Immunoglobulins-structure and function of each, complement- various pathways and components, brief immunodeficiency disorders

1. Dr. M. Krishna Vasudev
Immunoglobulins:
Structure and Function

2. Immunoglobulin
Immunoglobulin is a glycoprotein that is made in
response to an antigen and can recognize and
bind to the antigen that caused its production.

3.  Are gamma globulins
 Synthesized by plasma cells
 Constitute 25-30 % of total serum proteins
 Antibodies are present in serum, tissue fluids and
mucosal surfaces.
 All antibodies are immunoglobulins, but all
immunoglobulins may not be antibodies

4. Basic structure
 Composed of 4 polypeptide
chains.
 2 identical light and 2 identical
heavy chains
 Linked by disulphide bonds
 Light chains similar in all
immunoglobulins
 Light chains occur in 2
varieties kappa and lambda
 Light and Heavy chains are
subdivided into variable and
constant region.
 Each heavy and light chain
contains amino terminal in
variable region carboxy
terminal in constant region

5.  Heavy chains are structurally
and antigenically distinct for
each class
 Each immunoglobulin peptide
chain has intra chain disulphide
bonds- form loops
 Each loop is compactly folded
to form a globular structure-
domain
 Light chain contains a single
variable domain (VL) and a
single constant domain (CL).
 Heavy chain contains one
variable domain (VH) and 3
constant domains (CH1, CH2,
CH3)
 Hinge region is the segment in
heavy chain - between CH1,
CH2

6. Digestion with proteolytic enzymes
Papain enzyme
 Peptide bonds in the
hinge region are broken
 Produces 3 fragments
 2 identical fragments
called Fab fragments –
antigen binding activity.
 Other fragment called Fc
fragment (Fraction
crystallizable)

7. Pepsin digestion
•Produce a single fragment composed of two Fab like subunits F(ab)2 binds antigen
•Fc fragment is not recovered- digested to small numerous peptides.

8. Classification
 Based on structure and antigenic nature of H
chain the immunoglobulins are classified into 5
classes.
 Ig G- (gamma)
 Ig A- (alpha)
 Ig M- (mu)
 Ig D- (delta)
 Ig E - (epsilon)

9. Immunoglobulin G (Ig G)
 Most abundant class in
serum
 Constitutes 80% total
immunoglobulin
 Present in blood, plasma
and tissue fluids
 Contains less
carbohydrate than other
immunoglobulins
 It has a half life of 23 days:
the longest of all of the
immunoglobulin isotypes

10.  Crosses placenta and provide
natural immunity to foetus and
neonate at birth
 Acts against bacteria and
viruses by opsonizing
 Neutralize toxin
 Activate complement by
classical pathway
 Catabolism of IgG is unique in
that it varies with its serum
concentration

11. Sub classes of Ig G
 Ig G1, Ig G2, Ig G3, Ig G4.

12. Biological function of subclasses
 IgG1, IgG3, IgG4 – cross placenta and protect foetus
 IgG3 activates complement
 IgG1 and IgG3 binds to Fc receptor on phagocytic
cells, monocytes and macrophages and mediate
opsinization.

13. Immunoglobulin A (Ig A)
 Constitutes 10-15 % of total
immunoglobulins
 Present in milk, saliva,
tears, mucous of respiratory
tract, digestive tract and
genitourinary tract.
 In serum exist as monomer
 In external secretions exist
as dimer called secretory
Immunoglobulin.
 Has ‘J’ chain and secretory
piece.
 Half life: 6-8 days

14. Formation of secretory Ig A
 Dimeric Ig A binds to the
receptor on the surface of
the epithelial cells -
endocytosed and
transported across the cell
to the luminal surface
 After reaching the surface,
the poly-Ig receptor is
cleaved
 The portion of the receptor
that remains attached to
the Ig A dimer – secretory
component
 Secretory piece protects Ig
A from digestive enzymes
and denaturation by

15. Functions
 Provides local immunity.
 Secretory Ig A binds to surface antigens of
microorganism and prevent its attachment and
invasion of the mucosal surfaces of respiratory and
digestive tract- immune elimination.
 Secretory IgA provides important line of defense
against salmonella, Vibrio cholerae, N. gonorrhoeae,
influenza virus and poliovirus.
 Secretory IgA present in breast milk protects newborn
during first months of life.
 Activates complement by the alternative pathway
 Promotes phagocytosis and intracellular killing of
microorganisms

16. Immunoglobulin M (Ig M)
 Accounts for 5-10% of
total serum proteins
 Polymer of five
monomeric units
(pentamer)
 Held together by disulfide
bonds and ‘J’ chain
 Mol. Wt. of 900,000-
10,00,000 (millionaire
molecule)
 Half life: 5 days

17.  Most of IgM (80%) present intravascularly
 Present in low concentration in intercellular tissue
fluids
 Cannot cross placenta
 Presence of IgM antibody in serum of newborn
indicate congenital infection.
 Earliest immunoglobulin to be synthesized by foetus
(20 weeks)
 First immunoglobulin to be produced in primary
response to antigen
 Relatively short-lived hence it’s demonstration in the
serum indicates recent infection
 Monomeric IgM appears on the surface of
unstimulated B lymphocytes and act as receptors

18. Functions
 It agglutinates bacteria
 Activates complement by classical pathway
 Causes opsonization and immune heamolysis
 Believed to be responsible for protection against
blood invasion by microorganisms

19. Immunoglobulin E (Ig E)
 Structure is similar to Ig G
 Has 4 constant region
domains.
 Mol. Wt. 1,90,000
 Half life: 2 days
 Heat labile (inactivated at
560C in 1 hour)
 Normal serum
concentration 0.3 ug/ml
 Mostly present extra
cellularly
 Does not cross placenta

20.  Produced in the lining of respiratory and
intestinal tract
 Known as reagin antibody
 Does not activate complement nor agglutinate
antigens
 Binds to the Fc receptors on the membranes of
blood basophils and tissue mast cells
 Mediates immediate hypersensitivity reaction
and P.K. reaction
 Responsible for symptoms of anaphylactic
shock, hay fever and asthma.
 Play a role in immunity against helminthic
parasites

21.  IgE binds to Fc receptors on
the membrane of blood
basophils and tissue mast
cells.
 When two IgE molecules on
the surface of these cells
are cross linked by binding
of the same antigen- cells
degranulates.
 Release histamine and
pharmacological mediators
of anaphylaxis from cell.
 The physiological role of IgE
appears to be protection
against pathogens by mast
cell degranulation and
release of inflammatory
mediators

22.  Mediates P.K. reaction (PRAUSNITZ &
KUSTNER)
 The presence of a serum component responsible
for allergic reaction was first demonstrated by
Prausnitz and Kustner in 1921.
 Kustner was suffering from atopic hypersensitivity
to certain species of fish
 Kustner’s serum was injected intracutaneously in
Prausnitz
 After 24 hrs small quantity of cooked fish antigen
was injected at the same site
 A wheal and flare reaction occurred within
minutes.

23. Immunoglobulin D (Ig D)
 Structure is similar to IgG
 Serum concentration 30
micrograms per ml
 Constitutes 0.2% of total
immunoglobulins
 Half life: 3 days
 IgD together with IgM is
major membrane bound
immunoglobulin on
unstimulated B
lymphocytes-acts as
recognition receptors for
antigens

24. Properties and biological activities of Immunoglobulins
Ig G Ig A Ig M Ig D Ig E
1. Structure Monomer Monomer in
serum
Dimer in
secretion
Pentamer Monomer Monomer
2. Heavy chain
CH domain
Gamma
Three
Alfa
Three
Mu
Four
Delta
Three
Epsilon
Four
3. Mol. Wt. 1,50,000 1,60,000 9,00,000 1,80,000 1,90,000
4. Serum concentration (mg/ml) 12 2 1.2 0.03 0.00004
5. Present on membrane of mature B
cell
_ _ + + _
5. Intravascular
Distribution (%)
45 42 80 75 50
6. Crosses placenta + - - - -
7. Present in milk + + - - -
8. Selective secretion by seromucous
glands
- + - - -
9. Activation of complement
Classical
Alternate
+
-
-
+
+
-
-
-
-
-
10 Binds to FC receptor of phagocytes + - - - -
11 Induces mast cell degranulation - - - - +

25. Complement System

26. • Consists of approx. 20 proteins that are present
in normal human serum  synthesized mainly
by liver
• Heat-labile  inactivated by heating serum at
560C for 30 minutes
• Able to augment the effects of other components
of the immune system
• Important component of innate host defenses

27. • Three main effects:
1. Lysis of cells (bacteria, allografts, tumor cells)
2. Generation of mediators of inflammation
3. Opsonization – enhancement of
phagocytosis

28. • Sequential activation of complement
components occurs via one of three pathways:
1. Classic pathway
2. Lectin pathway
3. Alternative or Properdin pathway
• Lectin and alternative pathways are more
important the first time we are infected by
microorganisms because antibody required to
activate the classic pathway is not yet present

29. • Part of acquired or adaptive immunity
• Activated by Ag-Ab complexes
• Immunoglobulins involved: IgM and IgG (except
IgG4)
• Involves activation of C1
 Composed of C1q, C1r, and C1s  binds to
Fc portion of IgG and IgM
 Requires calcium for activation
Classic Pathway

30. • Other activators include:
1. Viruses – Murine and Retroviruses
2. Bacteria – Mycoplasma
3. Polyanions, especially bound to cations
a. PO4
3- - DNA, lipid A, cardiolipin
b. SO4
2- - dextran, heparin, chondroitin
4. Arrays of terminal mannan groups
Classic Pathway

31. Classic Pathway
Components of the Classical Pathway
Native component Active component(s) Function(s)
C1(q,r,s)
C1q
Binds to antibody that has bound
antigen, activates C1r.
C1r
Cleaves C1s to activate protease
function.
C1s Cleaves C2 and C4.
C2
C2a Unknown.
C2b
Active enzyme of classical
pathway; cleaves C3 and C5.
C3
C3a
Mediates inflammation;
anaphylatoxin.
C3b
Binds C5 for cleavage by C2b.
Binds cell surfaces for
opsonization and activation of
alternate pathway.
C4
C4a Mediates inflammation.
C4b
Binds C2 for cleavage by C1s.
Binds cell surfaces for
opsonization.

32. Classic Pathway
Components of the Membrane-Attack Complex
Native component
Active
component(s)
Function(s)
C5
C5a
Mediates inflammation;
anaphylatoxin, chemotaxin.
C5b
Initiates assembly of the
membrane-attack complex
(MAC).
C6 C6
Binds C5b, forms acceptor
for C7.
C7 C7
Binds C5b6, inserts into
membrane, forms acceptor
for C8.
C8 C8
Binds C5b67, initiates C9
polymerization.
C9 C9n
Polymerizes around
C5b678 to form channel
that causes cell lysis.

33. Classic Pathway

34. Alternative Pathway
• Also known as the Properdin Pathway
• Part of innate immunity
• Bypasses C1, C4, and C2
• Does not require an antigen-binding protein
• Does not wait for antibody to be formed for
activation
• Acts synergistically with the classical pathway

35. Alternative Pathway
• Usually activated by products of micro-organisms
like endotoxin
• Other activators include:
1. Complexes containing IgA
2. Some virus-infected cells (e.g. EBV)
3. Many gram negative and gram positive
organisms
4. Parasites – Trypanosomes, Leishmania
5. Dextran SO4
6. Erythrocytes
7. Carbohydrates (agarose)

36. Alternative Pathway
Components of the Alternate Pathway
Native component
Active
component(s)
Function(s)
C3
C3a
Mediates inflammation;
anaphylatoxin.
C3b
Binds cell surfaces for
opsonization and activation
of alternate pathway.
Factor B
B
Binds membrane bound
C3b. Cleaved by Factor D.
Ba Unknown.
Bb
Cleaved form stabilized by
P produces C3 convertase.
Factor D D
Cleaves Factor B when
bound to C3b.
Properdin P
Binds and stabilizes
membrane bound C3bBb.

37. Alternative Pathway

38. Lectin Pathway
• Also known as the MBL Pathway
• Activated by binding of mannose-binding
lectin (or mannose-binding protein) to surface
of microbes bearing mannan (polymer of the
sugar mannose) in a calcium dependent
manner
• Binding causes activation of MASP (MBP-
associated serine proteases)  cleave C2
and C4

39. Lectin Pathway

40. • All three pathways lead to production of C3b 
central molecule of complement cascade
• Presence of C3b on surface of a microbe marks
it as foreign and targets it for destruction
• C3b with two important functions:
1. Combines with other complement
components to generate C5 convertase
2. Opsonizes bacteria

43. Biologic Effects:
1. Opsonization
• C3b & C1q; enhance phagocytosis
2. Chemotaxis
• C5a and C5,6,7 complex  attract neutrophils
• C5a – enhance adhesiveness of neutrophils to
the endothelium
3. Anaphylatoxin (C3a, C4a, C5a)
• Cause degranulation of mast cells
• Bind directly to smooth muscles of bronchioles
 bronchospasm

44. Biologic Effects:
4. Cytolysis (MAC)
• Disrupt the membrane & the entry of water
and electrolytes into the cell
5. Enhancement of antibody production
• Binding of C3b to its receptors on the
surface of activated B cells  enhanced
antibody production

46. Regulation of Complement System
1. C1 inhibitor (C1-INH)
• Important regulator of classic pathway
• A serine protease inhibitor (serpin)
• Irreversibly binds to and inactivates C1r and
C1s, as well as MASP in lectin pathway
2. Factor H
• Regulate alternative pathway
• Reduce amount of C5 convertase available
• With both cofactor activity for the factor I-
mediated C3b cleavage, and decay
accelerating activity against C3bBb (C3
convertase)

47. Regulation of Complement System
3. Properdin
• Protects C3b and stabilizes C3 convertase
4. Factor I
• Cleaves cell-bound or fluid phase C3b and
C4b  inactivates C3b and C4b
5. Decay accelerating factor (DAF)
• Glycoprotein on surface of human cells
• Prevents assembly of C3bBb or accelerates
disassembly of preformed convertase  no
formation of MAC
• Acts on both classical and alternative

48. Regulation of Complement System
6. C4b-binding protein (C4BP)
• Inhibits the action of C4b in classical pathway
• Splits C4 convertase and is a cofactor for
factor I
7. Complement Receptor 1 (CR-1)
• Co-factor for factor I, together with CD46
8. Protectin (CD59) and Vitronectin (S protein)
• Inhibits formation of MAC by binding C5b678
• Present on “self” cells to prevent complement
from damaging them

50. Clinical Aspects
1. Deficiency of C5-C8 & Mannan-binding lectin
• Predispose to severe Neisseria bacteremia
2. Deficiency of C3
• Severe, recurrent pyogenic sinus & resp.
tract infections
3. Deficiency of C1 esterase inhibitor
• Angioedema inc. capillary permeability and
edema
4. Deficiency of DAF
• Increased complement-mediated hemolysis
 paroxysmal nocturnal hemoglobinuria

51. Clinical Aspects
5. Transfusion mismatches
• Activation of complement  generate large
amounts of anaphylatoxins & MAC  red cell
hemolysis
6. Autoimmune diseases
• Immune complexes bind complement  low
complement levels + activate inflammation 
tissue damage
7. Severe liver disease
• Deficient complement proteins  predispose
to infection with pyogenic bacteria

52. Clinical Aspects
8. Factor I deficiency
• Low levels of C3 in plasma due to
unregulated activation of alternative pathway
 recurrent bacterial infections in children
• Mutations in factor I gene  implicated in
development of Hemolytic Uremic Syndrome

53. Dr. M. Krishna Vasudev
Dept of General Medicine
Immunodeficiency
disorders

54. Types
Primary-usually genetic, congenital
Secondary-Acquired

55. Mechanisms of Immunodeficiency
Loss or reduction of:
 Cell type
 Cell numbers
 Cell function

56. Loss of Cell Function
 Receptors
 Cell signaling
 Cytokine production
 Ig production
 Co stimulation impairment
 Intracellular killing
 Extravasation impairment

58. Primary Immunodeficiency
 Myeloid lineage
 Congenital agranulocytosis
 Leukocyte-adhesion deficiency
 Lymphoid lineage
 Severe combined immunodeficiency (SCID)
 B cells
 Agammaglobulinemia
 Hypogammaglobulinemia
 Specific Ig Deficiencies
 T cells
 DiGeorge Syndrome
 Wiskott Aldrich Syndrome

60. Secondary Immunodeficiency
 Drug related
 Disease related
 Cancer
 AIDS
 HIV
 T helper cell as target

61. SCID
 Various genetic defects
 No TCR or defective TCR
 Defective cell signaling
 Defective IL 2
 Recurrent infections
 Death at early age

62. Wiskott Aldrich Syndrome
 X linked disorder
 Affects platelet numbers/function
 Affects T cell function
 Cytoskeleton of lymphocytes affected
 Lower amounts of IgM
 Increased susceptibility to certain bacterial
infections

63. Bruton’s Agammaglobulinemia
 Low levels of IgG
 B cell signal transduction affected
 Defective BCR
 Recurrent bacterial diseases starting at end of
first year of life
 Short life span

64. DiGeorge Syndrome
 Poorly developed or functioning thymus
 Associated with other developmental conditions
 Depression of T cell numbers
 Absence of T cell response
 Humoral response to T independent antigens only

66. Secondary Immunodeficiency
 Drugs
 Irradiation
 Cancer
 AIDS

67. AIDS
 HIV as agent
 T helper as target cell
 Attacked by own CD8 cells
 Susceptible to opportunistic infections
 Kaposi’s sarcoma
 CMV
 Candida
 Pneumocystis carinii