Immunology Module 5: Structure and Function of Antibodies

Mature into plasma cells and secrete antibody molecules that bind to the pathogen and neutralize it

B-cell membrane immunoglobulins (Ig) bind to epitope and causes it to

4 polypeptides —> 2 identical longer heavy chains and 2 identical shorter light chains

Immunoglobulin (antibody) molecules consists of

Disulfide bonds

The various immunoglobulin (antibody) molecule chains are bridged by

Variable domain heavy chains (VH) and variable domain light chains (VL)

N-terminal domains in each immunoglobulin (antibody) polypeptide chain have

VH and VL domains

Antigen binding site is formed by

Gamma fraction of serum contains immunoglobulins that bind to antigens

1939 Tinelius and Kabat Experiment demonstrated

1939 Tinelius and Kabat Experiment explained

• Injected rabbits with ovalbumin antigen and collected the serum dividing it into tubes A & B

• Added ovalbumin to test tube B causing it to precipitate

• Removed the precipitate and subjected the serums from each tube to electrophoresis

• Absorbency results were plotted against migration distance from the initial point of application

• Comparison between the electrophoresis profiles of 2 aliquots showed decreased amounts of gamma-globulin fraction in the aliquot that reacted with antigen ovalbumin

Gel filtration and proteins are separated by molecular weight

Gamma globulins are characterized by

Appox. 150,000 daltons

Molecular weight of IgG

Meracaptoethanol, which reduces disulfide bonds

To determine if IgG was composed of polypeptide subunits it was subjected to

50 Kd heavy chains and 25 Kd light chains - to account for the naive molecular weight of IgG it was determined that there must be 2 of each peptide

2 protein peaks revealed by gel filtration after reduction and denaturation

The structure of immunoglobulins

Edelman and Porter experiments deciphered

Edelman and Porter experiments explained

Elucidated IgG strutures using enzyme (Papain) digestion to produce 2 identical 50 Kd fragments with antigen-binding activity and 1 50 Kd fraction without antigen binding activity

Fab

Meaning fragment antigen binding, were fragments with antigen binding activity

Fc

Meaning fragment crystallizable, were the elucidated single 50 Kd fragment that was observed to crystallize in cold storage

Pepsin, into a single 100 Kd fragment with 2 antigen binding sites (F(ab)2)

What enzyme digested the Fab fragment and what was it digested into

Pepsin, into multiple small fragments

What enzyme digested the Fab fragment and what was it digested into

Reacted with heavy and light chain antibodies indicating that the Ag binding site was composed of 1 heavy and 1 light chain

When antibodies were raised against heavy and light chains and tested against Fab and Fc fragments, Fab:

Reacted with the heavy chain antibodies only indicating antigen binding site was composed only of heavy chains

When antibodies were raised against heavy and light chains and tested against Fab and Fc fragments, Fc:

Amino acid sequencing

Critical to understanding the nature of the antigen binding site of immunoglobulins

Due to the heterogeneity of antibodies in serum (the antiserum) because amino acids have a variety of molecular masses and reflect antibody specificities to immunoglobulin array of different antigens

Why is amino acid sequencing difficult?

A pure single species of immunoglobulin molecules

What is needed to elucidate the antigen binding site of the antibody?

Patients with multiple myeloma

Who was the pure source of immunoglobulin is obtained from?

MM patients have a massive quantity of 1 type (type varies with individual patients) of immunoglobulin in their serum

Why were multiple myeloma patients used to obtain a pure source of immunoglobulins

Intact light chains (Bence-Jones protein) in their urine and by heating urine the light chain is precipitated resulting in the pure protein that could be sequence

MM patients excrete

Amino terminal portion of a sequence was variable & the carboxyl-terminal portion was conserved in a sequence

Light chain AA sequencing revealed that

2 types: kappa and lambda

Conserved sequences present in carboxyl-terminus of light chain

Variable domain (VL)

Amino terminal half of light chain

Constant domain (CL)

Carboxyl terminal half of light chain

Variability of AA sequences in positions 1-117 & the rest of the sequence was constant

Heavy chain sequence analysis of Ig from MM patients showed

Mu, Delta, Gamma, Epsilon, Alpha

5 types of heavy chain sequencing patterns (isotypes that determine antibody class

IgM, IgD, IgD, IgE, IgA

Isotype antibodies of Mu, Delta, Gamma, Epsilon, Alpha

IgG, IgA, IgD

Isotypes that have hinge region between CH1 and CH2 domains - 330 AAs in constant region

Proline, cysteine

Hinge region is rich in ______, ______ which allows the antigen binding site to be flexible

2 Fab arms (connected by disulfide bonds) to bind at various different angles

Hinge’s flexibility allows

IgM, IgE

Isotypes that do not have hinge region - 440 AAs in the constant region

Subclass IgA1, subclass IgA2

IgA is further classified into 2 sub-isotypes due to minor variations in alpha regions

Subclass IgG1, subclass IgG2, subclass IgG3, subclass IgG4

IgG is further classified into 4 sub-isotypes due to minor variations in gamma regions

Papain and pepsin cleavages

Hinge region exist between

Domains

Distinct functional and structural units in an immunoglobulin; Conserved molecular structure within a protein that confer a unique motif

IgG domain

In immunoglobulin molecules light and heavy chains contain a homologous unit of 110 AA with an internal disulfide bond

1 variable domain (VL) & 1 constant domain (CL)

How man variable domain(s) and constant domain(s) does a light chain have?

1 variable domain (VH) and 3-4 constant domains (CH)

How man variable domain(s) and constant domain(s) does a heavy chain have?

Ig superfamily

Many immune system proteins contain Ig domains which classifies them as part of the

Complementary determining regions (CDR) aka hypervariable regions

Variable regions that contain most of the sequence differences in antibodies that is confined to 3 short regions of the heavy & light chains

All 3 CDRs

Which complementary determining regions are involved in epitope binding (exemplified by Vitamin K)

Paratope

Binding site present in the antibody

Opsonization, triggers mast cell degranulation, activates B-lymphocytes, complement activation, antibody-dependent cell-mediated cytoxicity (ADCC), or transcytosis

Once antibody binds to antigen, the Fc region undergoes conformational changes causing different effector functions including

Opsonization

The process by which particulate antigens are rendered susceptible to phagocytosis & leads to enhanced phagocytosis

Complement system

Collection of serum glycoproteins in a cascade mechanism that destroy pathogens & remove antigen when activated

Antibody-dependent cell mediated cytotoxicity (ADCC)

A cell-mediated reaction in which non-specific cytotoxic cells (like neutrophils, natural killer cells, eosinophils, & macrophages recognize bound antibodies on a target cell and destroy the target cell

Dumping toxic substances (like lytic enzymes, preforin, granzyme, and TNF) into the cell

ADCC - once Fc receptor binds to Fc portion of the target-bound antibody the cell kills that target cell by ____ which occurs because of the engagement of the Fc region on the antibody that is bound to the target and Fc receptor of the cells

Transicytosis

Transfer of immunoglobulins across an epithelial layer

Passive immunization

Transfer of IgG from mother to fetus (fetus acquires a repertoire of pre-formed antibodies from the mother that protects the fetus from pathogens)

IgG

Most abundant class of Ig that makes up approx. 80% of the total immunoglobulins

Size of the hinge region and the number and position of the interchain disulfide bonds between the heavy chains

4 sub-types of IgG are distinguished from each other based on

IgG1, IgG3, IgG3

Subtypes of IgG that cross the placenta and protect the developing fetus

IgG3

IgG that is a potent complement activator and binds with high affinity to Fc recptor

IgG2

IgG that binds with low affinity to Fc receptor & a less efficient as a potent complement activator

IgG1

IgG that is less efficient as a potent complement activator & binds with high affinity to Fc

IgG4

IgG that binds with intermediate affinity to Fc receptor but not to a complement activator

IgM

The 1st Ig synthesized during the primary immune response and the 1st Ig to be synthesized in newborns (allows newborns to mount a primary immune response when exposed to antigens for the 1st time)

IgM

Ig that is very efficient in binding complex antigens like virus particles (bc it has 10 binding sites) and in activating complement

Monomeric IgM

Membrane bound form of IgM that acts as receptors on B-cells

Pentameric IgM

Secreted form of IgM that has disulfide bonds liking CH3 and CH4 domains of adjacent molecules & has a J-peptide that helps to stabilize it

J-chain

What helps to stabilize the IgM pentameter and allows IgM to be transported across the epithelium

IgA

Ig that is the predominant immunoglobulin in external secretions (like breast milk, saliva, tears, mucus, etc.)

Secretory IgA

Consists of dimers or tetramers together with a J -chain and a secretory component

Secretory IgA and pentameric IgM

What Igs have J-chains

Seceratory component

Peptide chain that functions to protect the IgA from proteases (protease digestion) present in mucosal surface

Secretory component

Fc region of dimeric IgA binds to poly-Ig receptor on the basolateral side of epithelial cells & the bound dimeric IgA with poly-Ig receptor is transported to luminal surface of epithelial cells where poly-Ig is cleaved to form

IgE

Ig responsible for immediate hypersensitivity reactions (like hay fever, asthma, hives, anaphylactic shock)

Degranulation

IgE binds to Fc receptors on basophils and mast cells, and this interaction induces

Granules

What have pharmacologically active mediators that are responsible for allergy symptoms

P-K reaction (Prausnitz-Kustner)

Immunologic tests formally used to determine if a patient has an allergic reaction, has been replaced with skin allergy/skin prick test (SPT)

IgD

Ig that is a major membrane-bound Ig expressed in mature B-cells, but doesn’t otherwise have a biological function that has been identified

IgG3, IgM, IgG1

Ig that activates the classical complement pathway

IgM, IgD

Igs present on the membranes of mature B-cells

IgG1, IgG3, IgG4

Igs that bind Fc receptors of phagocytes

IgA1, IgA2, IgM

Igs involved in mucosal transport

Isotype

Constant region determinants that define each heavy chain class and sub-class & each light chain type & sub-type

Allotypic determinants

Minor variations in amino acid difference in conserved portion of Ig molecules

Allotype

Allotypic determinants expressed by the Ig

Idiotope

An antigenic determinant of the variable region

Idiotype

Antibody molecules that share an idiotope belong to the same

B-cell receptor

Comprises a membrane bound Ig molecule and its 2 associated signal-transducing molecules (Ig alpha-beta heterodimer)