October 5, 2004. IMMUNITY ADAPTIVEINNATE CELL MEDIATEDHUMORAL.

October 5, 2004

IMMUNITY

ADAPTIVE INNATE

CELL MEDIATED HUMORAL

IMMUNITY

ADAPTIVE INNATE

CELL MEDIATED HUMORAL

ANTIBODIESStructureSequence diversityNature of reaction with AgDifferent functional properties

If bacteria are injected into blood specific substances are found that exhibit different biologic properties

Agglutinin - specifically clump only the bacteria used for immunization

Opsonin - facilitate engulfment

Antitoxin - neutralize toxin associated with the immunizing bacteriaCytolysis - lyse bacteria

Precipitins - form flocculate precipitates when added to supernatant from the bacteria

A n t i b o d y ( A b )

A m o l e c u l e p r o d u c e d b y a n i m a l i n r e s p o n s e t o

f o r e i g n s u b s t a n c e ( A g ) w h i c h h a s t h e p r o p e r t y o f

b i n d i n g t h e e l i c i t i n g a g e n t

I m m u n o g l o b u l i n ( I g )

A n t i b o d y ( A b )

A m o l e c u l e p r o d u c e d b y a n i m a l i n r e s p o n s e t o

f o r e i g n s u b s t a n c e ( A g ) w h i c h h a s t h e p r o p e r t y o f

b i n d i n g t h e e l i c i t i n g a g e n t

I m m u n o g l o b u l i n ( I g )

Antibodies are found both as cell-associated receptors (BCR) and as circulating effector

molecules

A n t i g e n ( A g )

A n y m o l e c u l e c a p a b l e o f b e i n g b o u n d b y t h e

c o m b i n i n g s i t e o f a n A b o r t h e T - c e l l r e c e p t o r

I m m u n o g e n

A s u b s t a n c e c a p a b l e o f e l i c i t i n g a n i m m u n e

r e s p o n s e ; a l l i m m u n o g e n s a r e a n t i g e n s b u t n o t a l l

a n t i g e n s a r e i m m u n o g e n s .

We will now consider the experiments that were done to determine antibody atructure

albumin

αβ

γ

Electrophoretic migration

Absorbance

Immune serum from rabbits before and after the antibodies were removed by precipitation with Ag(ovalbumin)

albumin

αβ

γ


Absorbance

The antibodies migrated as a γ-globulinShowed they w

g a m m a g l o b u l i n

When purified antibodies (produced by dissolving immunoprecipiates) were analyzed in an ultracentrifuge they ran with a 7S sedimentation co-efficient indicating a m.w. of approx. 150,000

gamma globulin

7S = 150,000 molecular weight

gamma globulin


Molecular analysis of

precipitates showed

the valence = 2

Two molecules of Ag were precipitated by each Ab

gamma globulin


valence = 2

Digestion of antibodies with the enzyme papain yielded two fragments, the Fab (fragment antigen binding), and the Fc (fragment crystallizable)

Ratio: 2 Fab and 1 Fc

gamma globulin


valence = 2

papain --> 2Fab + 1Fc

Fab binds Ag but no precipitate

Fc forms crystals

MW of Fab and Fc = 50,000

1 ( F a b ' )

2

o f 1 0 0 , 0 0 0

b i n d s 2 m o l e c u l e s o f A g

c a n p r e c i p i t a t e

( F a b ' )

2 p e p s i n

b r e a k S S - - > 2 F a b

gamma globulin


valence = 2


pepsin--> 1 (Fab')

2

of 100,000

binds 2 molecules of Ag

can precipitate

(Fab')

2 break SS-->2Fab

If break disulfide bonds and separate on the basis of size you get two products

gamma globulin


valence = 2


Ab--> cleave disulfides-->

2H(50000) + 2L(25000)

Anti-H reacts with Fab and Fc

Anti-L reacts only with Fab

Anti-Fab reacts with H and L

Antiserum

pepsin--> 1 (Fab')

2

of 100,000

gamma globulin


valence = 2



2H(50000) + 2L(25000)




Antiserum

pepsin--> 1 (Fab')

2

of 100,000

gamma globulin


valence = 2



2H(50000) + 2L(25000)




Antiserum

pepsin--> 1 (Fab')

2

of 100,000

gamma globulin


valence = 2



2H(50000) + 2L(25000)




Antiserum

pepsin--> 1 (Fab')

2

of 100,000

Papain

DigestionPepsin

Disulfide

Reduction

L

H

Fc

Fab Fab F(ab')

2

+

Small Fragments

L chain

H chain

NH

3

+

COO

-

NH

3

+

gamma globulin


valence = 2



2H(50000) + 2L(25000)




Antiserum

pepsin--> 1 (Fab')

2

of 100,000

Papain

DigestionPepsin

Disulfide

Reduction

L

H

Fc

Fab Fab F(ab')

2

+

Small Fragments

L chain

H chain

NH

3

+

COO

-

NH

3

+

This is the structure of the fundamental building block of an Ab molecule. Some antibodies (such as IgG) are made of one of these building blocks. Others (IgA and IgM) are made of multiple copies of this basic building block.

albumin

αβ

γ


Absorbance

One problem with further analysis was the heterogeneity of antibodies: note the narrow peak of the homogenous protein albumin compared to the broad peak of the Ig

It was a major breakthrough when it was realized that multiple myeloma is a tumor of plasma cells generally synthesizing a single species of Ig and that the myeloma proteins are homogeneous Abs produced by the plasma cells

Bence-Jones proteins are monoclonal (homogeneous) L chains in patients with multiple myeloma

How are antibodies capable of

binding many different

antigens?

Light chains are of two types:

κ λ

anti - κ

anti - λ

++++

++++

-

-


κ λ

anti - κ

anti - λ

++++

++++

-

-

Take two kappa (or lambda) lights chains from two different patients with multiple myeloma and produce proteolytic fragments


κ λ

anti - κ

anti - λ

++++

++++

-

-

Take two kappa (or lambda) lights chains from two different patients with multiple myeloma, produce proteolytic fragments.

Analyze the products by 2-D peptide map (chromatography in one dimension, electrophoresis in the other).

You find that about half of the cleavage products are identical between the two chains while the other half differ.

electrophoresis-->

chromatography-->

The shaded spots indicate peptides shared by the two κ or λ chains. Note the difference between κ and λ.The conclusion is that the lights chains have a portion that is variable and a portion that is constant.

electrophoresis-->

chromatography-->

V

L

C

L

V

H

C

H

( κ or λ )

( )many different options

Amino acid sequence analysis shows that the variable portions is at one end of the light (and heavy) chains

V

L

C

L

V

H

C

H

( κ or λ )

( )many different options

More extensive analysis of the sequences of variable regions shows that they in fact contain relatively conserved regions and other regionswhich are hypervariable

More extensive analysis of the sequences of variable regions shows that they in fact contain relatively conserved regions and other regionswhich are hypervariable.

There are 20 different amino acids.If all are found with equal frequencyvariability is 20/.05 = 400.If only one amino acid is found thevariability is 1/1 =1/

There are 20 different amino acids.If all are found with equal frequencyvariability is 20/.05 = 400.If only one amino acid is found thevariability is 1/1 =1/

Hypervariable regions are also called complementarity determining regions (CDRs). The prediction is that the

CDRs contact antigen.

Hypervariable regions are also called complementarity determining regions (CDRs). The prediction is that the

CDRs contact antigen.

X-crystallography shows that the Ab molecule folds into compact domains. The CDRs are loops extending from the variable regions so that they are easily accessible for interaction with Ag. The other amino acids in the variable region are the “framework” amino acids and providea scaffold to maintain the CDRs in the proper orientation.

Light chains have two domains: one variable and one constantHeavy chains have four or five domains, one of which is variable with the remains constantAll heavy chains have at least one carbohydrate moiety attached

The forces are weak and operate at short distances

Therefore the closer the approach (the better the fit) the stronger the interaction

A complex of an Ab and a

protein Ag shows:

the reaction occurs between two

interacting surfaces

the better the fit, the stronger the interaction

most but not all of the interaction with Ag

occurs through the CDRs

on a protein Ag the amino acids compri s ing

the e pi topes are adjacent in 3D space but not

necessarily in linear sequence

Abs make contact with a protein Ag across a large planar face

usually 15-22 amino acids on Ag contact a similar number on the Ab giving a complementary surface of 650-900 A2

epitopes are exposed on the surfacemost Abs are elicited by proteins in their native statenote that Abs are designed to react with Ags in solution; the TCR reacts with Ags bound to cell surfaces

Another characteristic of the antibody molecule is a flexible hinge

D N P l i g a n d

When a divalent hapten reacts with Abs, trimers, tetramers and other large complexes form. Because of the flexibility of the hinge region the angle between the arms of the Abs vary.

D N P l i g a n d

Digestion with papain removes the Fc

Classes Heavy Chain Subclasses (human)

IgM μ

IgE ε

IgD δ

IgG γ 1, 2, 3, 4IgG IgG IgG IgG

IgA α 1, 2IgA IgA

+ = Classes subclasses isotypes

: Light chains have two isotypes κ and λ

Each isotype is encoded by a separate gene

There are several different possible structure of the constant regions of the heavy chains

IgMH chain has four constant region domains and no hinge

In addition to H and L also contains J chain

(H2L2)5J or (H2L2)6

Usually the first Ig made in response to antigen

Increased valence leads to increased avidity

IgG

IgGIgG

Most abundant serum immunoglobulin

J

SC

Monomer Dimeric IgA Secretory IgA

IgA

Present in secretions where it provides protection at epithelial surfaces

Frequently found as a polymer with J chain

In secretions it also has a fourth chain, secretory component, a product of the epithelial cells

Submucosa

Plasma

cell

Dimeric

IgA

IgA

receptor

Lumen

Epithelial

cells

Vesicle Enzymatic

cleavage

Secretory

component

IgE

Present at low concentrations

Responsible for allergy

Binds to receptors on mast cells

IgD

Principle role seems to be as a membrane Ig

All isotypes can exist as both secreted

and membrane bound antibodies.

The two forms differ in their carboxy

terminal sequence.

Secreted antibodies have a hydrophilic

terminus.

Membrane antibodies have a hydrophobic

sequence which inserts into the plasma

membrane and a short cytoplasmic tail.

All isotypes can exist as both secreted and membrane bound antibodies

October 5, 2004. IMMUNITY ADAPTIVEINNATE CELL MEDIATEDHUMORAL.

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Transcript of October 5, 2004. IMMUNITY ADAPTIVEINNATE CELL MEDIATEDHUMORAL.