Antibodies are Y-shaped proteins that recognize antigens with high specificity. They are composed of two light chains and two heavy chains connected by disulfide bonds. The variable regions at the tips of the Y, known as the antigen binding sites, contain complementarity determining regions that bind to antigens. There are five classes of antibodies (IgG, IgM, IgA, IgD, IgE) that differ in structure and function. The Fc region mediates effector functions like activation of complement and binding to immune cells.

1. Lecture 5
Protein Structure and Function II

2. Antibodies
 Proteins that recognize and bind to a
particular antigen with very high specificity.
 Belong to a group of serum proteins called
immunoglobulins (Igs).
 Each antibody has at least two identical sites
that bind antigen: Antigen binding sites.

3. Antibody Structure
 Antibodies are made up
of:
 2 Light Chains (identical)
~25 KDa
 2 Heavy Chains (identical)
~50 KDa
 Each light chain bound to
heavy chain by disulfide
(H-L)
 Heavy chain bound to
heavy chain (H-H)

5. Fb
CH2
CH3
Fv
Fv
Fv
Fb
Fv
Fb
Hinge
Elbow
CH2
CH3
Fv
Flexibility and
motion of
immunoglobulins

6. Hinge
CH1
CH2
CH3
VH1
CL VL

7.  First 100 a/a of amino terminal vary of both H
and L chain are variable
 Referred to as VL , VH, CH And CL
 CDR (Complementarity Determining
Regions) are what bind Ag
 Remaining regions are very similar within
same class

8. CDR Are Hypevariable

9. Non-covalent forces in
antibody - antigen interactions
Electrostatic forces Attraction between opposite charges
Hydrogen bonds Hydrogens shared between electronegative atoms
Van der Waal’s forces Fluctuations in electron clouds around molecules
oppositely polarise neighbouring atoms
Hydrophobic forces Hydrophobic groups pack together to exclude
water (involves Van der Waal’s forces)

10. Five classes of antibodies:
 IgG
 IgM
 IgA
 IgD
 IgE

11. Sequencing Of Heavy Chains
 Sequencing Of Several Immunoglobulins Revealed
 Five Basic Sequence Patterns
 a,g, d, e, m
 IgA, IgG, IgD, IgE and IgM
 The Above Classes Are Called Isotype
 Each class can have either k or l light chains
 Minor Differences Led To Sub-classes For IgA and IgG
 IgA1, IgGA2 and IgG1, IgG2, IgG3, IgG4

12. IgG
 Structure: Monomer
 Percentage serum antibodies: 80%
 Location: Blood, lymph, intestine
 Half-life in serum: 23 days
 Placental Transfer: Yes
 Known Functions: Enhances phagocytosis,
neutralizes toxins and viruses, protects fetus
and newborn.

13. IgM
 Structure: Pentamer
 Percentage serum antibodies: 5-10%
 Location: Blood, lymph, B cell surface (monomer)
 Half-life in serum: 5 days
 Placental Transfer: No
 Known Functions: First antibodies produced during
an infection.

14. IgA
 Structure: Dimer
 Percentage serum antibodies: 10-15%
 Location: Secretions (tears, saliva, intestine, milk),
blood and lymph.
 Half-life in serum: 6 days
 Placental Transfer: No
 Known Functions: Localized protection of mucosal
surfaces. Provides immunity to infant digestive
tract.

15. IgD
 Structure: Monomer
 Percentage serum antibodies: 0.2%
 Location: B-cell surface, blood, and lymph
 Half-life in serum: 3 days
 Placental Transfer: No
 Known Functions: In serum function is
unknown. On B cell surface, initiate immune
response.

16. IgE
 Structure: Monomer
 Percentage serum antibodies: 0.002%
 Location: Bound to mast cells and basophils
throughout body. Blood.
 Half-life in serum: 2 days
 Placental Transfer: No
 Known Functions: Allergic reactions.

17. Antibody Structure
 Repeating Domains of ~110 a/a
 Intrachain disulfide bonds within each domain
 Heavy chains
 1 VH and either 3 or 4 CH (CH1, CH2, CH3, CH4)
 Light chains
 1 VL and 1 CL
 Hinge Region
 Rich in proline residues (flexible)
 Hinge found in IgG, IgA and IgD
 Proline residues are target for proteolytic digestion (papain and
pepsin)
 Rich in cysteine residues (disulfide bonds)
 IgM and IgE lack hinge region
 They instead have extra CH4 Domain

18. Enzymatic Digestion Of Antibodies
 Digestion With Papain Yields
 3 Fragments
 2 identical Fab and 1 Fc
 Fab Because Fragment That is Antigen Binding
 Fc Because Found To Crystallize In Cold Storage
 Pepsin Digestion
 F(ab`)2
 No Fc Recovery, Digested Entirely
Mercaptoethanol Reduction (Eliminates
Disulfide Bonds) And Alkylation Showed

20. Why do antibodies need an Fc region?
The (Fab)2 fragment can -
•Detect antigen
•Precipitate antigen
•Block the active sites of toxins or pathogen-associated
molecules
•Block interactions between host and pathogen-associated
molecules
but can not activate
•Inflammatory and effector functions associated with cells
•Inflammatory and effector functions of complement
•The trafficking of antigens into the antigen processing
pathways

21. Structure and function of the Fc region
C H3
CH2
IgA IgD IgG
C H4
CH3
CH2
IgE IgM
The hinge region is
replaced by an additional Ig
domain
Fc structure is common to all specificities of antibody within an ISOTYPE
(although there are allotypes)
The structure acts as a receptor for complement proteins and a ligand
for cellular binding sites

23. Monoclonal Antibodies
 Immunize Animal With Antigen
 Multiple Clones Are Generated, Good For In Vivo
 For Clinical Diagnosis, Research, One Clone
That Reacts To Single Epitope Is Preferred
 Solution By Kohler and Milstein
 Fuse A Myeloma Cell (Cancerous) With A Normal
Plasma Cells
 Resulting Clones Can Be Cultured Indefinitely
 Produces An Antibody Recognizing One Epitope

24. Fc Receptors (FcR) Functions
 To Transport Abs Across Membranes
 Secretion of IgA Across Epithelium into lumen
 Transport of maternal Abs Across Placenta (IgG)
 Many Cell Types Use FcR
 Ex. Mast Cells, Macrophages, Neutrophils, B, T, NK
 Poly IgR
 Transport of IgA across epithelium
 FcRN
 Transport of maternal IgG to fetus

25. IgA Antibody Transport Across Cell
(Transcytosis)

26. Protein section review
 Proteins are polymers composed of L-amino
acids. Amino acids are 20 in number

27. Protein section review
 Amino acids possess two functional groups
namely carboxyl (-COOH) and amino (-NH2).
 In the physiological system, they exist as
zwitterions.

28.  The structure of protein is divided into four
levels of organization.
 The tertiary and quaternary structures ol
protein are stabilized by non-covalent bonds
such as hydrogen bonds, hydrophobic
interactions, ionic bonds etc.

29. Protein section review
 The determination of primary structure of a
protein: chemical and enzymatic methods are
employed.

30. Sample question
 A heptapeptide (2 M, D, R, K, F, G) was isolated from the
urine of a three-toed sloth.
 Reaction of the heptapeptide with FDNB gave DNP-M.
 Limited proteolysis with carboxypeptidase indicated that M was the first amino
acid released
 Cyanogen bromide (CNBr) reaction with the heptapeptide released one
equivalent of free homoserine lactone
 Chymotrypsin digestion of the heptapeptide yielded a pentapeptide and a
dipeptide. Reaction of the pentapeptide with dansyl-Cl gave dansyl-M.
 Trypsin digestion yielded two M-containing tripeptides and free R.
 Digestion of the heptapeptide with pepsin gave a tetrapeptide (containing M, R, K
and D) and a tripeptide (M, F, and G).
 DNFB & carboxypeptidase (Identify N- and C-terminal residues)
 CNBr acts only on methionine residues
 CNBr cleaves peptide chains on the carboxy side of M residues and converts them
into homoserine lactone
 Trypsin - cleavage on the C-side of Lys, Arg
 Chymotrypsin - C-side of Phe, Tyr, Trp
 Clostripain - like trypsin, but attacks Arg Pepsin hydrolyzes peptide chains on the N-side
of aromatic AA

31. Answer
 N-terminal AA is M
 C-terminal AA is M
 CNBr cleaves peptide chains on the carboxy side of M residues
and converts them into homoserine lactone) - M-?-?-?-?-?-M
 Chymotrypsin (carboxy side of aromatic AA's) - pentapeptide
- M-?-?-?-F - dipeptide - ?-M
 Trypsin digestion - since we know that M is at both the C-terminal
and the N-terminal, this places K as the 3rd AA
residue and R as the 4th - M-?-K-R-?-?-M
 Pepsin digestion - tetrapeptide (M,R,K & D) - sequence must be
- M-D-K-R - tripeptide (M,F & G) - sequence must be - F-G-M
 The sequence of the heptapeptide must be: M-D-K-R-F-G-M

32. Sample questions
 The imino acid found in protein structure
 (a) Arginine (b) Proline (c) Histidine (d) Lysin
 The bonds in protein structure that are not
broken on denaturation.
 (a) Hydrogen bonds (b) Peptide bonds (c)
lonic bond (d) Disulfide bonds