antibody struc

8/9/2019 Antibody Struc

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Antibody structureOverview

1. Antibodies belong to a class of proteins called

immunoglobulins

2. Antibody molecules belong to one of five classesi.e. IgG, IgM, IgA, IgD & IgE

3. Immunoglobulins are Y shaped proteins. The

arms of the Y bind antigens. The tail of the

Y is responsible for biological activity eg. Cactivity or binding to cells

4. Ability of immunoglobulins to bind antigen determ.

by AA sequence in variable region


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General Characteristics of

antibody

Antigen-specific products of B cells

First of the molecules participating in immune response

to be characterized, best understood

Basic building block, immunoglobulin domain, isused in molecules of both immune system & other

biological recognition systems

Antibody molecule has 2 separable functions:1) specific binding to antigen eliciting response

2) biological activity - recruit cells & components

designed to destroy agent to which ab developed


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Serum and plasma

Blood

Blood +

anticoagulant Cells

Plasma

Serum

Clot


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Isolation & characterizationChromatography

Molecular sieving

Ion exhange

Affinity

Salting out/ Dehydration

Ammonium sulfate

Alcohol precipitation

Ultracentrifugation

Immunochemical


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Chromatography

Molecular sieving

+ +- ++

++ +

-Ion exchange

Affinity


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Salting outAmmonium sulfate (half saturated)

Ethanol (90%)


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Density gradient

ultracentrifugation

Centri

fuga

tion

60,000

-100,00X

G

Displacement = Svedberg


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Kabat & Tiselius (1939)

Albumin

+ -

Globulins

-Discovered that hyperimmunizing rabbits resulted in increased -Purification revealed antibody activity resided in this serum portion


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Electrophoretic analysis of

serum

+ -

Sample application

Anode Cathode

+ -

Separation by charge

Albumin

+ -

Globulins


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Characterizing chainsAlbumin

1 2 + -P

rotein

conce

ntra

tion

Broad peak of = heterogenous proteins

multiple myeloma

+ -Pro

tein

concentra

tion

normal Need pure Ig to chemically analyze

Narrow peak of = homogenous proteins

Bence-Jones proteins in urine of multiplemyeloma pts. = dimers of immunoglobulin

light chains, or


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Porter (England)

SSS S

SS

Fab Fab

Fc

Treatment with proteolytic enzyme,papain,

resulted in three approximately equal sized fragments:

2 capable of ag rx (fragment antigen binding)

1 could be crystallized (fragment- crystallizable)

Fab specifically bind antigen, univalent (cant ppt)

one binding site each, identical to each other

Fc crystallizable (thus homogenous) cant bind ag

responsible for biological activity of molecule after

ag bound to Fab portions


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Eddleman (USA)

SH HSSHHSHS SH

Treatment with mercaptoethanol = 4 chains:

(mercaptoethanol breaks S-S bonds)

2 chains = 53,000 daltons

2 chains = 22,000 daltons

All immunoglobulins basic unit consisting of 4 polypeptides i.e.

2 H, 2 L


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Proteolytic enzyme digestion

reveals N- & C-terminal

Fc

SSSS S S

Papain digestion results

in cleavage @ N terminusin proline hinge region @

disulfide bridge

SSS SSS

Fab FabN-terminus

Pepsin digestion results

in cleavage @ C terminal

portion, resulting in a divalent

fragment (Fab2) joins by S-S

& several Fc fragments

Fab2

C-terminus


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Cleavage of IgSH HSSH

HS

HS

SH

2 Fragment ag binding

1 Fragment crystallizable

1 Fab2

Several small pieces

Fc

Pepsin

Papain

Reduce

dwME

SSSS S S

SSS SSS

Fab Fab

4polypeptide chains


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Light chain sequences

SSS SSS

Fab FabN-terminus

C terminus

N-terminus

C terminus

214 AA in

two domains

Analysis from

several myelomas

Identical

sequences

in C-termin.

domain

Different

sequences

in N-termin.domain

Variable (VL

)

Constant (CL)


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Heavy-chain sequence

SSS SSS

Fab FabN-terminus

C terminus

Heavy chain consists of 445 AA

in 4 domains

Flexible

Hinge

Region

N-terminal domain varies

(HV)

Other 3 domains constant

(HC)


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Sequence variability not distributed

evenly in variable regionVariation restricted to 3 regions

Hypervariable regions (HV1,HV2,HV3)

Location of antigen binding siteAA sequence det. shape of ag binding site

(paratope)

Determine epitopes to which Ig binds =

complementary- determining regions (CDR)

Framework regions = regions where AA seq.

is relatively constant (FR)

CDR = 6-10 AACDR 1 = 24 - 34

CDR 2 = 50 - 56

CDR 3 = 89 -97

Wu & Kabat plot

Heavy chain CDR

CDR 1 = 31-35

CDR 2 = 50-65

CDR 3 = 95-102

CDR 1

CDR 2

CDR 3

%

var

%var

St t f i bl &


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Structure of variable &

constant domains (X-ray

crystallography)Two layers, linked by

disulfide bonds

Each layer formedseveral stretches

conformation = stranLayers = sheet

Order of strandsis characteristic for

each sheet

3 D structure = Ig fold

Light chain

V domain

Light chain

C domain

N terminus

C terminus

strands strands

Disulfide bonds

CDR 1

CDR 2

CDR 3


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Opening to reveal strandscomprising each sheet

V domainC domain

Principal difference between C & V domains

V domain has 2 more strains forming extraloop

unique strands

strands lettered sequentially according to occurrence in AA sequence in dom.

Order & orientation

characteristic for each

domain


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Location of CDR & FR in Ig L

& H chains

FR1 FR2 FR3 FR4

FR1 FR2 FR3 FR4

CL

CH1 H

1 2 3

1 2 3

CDR

CDR


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L & H chain folding to yield 3

CDR in each chain to form

walls of ag binding groove


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Immunoglobulin consists of 4

polypeptide chains

C terminus

Constant

region

N terminus

Variable regions

Disulfide

bonds

Ag binding site Ag binding site

VHVL

CH1

CL

CH2

CH3 Light chain

Heavy chain

EM Rabbit Ig

X 2,042,500

I l i l i f


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Immunologic analysis of

immunoglobulins

Ig (like most other proteins) stimulate ab in otheranimal species

ll species have two major classes of L chains i.e. ,individual of species produces both types; ratio of

: varies by species (e.g. mouse 95% ; human60% )

in any Ig molecule, both L chains = either or ,never one of each

Ig of all species consist of 5 classes (isotypes) differ instructure of H chains

H chains among isotypes differ serologically, CHO

content, size & biological function


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H chains

Immunoglobulin isotype Heavy chain

IgM IgG IgA IgD IgE

Individual of species produces all H chains, in proportion

characteristic for species, but ab molecule H chains are

identical ( i.e., 2 , 2 etc.)

H chain confers unique biologic properties of molecule e.g.

1/2 life, receptor binding , enzyme-, C activation with ag


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Isotypic (Class) structure of Ig

gG isotype = 2 2 or 2 2 , but not 1 1 1 1IgE isotype = 2 2 or 2 2

.etc...

L H L H

L HL H


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H chains

Immunoglobulin isotype Heavy chain

IgM IgG IgA IgD IgE

Individual of species produces all H chains, in proportion

characteristic for species, but ab molecule H chains are

identical ( i.e., 2 , 2 etc.)

H chain confers unique biologic properties of molecule e.g.

1/2 life, receptor binding , enzyme-, C activation with ag


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Immunoglobulin consists of 4

polypeptide chains

C terminus

Constant

region

N terminus

Variable regions

Disulfide

bonds

Ag binding site Ag binding site

VH VL

CH1

CL

CH2

CH3 Light chain

Heavy chain

EM Rabbit Ig

X 2,042,500


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Isotypic (Class) structure of Ig

gG isotype = 2 2 or 2 2 , but not 1 1 1 1IgE isotype = 2 2 or 2 2

.etc...

L H L H

L HL H


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Hinge region

Angle = 0o

Angle = 600Angle = 900

Consists of approx 12 AA between CH1 & CH2

No homology between hinge & other Heavy chain domains

AA sequence unique for each class& subclass


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AA sequence in hinge region

Cys-Asp-Lys-Thr-His-Thr-Cys-Pro-Pro-Cys -Pro-Ala-Pro-Glu--Arg-Val-Glu-Pro-Lys-Ser-

Light Chain

Cys

Light Chain

Cys

Papain

Fab region Fc region

Cys-Asp-Lys-Thr-His-Thr-Cys-Pro-Pro-Cys -Pro-Ala-Pro-Glu--Arg-Val-Glu-Pro-Lys-Ser-

Hinge region


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Characteristics of hinge regionImmunoglobulins (with possible exception of IgM & IgE) contain hinge between

CH1 & CH2

No homology between AA sequence of hinge & heavy chains

AA sequence differs with different classes

Comprised of many cysteine andproline residues

Cysteine involved in formation of interchain disulfide bonds

Proline prevents folding in a globular structure, allowing flexibility between

two Fab arms of the Y-shaped antibody; allows open & close to accommodate

binding to two epitopes; because it is open, it can be cleaved by proteases

(e.g. papain) to generate the Fab & Fc fragments


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IgG

IgG1 IgG2

I G3 I G4

Subclasses show close overall relation

Heavy chains = 1, 2, 3,

Highest concentration in serum

Plays major role in immune dfns.

MW approx. 150 kDa

Small size ppt in surfaces

(e.g. cross placental barrier)

Opsinize, aggl. & ppt

agOnly activates classical C pway

All normal indiv. have all

IgG1>IgG2>IgG3>IgG4

IgG3 has shortest 1/2 life,

highest catabolic rate,

highest # S-SIgG4 = monoval. no aggl

ppt rx., autoab to clot fac

Autoab to DNA = IgG1,IgG


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Structural features of IgG

HV

Constant

Variable

Two H chains, Two L chains(either or but not both)Each H chain = 50 kD

Each L chain = 25 kD

150 kD, 7S, globulinLeast anodic of all serum proteins

+ -

Anode Cathode


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Structural features of IgMPentamer (5)

First Ig produced following immun.

Macroglobulin (M)

900 kD, 19S

SSS S

SS

SS

SS

SS

SS

SS

S

S

SS

S

S

S

S

SS

SS

SS

J chain Doesnt have hinge region

has additional H domain

Has a J chain (one of 2 Ig isotypes)

15 kD


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Structural features of IgA

S

S

SS

S

S

SS

SS

S

S

Secretory

piece

J chain

Principal Ig in external secretions e.g.saliva, mucus,sweat, gastric fluid & tears

Major Ig of colostrum & milk, provides

neonate with major source of intestinal

protection against pathogens

165 kD, 7S, migrates as fast Plasma cell forms basic IgA molecule with

J chain which form dimers (second Ig to

contain J chain)

When released from plasma cell, bind to

basal membranes of epithelia via

secretory pieceIn serum, primarily a monomer, no

secretory piece

Secretory component protects IgA from

proteolytic digestion


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Structural features of IgESometimes called reaginic ag

Mediates allergies (Type Ihypersensitivies)

190 kDa, 8S, migrates as fast Contains an extra domain (CH4)

which binds to mast cells

& basophils

May remain attached for long time

when ag reappears, cross links

IgE on mast cell surface, release

mast-cell granules & signs of

anaphylaxis


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antibody struc

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