Classical, Alternative and Lectin Pathway of Complement System

Overview of pathways of complement activation

Classical Pathway of Complement System

  • The classical pathway is a chain of events in which complement components react in specific sequences as a cascade resulting in cell lysis.
  • It is activated by antibody bound to antigen but never by native or free antibody.

Activators of the classical pathway: Activators of classical pathways include the following:

  • Immunoglobulins IgM and IgG.
  • The IgG subclasses vary with regard to their efficiency in activating the complement.
  • IgG3 immunoglobulins are the most efficient, followed by IgG1 and IgG2. IgG4 immunoglobulins do not activate the classical pathway.
  • Native, free IgG or IgM do not activate the complement system.
  • A single, native IgG molecule will not bind and activate the complement pathway.
  • However, if antibodies of the IgG class are aggregated by antigen binding, this will result in complement fixation and activation.
  • The formation of an antigen–antibody complex induces conformational changes in the Fc portion of the IgM molecule that expose a binding site for the C1 component of the complement system.
  • Staphylococcal protein A
  • C-reactive protein
  • DNA

Steps of activation of classical pathway:

  • The classical pathway of complement activation usually begins with the formation of soluble antigen–antibody complexes (immune complexes) or with the binding of antibody to antigen on a suitable target, such as a bacterial cell.

Following are the sequential steps in the classical pathway:


  • Activation of C1 is the first step in the cascade of classical pathway activation.
  • The C1 actually is a complex of three different types of molecules: C1q, C1r, and C1s. C1q first combines with the Fc portion of the bound antibody, IgM or IgG.
  • This results in the sequential activation of C4, C2, and C3.
  • For C1 to be activated, it must bind to at least two adjacent Fc regions.
  • This means that the concentration of antibody of the IgG class must be relatively high and that the specific antigenic determinants recognized by the IgG antibody must be in close proximity.
  • When pentameric IgM is bound to antigen on a target surface, it assumes the so-called stable configuration, in which at least three binding sites for the C1q are exposed.
  • Since IgG molecules have a lower valency, about 1000 of them are needed to ensure the initiation of the complement pathway as against only one IgM molecule.

The classical pathway of complement


  • C1q binding in the presence of calcium ions leads to activation of C1r and C1s.
  • Activated C1s is an esterase that splits C4 into two fragments: a small soluble fragment (C4a) and a larger fragment (C4b).
  • C4a has anaphylatoxin activity, and C4b binds to cell membrane along with C1. C4b in the presence of Mg2+ splits C2 into C2a and C2b.
  • The smaller fragment (C2b) diffuses away, while the larger fragment (C2a) remains attached to C4b.
  • The resulting C4b2a complex possesses enzymatic activity and is called C3 convertase, which converts C3 into an active form.


  • The C3 convertase activate thousands of C3 molecules and splits these molecules into C3a and C3b.
  • A single C3 convertase molecule can generate over 200 molecules of C3b, resulting in tremendous amplification at this step of the sequence.
  • The biological importance of activated C3b as well as C4b is that they are able to bind to C3b/C4b receptors (currently designated as CR1 receptors) present on almost all host cells, most notably phagocytes.
  • The increased affinity of phagocytic cells for C3b (or iC3b)/C4b-coated particles is known as immune adherence.
  • The latter is responsible for a significant enhancement of phagocytosis, which is one of the main defense mechanisms of the body.


  • Some of the C3b binds to C4b2a to form a trimolecular complex C4b2a3b called C5 convertase.
  • The C5 convertase splits C5 into C5a and C5b.
  • C5a diffuses away, while C5b attaches to C6 and initiates formation of C5b–9 complex otherwise known as membrane attack complex (MAC).
  • Released C5b67 complexes can insert into the membrane of nearby cells and mediate “innocent-bystander” lysis.
  • Regulator proteins in human sera normally prevent this from occurring, but in certain diseases cell and tissue damage may occur due to this process of innocent-bystander lysis.
  • The membrane-bound C5b–6–7 complex acts as a receptor for C8 and C9.
  • C8, on binding to the complex, stabilizes the attachment of the complex to the foreign cell
  • The C5b–8 complex acts as a catalyst for C9, which is a single chain glycoprotein with a tendency to
    polymerize spontaneously.


  • The C5b–8 complex on binding to C9 molecules undergoes polymerization, which finally ends in the formation of C5b–9 complex also known as MAC.
  • The MAC forms a transmembrane channel of 100 Å diameter in the cell.
  • This transmembrane channel allows the free exchange of ions between the cell and the surrounding medium.
  • Due to the rapid influx of ions into the cell and their association with cytoplasmic proteins, the osmotic
    pressure rapidly increases inside the cell.
  • This results in an influx of water, swelling of the cell, and, for certain
    cell types, rupture of the cell membrane and finally lysis.

Biological effects of complement activation

Membrane attack complex:

  • The formation of the MAC is the terminal sequence of all three different pathways including the classical pathway.
  • All the three pathways converge at the step involving the formation of the MAC.
  • The formation of the MAC involves the participation of the complement components C5b, C6, C7, C8, and C9.
  • A complex of C5b, C6, and C7 is first formed in the soluble phase and then attaches to the cell membrane through the hydrophobic amino acid groups of C7.
  • The C7 becomes exposed as a consequence of the binding of C7 to the C5b–C6 complex.

Action of membrane attack complex

Although the classical pathway is generally activated by the antigen-antibody complex or aggregated immunoglobulin, activation may also be due to other stimuli, such as DNA, C reactive protein, trypsin-like enzymes or some retroviruses.

Alternative Pathway of Complement System

  • The alternative pathway was first described by Pillemer in 1954.
  • It differs from the classical pathway in (a) the nature of activating substances and (b) the sequence of events itself.
  • The alternative pathway is unique in not requiring antigen–antibody complexes to activate the complement.
  • This pathway does not depend on antibody and does not involve the early complement components (C1, C2, and C4) for activation of the complement.
  • It, therefore, can be activated before the establishment of an immune response to the infecting pathogen.

Activators of the alternative pathway:

Activators of the alternative pathway of complement activation include:

(a) IgA

(b) IgD

(c) bacterial endotoxin

(d) cobra venom factor

(e) nephritic factor.

Steps of activation of alternative pathway:

The initial component of the alternative pathway involves four serum proteins:

C3b, factor B, factor D, and properdin.


  • The C3b binds with factor B to form C3bB complex.
  • The interaction between C3b and factor B is stabilized by Mg2+, which is the only ion required for functional activation of the alternative pathway.
  • Therefore, tests to discriminate between the two complement activation pathways are often based on the selective chelation of Ca2+ (to disrupt C1q, C1r2, and C1s2) and the addition of sufficient Mg2+ to allow activation of the alternative pathway.


  • The C3bB is split into two fragments, Ba and Bb, by another serum protein called factor D or C3 proactive convertase.
  • Since factor D has never been isolated in its proenzyme form, it is generally believed to be activated immediately upon leaving the hepatocyte where it is synthesized.
  • The Ba is released into the medium and the Bb binds to C3b forming the C3bBb complex, which possesses the C3 convertase activity.


  • The C3bBb complex activates more C3, leading to the formation of more C3bBb, which in turn is capable of activating C5 and the MAC.
  • The C3bBb complex has a half-life of only 5 minutes, but by binding with properdin it forms PC3bBb complex, which is relatively heat stable.


  • The alternative pathway then proceeds from C3 to produce finally the MAC, in the same way as occurs in the classical pathway.

Alternative pathway of complement

Lectin Pathway of Complement Activation

  • The lectin pathway, as the name suggests, is triggered by lectins. 
  • Lectins are the proteins that recognize and bind to specific carbohydrate targets.
  • The mannose-binding lectin (MBL) is one such protein that takes part in the lectin pathway of complement activation.
  • MBL is a large serum protein that binds to nonreduced mannose, fructose, and glucosamine on bacterial and other cell surfaces with mannose-containing polysaccharides.
  • The binding of MBL to a pathogen results in the secretion of two MBL-associated serine proteases: MASP-1 and MASP-2.
  • MASP-1 and MASP-2 are similar to C1r and C1s, respectively, and MBL is similar to C1q.
  • Formation of the MBL/MASP-1/ MASP-2 trimolecular complex results in activation of MASPs and subsequent cleavage of C4 into C4a and C4b.
  • Subsequently, it proceeds to produce MAC in the same way as that occurs in the classical and alternative pathways.

Overview of pathways of complement activation

Classical, Alternative and Lectin Pathway of Complement System

Also Read: 

Complement System: Properties, Components and Activation


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