Organization And Expression Of Immunoglobulin Genes

Structural Biology and Functions of Immunoglobulin's

Organization and Expression of Immunoglobulin Genes Kasi.M.Kannan Lecturer in Microbiology V.H.N.S.N.College Virudhunagar

Antibodies: Structure and Function

• Antibodies are the antigen binding proteins present on the B-cell membrane and secreted by plasma cells. • Membrane-bound antibody confers antigenic specificity on B cells; antigen-specific proliferation of B-cell clones is elicted by the interaction of membrane antibody with antigen. • Secreted antibodies circulate in the blood, where they serve as the effectors of humoral immunity by searching out and neutralizing antigens or marking them for elimination. • All antibodies share structural features, bind to antigen, and participate in a limited

Basic Structure of Antibodies • The first evidence that antibodies were contained in particular serum protein fractions came from a classic experiment by A. Tiselius and E. A.Kabat, in 1939. • They immunized rabbits with the protein ovalbumin (the albumin of egg whites) and then divided the immunized rabbits’ serum into two aliquots. • Electrophoresis of one serum aliquot revealed four peaks corresponding to albumin and the

• The other serum aliquot was reacted with ovalbumin, and the precipitate that formed was removed; the remaining serum proteins, which did not react with the antigen, were then electrophoresed. • A comparison of the electrophoretic profiles of these two serum aliquots revealed that there was a significant drop in the γ –globulin peak in the aliquot that had been reacted with antigen • Thus, the globulin fraction was identified as containing serum antibodies, which were called immunoglobulins, to distinguish them from any other proteins that might be contained in the γ -globulin fraction.

• Experimental demonstration that most antibodies are in the γ -globulin fraction of serum proteins. • After rabbits were immunized with ovalbumin (OVA), their antisera were pooled and electrophoresed, which separated the serum proteins according to their electric charge and mass. • The blue line shows the electrophoretic pattern of untreated antiserum. The black line shows the pattern of antiserum that was incubated with OVA to remove anti-OVA antibody and then electrophoresed

Structure of Immunoglobins • IgG Y shaped • Antigen binding site located on tip of the Y arms • Fab arms connected to Fc stem domain via a flexible hinge • Two identical H chains and L chains • Each chain has a N-terminal VH and VL domain that together form the antigen binding site • Covalent disulfide bridges between H and H-L

Structure of antibody

Antibody Structure

Antibody Structure

•

Constant and Variable regions of C Antibodies terminus of both H and L chains • invariant and defined as C (constant) region. • Length ~ 330 amino acids in H chains and ~ 110 amino acids in L chains

• N terminal segment • Substantially different in different antibodies, thus named V (variable) region • V region ~ 110 amino acids in length • Contains three subregions that show maximal variation between different antibodies • Defined as hypervariable regions • Designated as complementarity determining regions (CDRs)

The Immunoglobin Fold

Immunoglobin Fold • V and C domains share the basic Ig fold • Differences domains

between

the

two

• C domain is built of seven β -strands arranged so that four strands form one sheet and three strands form a second sheet. • The strands are closely packed together and joined by a single disulphide bond • Most of the invariant residues of the constant domain are in the sheets • Overall structure of the V domain

Variable and Constant Folds

Domain Structure of Immunoglobulin's Domains are folded, compact, protease resistant structures Fab

Fc S S

C

L

VL

Light chain C domains

C

H

3

S S S S

CH2

Heavy chain C domains

CH1 S

VH

S

F(ab)2 Pepsin cleavage sites Papain cleavage sites

- 1 x (Fab)2 & 1 x Fc - 2 x Fab 1 x Fc

CH3

CH2

CH3

CH1

CH2

CH3

VH1 CH1

CH2

CH3

VH1 CH1 VL

CH2

CH3

VH1 CH1 VL

CH2

CH3

CL

VH1 CH1 VL

CH2

CH3

CL

VH1 CH1 CL

VL

CH2 Elbow Hinge

CH3

Fv

Fv

Fv

Flexibility and motion of immunoglobuli ns

Fb

Fb

Elbow

Fb

CH2 3

C

H

C

H

3

CH2

Hinge

Fv

Fv

Fv VH1 CH1

Fb VL

CL

Fab CH2 Elbow Hinge Fc

Carbohydrate CH3

The Immunoglobulin Fold The characteristic structural motif of all Ig domains A β barrel of 7 (CL) or 8 (VL) polypeptide strands connected by loops and arranged to enclose a hydrophobic interior

Single VL domain

A barrel made of a sheet of staves arranged in a folded over sheet

Barrel under construction

The Immunoglobulin Fold

COOH

S S

NH2

Unfolded VL region showing 8 antiparallelβ -pleated sheets connected by loops.

mmunoglobulins are Bifunctional Proteins •Immunoglobulins must interact with a finite number of specialised molecules Easily explained by a common Fc region irrespective of specificity

•- whilst simultaneously recognising an infinite array of antigenic determinants.

In immunoglobulins, what is the structural basis for the infinite diversity needed to match the antigenic universe?

ariability of amino acids in related proteins Wu & Kabat 1970 100

Variability

80

Cytochromes C

60 40 20 20

40

60

80

100

120

Amino acid No. 100

Variability

Human Ig heavy chains

80 60

40 20 20

40

60

80

100

120

Amino acid No.

Framework and Hypervariable regions •Distinct regions of high variability and conservation led to the concept of a FRAMEWORK (FR), on which hyper variable regions were suspended. •Most hypervariable regions coincided with antigen contact points - the COMPLEMENTARITY DETERMINING REGIONS (CDRs)

FR1

CDR1 FR2CDR2

FR3

CDR3

FR4

100

Variability

80 60

40 20 20

40

60

80

100

120

Amino acid No.

Hypervariable CDRs are located on loops at the end of the Fv regions

Hypervariable regions

Space-filling model of (Fab)2, viewed from above, illustrating the surface location of CDR loops

Light chains brown Heavy chains

Green and Cyan and blue

Hypervariable loops and framework: Summary •The framework supports the hyper variable loops it forms a compact β barrel/sandwich with a hydrophobic core •The hypervariable loops join, and are more flexible than, the β strands •The sequences of the hypervariable loops are highly variable amongst antibodies of different specificities •The variable sequences of the hypervariable loops influences the shape, hydrophobicity and charge at the tip of the antibody

Antigens vary in size and complexity

Protein: Influenza haemagglutinin

Hapten: 5-(para-nitrophenyl phosphonate)-pentanoic acid.

Antibodies interact with antigens in a variety of ways Antigen inserts into a pocket in the antibody

Antigen interacts with an extended antibody surface or a groove in the antibody surface

he Complementarity Determining Region • Six loops of the VH (H1, H2 and H3) and VL (L1, L2 and L3) domains create a great variety of surfaces • Deep binding cavities: such as those seen in some antibody-hapten complexes • Wide pockets : seen in certain antibody-peptide complexes • Flat surfaces : seen in antibody-protein interactions • H3 is the most variable of the loops and in all crystallographically solved antibody-antigen

What Do Antibodies Recognize?

• • • • •

Proteins (conformational determinants, denatured or proteolyzed determinants) Nucleic acids Polysaccharides Some lipids Small chemicals (haptens)

Antigen:Antibody complex • Antibodies bind to antigens by recognizing a large surface, and through surface complementarity. • Thus, these complexes have a very high affinity for each other.

Ig gene Super family - IgSF • The genes encoding Ig domains are not restricted to Ig genes. • Although first discovered in immunoglobulins, they are found in a superfamily of related genes, particularly those encoding proteins crucial to cellcell interactions and molecular recognition systems. • IgSF molecules are found in most cell

• Large numbers of membrane proteins have been shown to possess one or more regions homologous to an Ig domain. • Each of these membrane proteins is classified as a member of the immunoglobulin superfamily. • The term superfamily is used to denote proteins whose corresponding genes derived from a common primordial gene encoding the basic domain structure. • These genes have evolved independently and do not share genetic linkage or function. • The following proteins, in addition to the Ig themselves, are representative members of the immunoglobulin superfamily

• • • • • • • •

Ig-/Ig- heterodimer, part of the B-cell receptor Poly-Ig receptor, which contributes the secretory component to secretory IgA and IgM T-cell receptor T-cell accessory proteins, including CD2, CD4, CD8, CD28, and the , , and chains of CD3 Class I and class II MHC molecules Beta 2-microglobulin, an invariant protein associated with class I MHC molecules Various cell-adhesion molecules, including VCAM-1, ICAM-1, ICAM-2, and LFA-3 Platelet-derived growth factor Numerous other proteins, also belong to the immunoglobulin superfamily.

Expression of Immunoglobulin Genes

One of the most remarkable features of the vertebrate immune system is its ability to respond to an apparently limitless array of foreign antigens. As immunoglobulin (Ig) sequence data accumulated, virtually every antibody molecule studied was found to contain a unique amino acid sequence in its variable region but only one of a limited number of invariant sequences in its constant region. The genetic basis for this combination of constancy and tremendous variation in a single protein molecule lies in the organization of the immunoglobulin genes.

Generation of antibody diversity:

at gene, antibody, cell and organism level

Heavy chain variable region has 3 segments: VH, DH, JH Light chain variable region has 2 segments: VH, JH

1. Combinatorial joining – Utilize any of the multiple gene segments – Most 3’ is used most frequently – Variable gene segment selection

Typical gene coding transmembrane protein

Separate exons code for individual domains of Ig protein molecule

Genetics of Ig kappa light chain synthesis

DNA rearrangement for kappa light chain

”Looping out” unwanted genes

Construction of Ig light chain

Construction of Ig heavy chain

Genetics of Ig heavy chain synthesis

2. Junctional diversity – Variable nucleotide lengths of J region 3. Hypermutation (somatic) – Untemplated point mutations – Hot spots of mutation in binding region

Somatic hypermutation

(yields variation into rearranged Ig that is subject to pos & neg selection to yield improved antigen binding)

4. Somatic gene conversion (few species) – Translocation of short segments of V region (pseudogenes) to VH gene – Used when species has few VH genes or little variation – Chicken, rabbit

Somatic gene conversion (eg., chicken IgH) Pseudogene sequences copied into rearranged B-cell VDJ unit

Process of gene conversion: pseudo genes generate sequence diversity

Diversification of chicken Ig occurs through gene conversion

5. Different pairing of VH and VL chain – To form antigen-binding site – Combinatorial joining of chains (not gene segments) – Mechanism acting at the protein level

V-region genes constructed from gene segments

6. Class switching of immunoglobulin – – – – –

In specific cytokine environment VDJ is juxtaposed with different CH genes Same antigen-specificity, new function Occurs by removal of intervening DNA Structural variation in C regions confers functional specialization

Class switching in Ig synthesis

Mechanism of class switching

Isotype switching involves recombination between specific switch signals

Co-expression of IgD and IgM regulated by RNA processing

Different types of variation between immunoglobulins

Differences in inheritance among major immunoglobulin variants

Ig classes and subclasses in mammals

Synthesis, Assembly, and Secretion of Immunoglobulins • The heavy and light chains are synthesized on separate polyribosomes (polysomes). • The assembly of the chains to form the disulfide-linked immunoglobulin molecule occurs as the chains pass through the cisternae of the rough endoplasmic reticulum (RER) into the Golgi apparatus and then into secretory vesicles. • The main figure depicts assembly of a secreted antibody. • The inset depicts a membrane-bound antibody, which contains the carboxyl-terminal transmembrane segment. • This form becomes anchored in the membrane of secretory vesicles and then is inserted into the cell membrane when the vesicles fuse with

Synthesis, Assembly, and Secretion of Immunoglobulins

• Transcription of immunoglobulin genes is regulated by three types of DNA regulatory sequences: promoters, enhancers and silencers. • Growing knowledge of the molecular biology of immunoglobulin genes has made it possible to engineer antibodies for research and therapy. • The approaches include chimeric antibodies, bacteriophage-based combinatorial libraries of Ig-genes, and the transplantation of extensive