fv (ScFvScFv))Humanized antibodiesHumanized antibodiesImmunotoxinsImmunotoxins
CodaCoda
The function of antibodies, like the rest of The function of antibodies, like the rest of the body, is only understood with a firm the body, is only understood with a firm foundation in (molecular) anatomy.foundation in (molecular) anatomy.The 5 Ig isotypes each mediate specific The 5 Ig isotypes each mediate specific biological effects, due to different C region biological effects, due to different C region amino acid sequences in their respective H amino acid sequences in their respective H chain.chain.