Complement

1. Complement AHMAD SH. SILMI Haematology and Immunology ISLAMIC UNIVERSITY GAZA

2. Complement:history Discovered in 1894 by Bordet It represents lytic activity of fresh serum Its lytic activity destroyed when heated at 56C for 30 min

3. Introduction • 30 circulating & membrane – expressed proteins. • C` components are synthesized in the liver & by cells involved in the inflammatory response. • Conc. Of C = 3 mg/ml, C3 = lmg/ml, factors D & C2 in trace amount. • - Many of complement proteins are zymogens i.e pro- enzymes which require proteolytic cleavage to become active. • If C` activation is not regulated, the host can be damaged.

4. Complement functions • Important in both innate and Ab-mediated acquired immune responses. • Production of opsonins "enhance phagocytosis". • Production of anaphylatoxins  peptides inducing local & systemic inflammatory responses. • Direct killing of microorganisms. • Enhancing Ag-specific immune responses. • Maintaining homeostasis "the maintenance of stability within the body" by removing immune complexes & dying cells.

5. Definitions • C-activation: alteration of C proteins such that they interact with the next component • C-fixation: utilization of C by Ag-Ab complexes • Hemolytic units: dilution of serum which lyses a proportion (usually 50%) of Ab-coated r.b.c (CH50) • C-inactivation: denaturation (usually by heat) of an early C-component resulting in loss of hemolytic activity • Convertase/esterase: altered C-protein which acts as a proteolytic enzyme for another C-component

6. Proteins of the complementsystem (nomenclature) • C1(qrs), C2, C3, C4, C5, C6, C7, C8, C9 • factors B, D, H and I, properdin (P) • mannose binding lectin (MBL), MBL associated serine proteases (MASP-1 MASP-2) • C1 inhibitor (C1-INH, serpin), C4-binding protein (C4-BP), decay accelerating factor (DAF), • C1 receptor (CR1), protein-S (vitronectin)

7. Activated component are usually over-lined: e.g. C1qrs Activation product of complement proteins (nomenclature) When enzymatically cleaved, the larger moiety, binds to the activation complex or membrane and the smaller peptide is released in the microenvironment Letter “b” is usually added to the larger,membrane-binding, peptide and “a” to the smaller peptide (e.g., C3b/C3a, C4b/C4a, C5b/C5a), EXCEPT C2 (the larger, membrane-binding moiety is C2a; the smaller on is C2b)

8. antibody independent antibody dependent Activation of C3 and generation of C5 convertase activation of C5 LYTIC ATTACK PATHWAY Pathways of complement activation LECTIN PATHWAY ALTERNATIVE PATHWAY CLASSICAL PATHWAY

9. Complement Activation • Sequential activation "a cascade" of successive components. • Activation of one component induces enzymatic function that triggers the activation of the next component in sequence. • Upon activation, individual components are split into fragments designated by lower-case letters. • Complement proteins C3 and C4 possess an internal thioester bond which enables their covalent binding to amino and hydroxyl groups . This is a key step in the localization of complement activation to sites of inflammation .

10. The Classical Pathway • Activators of C. Pathway • Ag-Ab complexes. • Some viruses, necrotic cells and subcellular membranes (e.g. from mitochondria). • Aggregated immunoglobulins "Ig". • C-reactive protein.

11. Activation of the classical pathway • The classical pathway is initiated when C1 binds to the Ab in an Ag-Ab complex. C1 complex = C1qr2s2. "Clq is made up of 6 indentical subunits". • Globular head of Clq subunits bind to Clq specific receptors on FC of either IgM or IgG. • Cl becomes enzymatically active " C1qr2s2 esterase cleaves the next component, C4 into C4a, C4b. • C4b binds to bacterial surface.

12. Activation of the classical pathway • C4b then binds C2 which is cleaved by C1s which yields C2b and C2a. • C2a binds C4b to form complex C4b2a which is the classical pathway C3 convertase. • C3 convertase cleaves C3 into C3a "a fluid phase anaphylatoxin" and C3b which binds to cell surface. • C3 convertase with bound C3b are referred to as C5 convertase. • C5 convertases "C4b2a3b" cleaves C5 into C5a and C5b which binds to cell surface. • C5b binds C6, C7, C8, C9 to form a membrane attack complex "MAC" that results in cell lysis.

13. C1r C1s C1q Ca++ Components of the Classical Pathway C4 C2 C3 C1 complex

14. C4a C1r C1s C1q b Ca++ Classical Pathway Generation of C3-convertase C4

15. C2b C1r C1s a C1q Ca++ C2 a Classical Pathway Generation of C3-convertase C2 C4a Mg++ C4b2a is C3 convertase C4b

16. C1r C3a C1s C1q Ca++ C2 a b Classical Pathway Generation of C5-convertase C2b C4a C4b2a3b is C5 convertase; it leads into the Membrane Attack Pathway Mg++ C3 C4b

17. MBL Components of mannose-binding lectin pathway C4 MASP2 C2 MASP1

18. The Lectin Pathway • It is activated by terminal mannose residues of proteins and polysaccharides found on the surface of bacteria. • These mannose residues are not found on the surface of mammalian cells. • The lectin pathway of complement activation may be thought of as discriminating between self and non-self. • It is activated in absence of Ab, it is part of innate immune defense.

19. The Lectin Pathway • The bacterial mannose residues bind to a circulating complex of mannose-binding lectin "MBL; homologous to that of Clq in the classical pathway" and two associated proteases "mannose-associated serine protease 1-MASP-1 and MASP-2. • Binding activates MASP-1 to sequentially cleave the classical component pathway components C4 and C2 to form C4b2a "the classical C3 convertase" on the surface of the bacterium. • MASP-2 appears to be able to cleave C3 directly. • Thus the lectin pathway converges with the classical pathway at the activation of C3.

20. C2a C2a C2b C4a C4b C4b MBL Mannose-binding lectin pathway C4b2a is C3 convertase; it will lead to the generation of C5 convertase C4 C2 MASP2 MASP1

21. Components of thealternative pathway D C3 B P

22. The Alternative Pathway • This pathway is triggered by almost any foreign substance e.g. LPS, cell wall of some yeast, a protein present in the venom of cobra, some viruses, aggregated Ig and necrotic cells. • Activation of alternative pathway occurs in the absence of Ab. • It is an effector arm of the innate immune defenses. • Some components of this pathway are unique "the serum factor B and D and properdin, also known as factor P". • C3, C3b, C5, C6, C7, C8, C9 are common to those used in the classical pathway.

23. Activation of Alternative pathway • C3b is generated by the spontaneous cleavage of a reactive thiol group in C3. • C3b can bind to OH groups of proteins and carbohydrates expressed on cell surfaces which initiated alternative pathway. • Serum protein factor B combines with C3b on the cell surface to from a complex "C3bB". • Factor D then cleaves factor B in C3bB generating Ba + Bb "remains attached to C3b".

24. Activation of Alternative pathway • C3bBb is the alternative pathway C3 convertase which cleaves C3 into C3a and C3b. • C3bBb is stabilized by properdin binding. Thus C3bBb cleaves high levels of C3 in a short time. • Properdin binding to C3bBb creates an amplification loop in the alternative pathway. • C3bBb + C3 ------------------------- C3a + C3b • C3bBb + C3b------------------------- C3bBb3b "C5 covertase" • C3bBb3b +C5-------------------------C5a + C5b • C5b + C6,C7,C8,C9-------------------C5b6,7,8,9 "Membrane Attack Complex"

25. b i b C3a Spontaneous C3 activation Generation of C3 convertase D H2O B C3 C3 C3iBb complex has a very short half life

26. C3b b b C3a C3-activationthe amplification loop If spontaneously-generated C3b is not degraded D B C3

27. C3b b b Bb C3b C3a C3-activationthe amplification loop D B C3 C3a

28. Bb C3b C3b b b Bb C3b C3a C3-activationthe amplification loop D B C3 C3a C3a

29. Bb Bb C3b C3b C3b Bb C3b C3-activationthe amplification loop C3a C3a C3a

30. Bb Bb C3b C3b Bb C3b C3-activationthe amplification loop C3a C3a C3a

31. C3b DAF CR1 Autologous cell membrane Control of spontaneousC3 activation via DAF DAF prevents the binding of factor B to C3b B

32. B C3b b B b b DAF CR1 Autologous cell membrane Control of spontaneousC3 activation via DAF DAF dislodges C3b-bound factor Bb

33. DAF B b B b C3b C3b CR1 CR1 iC3b iC3b Control of spontaneousC3 activation via CR1 H I I DAF Autologous cell membrane

34. C3c C3c C3dg C3dg C3b C3b I I iC3b iC3b Degradation of spontaneously produced C3b

35. C3a C3b finds an activator (protector) membrane C3b b b C3b stabilization andC5 activation This is stable C5 convertase of the alternative pathway D P B C3

36. C3b regulation on self and activator surfaces C3b

37. C2a C3b C4b C5-convertase of the two pathways C5-convertase of the Classical and lectin Pathways C5-convertase of the Alternative Pathway Bb C3b C3b

38. Lytic pathway Generation of C5 convertase leads to the activation of the Lytic pathway

39. C8 C7 Components of the lytic pathway C6 C5 C 9

40. C5a b C3b C4b C2 a Lytic pathwayC5-activation C5

41. C7 C5 b Lytic pathwayassembly of the lytic complex C6

42. C8 C6 C7 C5 b Lytic pathway:insertion of lytic complex into cell membrane C 9 C 9 C 9 C 9 C 9 C 9 C 9 C 9 C 9

43. Regulation of Complement Pathway • The 1st step in the activation of the classical complement pathway is inhibited by C1 esterase inhibitor "ClINH" which binds to Clr2 and Cls2 causing them to dissociate from C1q & preventing C` activation. • ClINH also regulates the alternative pathway by inhibiting the function of C3bBb and the lectin pathway by inhibiting MASP-1 & MASP-2.

44. C1r C1r C1s C1s C1q C1qrs breakdown C1Inh

45. Regulation of Complement Pathway • Several proteins regulate the classical pathway C3 convertase "C4b2a" by binding to C4b and displacing C2a-the activated enzyme component -from the complex. These regulations are serum proteins known as C4b-binding protein "C4BP", decay-accelerating factor "DAF, CD55" and membrane cofactor protein MCP, CD46" and the cell surface component complement receptor 1 "CR1"; CD35 serum protein factor I cleaves C4b on the cell surface.

46. Regulation of Complement Pathway • Activation of the alternative pathway is also regulated. The serum protein factor H competes with factor B for binding to C3b on a cell surface. Factor H also binds to C3b in C3bBb "C3 convertase" and displaces Bb, preventing further activation of the complement cascade. • Alternative pathway convertase C3bBb is regulated by the same cell-surface regulatory molecules "DAF, MCP, CRl" that inhibit the function of the classical pathway C3 convertase.

47. Regulation of Complement Pathway • The terminal step in C` pathway is also regulated. CD59 "protectin" a cellular membrane protein prevents lysis by binding C5b-8 on cell surface and preventing C9 polymerization. Homologous restriction factor "HRF" is another protein which has similar action as CD59 but much weaker. S protein "vitronectin" and SP-40, "clusterin" bind the hydrophobic regions of C5b6, C5b67, C5b678, C5b6789 and prevent interaction with membranes.

48. Biological Activities of C` • Production of opsonins C3b & C4b are the major opsonins generated. • Production of Anaphylatoxins C3a, C4a, C5a are known as anaphylatoxins • Lysis of cells e.g. bacteria. • Enhancing B cell Responses to Antigens. • Removing Dead or Dying cells. • Responses to viruses • complement can distinguish self from nonself.

49. Biological effects of C5a

50. Opsonization and phagocytosis