The Complement System

The Complement System Adapted from the Presentation of Jean F. Regal, Ph.D. Medical School - Duluth

Learning Objectives • Explain the importance of the complement system in host defense and inflammation and the clinical consequences of complement deficiencies. • Describe the biochemistry of activation of the three different pathways including the initiators, sequence of reactions, important enzymes, and fragments. • List the proteins which control the complement system and where they act. • Describe the biological responses mediated by the different complement receptors. • Describe the biological effects of complement activation.

Megan Morris, age 5, is brought to your clinic by her mother, Mrs. Sarah Morris. Megan and her family have recently moved into your community and this is her first visit to see a physician in your community. Mrs. Morris has brought Megan to your clinic because Megan has been crying off-and-on since yesterday. Megan is cuddled tightly to her mother. • Why would a 5 year-old child cry so much? • What questions would you wish to ask the child and/or the mother?

In answer to your questions, Mrs. Morris indicates that, yesterday evening, Megan began crying and saying that her face hurt. Mrs. Morris indicates that this was an unusual behavior, as Megan is usually a happy child who rarely cries. This morning, Mrs. Morris noticed that Megan’s face was swollen. Initially, she thought that the facial swelling might have been from Megan’s crying. However, it seemed to be too great an amount of swelling for that to have been the cause. • What additional questions would you wish to ask the child and/or the mother?

At first, Mrs. Morris says that nothing like this has ever happened to Megan before. Then, as she continues to think about your question, she mentions that Megan did have some pain and swelling on her left hand about a year ago but that the swelling was mild. At the time, Mrs. Morris thought that Megan might have been bitten on the left hand by a mosquito, or was stung by a bee or other insect. The swelling went away overnight while Megan slept. Mrs. Morris asks if this past event was related to what is happening now to Megan. • What would you say to Mrs. Morris? • What additional questions would you wish to ask the child and/or the mother?

This is Megan’s appearance, after you ask her to look toward you. • What do you note about Megan’s appearance? • What additional questions would you wish to ask Megan and her mom?

Megan Morris • Considering that Megan has a lot of swelling, she may have diminished kidney function or reduced liver function. • However, the swelling seems to be limited to Megan’s face, reducing the likelihood of a systemic problem. • It appears that there is a local problem that has developed.

Complement:Location of Complement Proteins • Complement is not a single protein but a complex of proteins that are found constitutively in the plasma. • Complement proteins are present in secretions, such as bronchial fluids, where they protect portals of entry. • Complement proteins are present in interstitial fluids where they protect against agents that penetrate the protective barriers (skin, mucosal membranes, etc.).

Production of ComplementProteins • The molecular weights of complement proteins range widely from 24-400 kDa. • Complement proteins are synthesized • Primarily by liver hepatocytes and by tissue macrophages, • Secondarily by epithelial cells, fibroblasts and monocytes. • Concentration ranges in plasma: • 1 or 2 ug/ml – Mannose-Binding Lectin and Factor D • 300 ug/ml – C4 • 1200 ug/ml – C3

Roles of Complement • Complement proteins are activated on demand. • Complement proteins are activated in a cascade. • In these ways, complement proteins are similar to clotting proteins. • Complement proteins are non-specific proteins that play roles both in the innate immune system and in the adaptive immune system. • Destroy bacteria • Destroy fungi • Destroy viruses

Importance of Complement • The complement system is so important to our defense against microorganisms that there are several pathways by which the complement system can be activated. • Classical pathway • Alternative pathway • Mannose-binding lectin pathway (aka, lectin pathway)

Nomenclature of Complement Proteins • Complement proteins in the common portions of the Classical Pathway are • Denoted with the letter “C” followed by a number and are named C1 through C9. • Proteins in the Mannose-Binding Lectin Pathway are • Mannan-binding lectin (MBL) • MBL-associated serine protease-1 (MASP-1) • MBL-associated serine protease-2 (MASP-2) • Proteins in the Alternative Pathway that lead to the common portions of the classical complement pathway are • Denoted as factors (Factor B and Factor D).

Function of the Complement System • The complement system acts as an auxiliary system in immunity, both on its own and in conjunction with humoral immunity. • In its role in innate immunity, it is a primitive surveillance and defense system for microbes, independent of T cells and antibodies. • In its role in adaptive immunity, it is a major effector system for humoral immunity.

Specific Functions of the Complement System Chemotactic Agent Activator of Inflammation Complement also augments stimulation of B cells through complement receptor 2 (CR2/CD21) to increase the humoral immune response.

Activation of the Classical Pathway

Classical Pathway Lectin Pathway Alternative Pathway Antigen Antibody Complexes (IgG/IgM) Polysaccharides on Microbes; Also IgA Foreign Surfaces (LPS); Spontaneous (Nucleophiles) Activators C1q MBL MASP-1, MASP-2 C3 + H2O Factor B Factor D C1r2 C1s2 C4 C2 C3 convertase(C4b2a) C3b (Opsonin) C3 C3a C5 convertase(C4b2a3b) C5a C5 C5bC6C7C8C9 MembraneAttackComplex Terminal lyticPathway (Anaphylatoxins)

Classical Pathway Lectin Pathway Alternative Pathway Antigen Antibody Complexes (IgG/IgM) Polysaccharides on Microbes; Also IgA Foreign surfaces (LPS); Spontaneous (Nucleophiles) Activators C1q MBL MASP-1, MASP-2 C3 + H2O Factor B Factor D C1r2 C1s2 C4 C2 C3 convertase(C4b2a) C3b (Opsonin) C3 C3a C5 convertase(C4b2a3b) C5a C5 C5bC6C7C8C9 MembraneAttackComplex Terminal lyticPathway Complement Sensors (Anaphylatoxins)

Activation of C1 • C1 is present in plasma as an inactive C1qr2s2 complex • Binding of two arms of the complex to immunoglobulin (2 IgG or 1 pentamericIgM) causes conformational change in C1q. This initiates a cascade of events. • C1q conformational change  C1r conformational change • C1r conformational change C1r active enzyme • C1r active enzyme  C1s enzymatic cleavage • C1s enzymatic cleavage  C1s active enzyme • C1s active enzyme  C4 cleavage • This result of this cascade is often referred to as the C1 esterase cleavage of C4. • Cleavage of C4 is controlled by the C1 inhibitor (C1INH) • The absence or mutation of C1 inhibitor leads to hereditary angioedema (swelling of the face and respiratory airways, as well as abdominal cramps). 2 IgG/1 IgM C1q C1q C1r C1r C1s C1s C4 C4b Italics = conformational change Color = enzyme activity

Activation of C1 C1 esterase

Activation of C4 • C1 esterase cleaves C4. • C4a can act a chemoattractant • C4b has a thioester region which forms covalent bonds with molecules on the target surface. • C4b can act as an opsonin and interacts with complement receptors (CR1).

Activation of C2 • C2 interacts with C4b and is cleaved by C1s, forming a C4b2a complex on the surface. • C4b2a is the classical pathway’s C3 convertase. • Thus, C4b2a is an enzyme that cleaves C3 to C3a and C3b. Note: There is some disagreement among scientists about the nomenclature for the cleavage products for C2. For example, some scientists identify the C3 convertase as the C4b2b complex.

C3 activation • C4b2a cleaves C3, activating a labile thioester bond on C3b. • This thioester can bind COVALENTLY to free hydroxyl or amino groups, resulting in C3b covalently binding to target surfaces. • C3b bound to a surface acts as an opsonin. • Key points for the classical pathway • Activation occurs in conjunction with specific antibody • C3b and C4b covalently bind to target via thioester bonds • Because there is a series of enzymatic cleavage events, there is tremendous amplification of the signal as the signal progresses down the series.

Review of Activation of theClassical Pathway • The sequence of complement protein activation in the classical pathway is 1>4>2>3>5>6>7>8>9 • Note that 4b gets “before (b 4)” its expected place. • The classical pathway is triggered by antigen binding to (crosslinking) two IgG molecules or two subunit parts of one IgM molecule. • The cascade of proteolytic steps in the classical pathway are performed by serine esterases. • C4b and C3b bind covalently to surfaces via thioester bonds.

Sequential Enzymatic Cleavage Events in Complement Activation

Classical Pathway Lectin Pathway Alternative Pathway Antigen Antibody Complexes (IgG/IgM) Polysaccharides on Microbes; Also IgA Foreign surfaces (LPS); Spontaneous (Nucleophiles) Activators C1q MBL MASP-1, MASP-2 C3 + H2O Factor B Factor D C1r2 C1s2 C4 C2 C3 convertase(C4b2a) C3b (Opsonin) C3 C3a C5 convertase(C4b2a3b) C5a C5 C5bC6C7C8C9 MembraneAttackComplex Terminal lyticPathway Complement Sensors Enzymatic Cleavage Events (Anaphylatoxins)

Activation through C5 • Involves proteolytic cleavage steps, liberating smaller fragments from C2 through C5. The smaller fragments are soluble and can have biologic effects. The larger fragments remain bound in a complex required for the next activation step. • By convention, • Smaller fragments are denoted by the letter ‘a’ (e.g., C3a, C5a) • Larger fragments by ‘b’ (e.g., C3b, C5b) • Notable exception is C2 (C2a is the larger, active fragment). • Complexes with enzymatic activity are often denoted by a line over the top of the numbers or letters, as in • (C4b2a)

Activation of the Mannose-Binding Pathway

MBL Pathway • Activation of the MBL Pathway is primarily mediated by a protein constituent in the plasma called mannan-binding lectin (also called the mannose-binding lectin or MBL). • Activation of the MBL Pathway does not require specific antibody for activation. • Activation of the MBL Pathway occurs by a C1-independent mechanism. • Activation of the MBL pathway occurs when MBL binds to specific sugar residues like N-acetyl glucosamine or mannose that are present in the cell wall polysaccharides of microorganisms such as Salmonella, Listeria, Neisseria, Candida, etc. • MBL, which resembles C1q, interacts with MASP-1 and MASP-2 by a mechanism similar to C1q interaction with C1r and C1s, resulting in the formation of the classical pathway C3 convertase (C4b2a).

Activation of the Alternative Pathway

Alternative Pathway • Phylogenetically the oldest of the C3 activating pathways. • Does not require specific antibody/antigen binding for activation. • Can be triggered by a low level of spontaneous lysis of C3 by water to C3i that functions in a manner similar to C3b. • Can be amplified by C3b binding to foreign surface structures (LPS) or by additional cleavage by bacterial proteases.

Some Initiators or Activators of the Alternative Pathway of Complement Activation • Many Gram-negative and Gram-positive bacteria • LPS from Gram-negative bacteria • Teichoic acid from Gram-positive cell walls • Fungal and yeast cell walls (zymosan) • Some viruses and virus-infected cells • Some tumor cells • Some parasites • Human IgA, IgG and IgE in complexes • Anionic polymers (dextran sulfate) • Pure carbohydrates (agarose, inulin)

Formation of the Alternative Pathway C3 Convertase (C3bBb) • C3 tickover - spontaneous conformational change of a few C3 molecules, leading to water hydrolyzing the thiolester bond of C3 to form C3 H20 or C3i. • C3i is then deposited in a random and non-specific manner on the surfaces of host cells and pathogenic organisms alike. • On the normal host cell, bound C3i can inactivated by binding to Factor I and Factor H. • On the pathogenic organism, bound C3i can be further activated by binding to Factor B to form C3iB which is then cleaved by Factor D to form C3iBb (C3 convertase). • Properdin acts to stabilize the alternative pathway C3 convertase (C3bBb) • Surfaces rich in carbohydrate and deficient in sialic acid tend to be the best activators.

Activation and Inactivation of C3b C3 = Complement C3 FB = Factor B FD = Factor D FI = Factor I (in conjuction with Factor H, inactivates soluble C3b and C4b when deposited on the surface of a normal cell) FH = Factor H (cofactor of Factor I in mediating cleavage of C3b to its inactive form C3bi aka C3i Stablized by properdin Target Cell Membrane Normal Cell Membrane

Amplification of C3 Cleavage by Membrane-Bound C3bBb

Activation of C5 and the Terminal Complement Pathway • C5 is cleaved by either the Classical Pathway C5 convertase (C4b2aC3b) or by the Alternative Pathway C5 convertase (C3bBbC3b) into 2 fragments: C5a and C5b. • Cleavage of C5 is the last enzymatic step • C5b binds to a target and then interacts with C6, C7, C8 and C9 to form the Membrane Attack Complex in the lipid membrane. • The Membrane Attack Complex forms a transmembrane channel that allows passage of ions, compromises of the semi-permeable membrane, and causes lysisof the cell.

Activation of C5 • C5 is cleaved into 2 fragments (C5a and C5b) by either • The Alternative Pathway C5 convertase (C3bBbC3b) or • The Classical/LectinPathway C5 convertase (C4b2aC3b). • Cleavage of C5 is the last enzymatic step.

Classical Pathway Lectin Pathway Alternative Pathway Antigen Antibody Complexes (IgG/IgM) Polysaccharides on Microbes; Also IgA Foreign surfaces (LPS); Spontaneous (Nucleophiles) Activators C1q MBL MASP-1, MASP-2 C3 + H2O Factor B Factor D C1r2 C1s2 C4 C2 C3 convertase(C4b2a) C3b (Opsonin) C3 C3a C5 convertase(C4b2a3b) C5a C5 C5bC6C7C8C9 MembraneAttackComplex Terminal lyticPathway Complement Sensors (Anaphylatoxins) Non-Cleavage EventsInvolved in MAC Assembly

Non-Cleavage Events in Assembly of the Membrane Attack Complex • C5b then interacts with C6, C7, and C8. • Lysis can occur in the absence of binding of C9 but it is slower.

Classical Pathway Lectin Pathway Alternative Pathway Antigen Antibody Complexes (IgG/IgM) Polysaccharides on Microbes; Also IgA Foreign surfaces (LPS); Spontaneous (Nucleophiles) Activators C1q MBL MASP-1, MASP-2 C3 + H2O Factor B Factor D C1r2 C1s2 C4 C2 C3 convertase(C4b2a) C3b (Opsonin) C3 C3a C5 convertase(C4b2a3b) C5a C5 C5bC6C7C8C9 MembraneAttackComplex Terminal lyticPathway Complement Sensors (Anaphylatoxins) Punches Hole in Bacterial or Viral Membrane

Assembly of C9 Channel • If C9 molecules are bound to the C5bC6C7C8 complex, they form the Membrane Attack Complex that can punch a hole in the lipid membrane. • Since the Membrane Attack Complex is a transmembrane channel that allows passage of ions, it will compromise the semi-permeability of the membrane and result in lysis of the cell.

Notes on C9 Assembly • If the interaction with C5b through C9 occurs in proximity to a membrane, then the MAC assembly occurs in that membrane and lysis is the end result. • Alternatively, C5b-9 can bind to S protein in the fluid phase. In this case, lysis does not occur.

Summary of Pathways of Activation • Three Primary Pathways of Activation with different start signals • Classical – antigen antibody • Mannose binding lectin - mannose • Alternative – LPS, carbohydrates, etc • Proteolytic cleavages of complement components operate through C5 • Non-proteolytic events for assembly of C6789 membrane attack complex

Summary of Names You Need to Know Classical Pathway: C1q, C1r, C1s, C4, C2 Mannose Binding lectin pathway: MBL (mannose binding lectin) MASP-1 (MBL-associated serine protease) MASP-2 Alternative Pathway: Factor B Factor D Properdin Common to all pathways: C3 Terminal Lytic pathway: C5, C6, C7, C8, C9

Control What stops the activation? Or Why don’t we lyse all of our own cells?

Things That Limit Complement Activation • Short half life of the enzymes formed • Properties of non-activator surfaces • Inhibitors • Fluid phase inhibitors • So active fragments don’t go too far • Membrane bound inhibitors • On our own membranes • So C3b and C4b don’t attach or don’t lead to lysis of our own cells

Activation and Inactivation of C3b C3 = Complement C3 FB = Factor B FD = Factor D FI = Factor I ( inconjuction with Factor H, inactivates soluble C3b and C4b when deposited on the surface of a normal cell) FH = Factor H (cofactor of Factor I in mediating cleavage of C3b to its inactive form C3bi aka C3i Stablized by properdin Target Cell Membrane Normal Cell Membrane

The Complement System