Complement Membrane Attack Complex/MAC structure

Structure and Assembly Functions MAC Assays MAC in Disease Therapeutic Targeting

The membrane attack complex (MAC) is a crucial innate immune effector of the complement terminal pathway that forms cytotoxic pores on the surface of microbes. The complement system, composed of over 35 proteins found in the plasma or bound to host cells, forms an integral part of the early immune response. There are three major pathways of complement system activation, the classical, the alternative and the mannose-binding lectin pathways. They can activate the terminal pathway, including the formation of MAC, shown in the late steps of Fig.1. Activation of complement triggers assembly of MAC to form a multiprotein pore that inserts into and then directly lyses microbes.

Fig. 1 Pathways of complement system activation.^{1, 4}

Structure and Assembly of MAC

In response to complement system activation, MAC assembles from fluid-phase proteins to form pores in lipid bilayers. MAC assembly needs the sequential and irreversible association of complement proteins including C5b, C6, C7, C8, C9.

Molecular Composition

MAC comprises five complement proteins.

C5b: Initiates assembly after cleavage from C5.
C6 and C7: Stabilize membrane insertion.
C8 (α, β, γ subunits): Anchors the complex to the lipid bilayer.
C9 (10–18 monomers): Forms the transmembrane β-barrel pore.

All components share a conserved MACPF domain, evolutionarily related to bacterial cholesterol-dependent cytolysins.

Assembly Mechanism

The whole process of assembly of MAC is shown in Fig.2. The first step needs cleavage of C5 (purple) into the small anaphylatoxin C5a and the large fragment C5b via the C5 convertase (turquoise). Then, C6 (yellow) binds the labile C5b intermediate and forms the C5b6 complex. After that, C7 (green) binds C5b6 and the newly formed C5b7 complex is anchored to the membrane surface. Subsequently, C8, which contains a heterotrimeric protein composed of C8a (orange), C8b (red) and C8g (dark blue), is incorporated into the assembly precursor forming C5b8 and marking the first membrane penetration event. In the last step, multiple copies of C9 (light blue) take part in the assembly and span membrane to form the final MAC.

Fig. 2 Illustration of the stepwise MAC assembly pathway from soluble complement factors.^{2, 4}

Table 1 Basic structural features.

Features	Description
Split-washer shape	Asymmetric arrangement creates membrane distortion alongside pore formation.
Giant β-barrel	Irregular β-sheet architecture spans the membrane, distinct from α-helical models.
Hybrid pore	Combines rigid C8α-γ core with flexible C9 extensions for adaptive membrane penetration.

Functions of MAC

The MAC serves as the terminal effector of the complement system, combining direct pathogen elimination with immunomodulatory signaling. Its functions emerge from coordinated interactions among its components (C5b, C6, C7, C8, C9), which orchestrate both cytolytic and non-cytolytic mechanisms.

Fig. 3 Membrane attack complex (MAC) formation and the resultant consequences in target cell.^{3, 4}

Primary Functions

Cytolytic pathogen elimination

MAC disrupts target cell membranes through pore formation:

Osmotic lysis: Transmembrane channels (10–100 nm) allow uncontrolled ion/water flux, causing cell swelling and rupture.
Efficiency against pathogens: Particularly effective against Gram-negative bacteria, where MAC pores bypass protective lipopolysaccharide layers.

Non-cytolytic immune signaling

Sublytic MAC deposition activates intracellular pathways:

NF-κB activation: Drives proinflammatory cytokine production (IL-6, TNF-α).
Inflammasome assembly: MAC-induced K⁺ efflux and mitochondrial ROS trigger NLRP3 activation, promoting IL-1β/IL-18 secretion.
Cellular adhesion: Upregulates endothelial P-selectin, enhancing leukocyte recruitment.

Component-Specific Roles

Activation of the complement system of the immune system leads to the formation of the enzyme C5 convertase, production of C5b and assembly of the MAC. MAC consists of a complex of four complement proteins (C5b, C6, C7, and C8) that bind to the outer surface of the plasma membrane, and many copies of a fifth protein (C9) that hook up to one another, forming a ring in the membrane. Each complement protein plays an important role in the molecular assembly of MAC.

Table 2 The role of each complement protein of MAC.

Component	Function	Clinical Relevance
C5b	Initiates MAC assembly after C5 cleavage Reacts with C6 to form the stable C5b6 complex	Deficiency increases susceptibility to Neisseria infections.
C6	Stabilizes C5b and recruits C7 Critical for membrane insertion	C6 deficiency linked to recurrent meningococcal disease.
C7	Anchors MAC precursors to lipid bilayers via hydrophobic domains	Reduced C7 levels correlate with pyoderma gangrenosum and ovarian cancer progression.
C8	Bridges C5b-7 to C9 α-γ subunit penetrates membranes	C8β deficiency impairs MAC formation, increasing infection risk.
C9	Polymerizes (10–18 subunits) to form β-barrel pores	C9 deficiency delays pathogen lysis but reduces autoimmune pathology.

MAC Assays

Assays for the MAC are crucial for understanding its role in disease pathogenesis and for monitoring complement activation. These assays can detect MAC components or its soluble form, sC5b-9, which is a complement activation product.

Immunofluorescence and Immunohistochemistry (IHC)

These techniques are used to visualize MAC components (C5b-9) in tissue sections, helping to identify MAC deposition in specific diseases.
Tissue sections are stained with antibodies against MAC components (e.g., C9 and C5b-9) using protocols like multiplex IHC, which allows simultaneous detection of multiple markers.

ELISA Kits

ELISA kits are used for quantifying MAC or its soluble form, sC5b-9, in biological fluids.
These kits contain pre-coated plates with antibodies specific to MAC components. Samples are added, and bound MAC is detected using enzyme-conjugated secondary antibodies.

Surface-Enhanced Raman Spectroscopy (SERS)

SERS is a sensitive technique for detecting MAC components on extracellular vesicles (EVs).
EVs are captured using antibodies against tetraspanins (e.g., CD9, CD63, CD81), and then labeled with SERS nanotags targeting C9 or C5b-9. The Raman signal is used to quantify MAC-positive EVs.

Soluble Terminal Complement Complex (sC5b-9) Assays

These assays measure the level of sC5b-9 in plasma, which is a biomarker for complement activation.
Enzyme immunoassays (EIA) are commonly used to quantify sC5b-9 levels, providing insights into systemic complement activation.

MAC in Disease Pathogenesis

MAC plays a pivotal role in the pathogenesis of various diseases, primarily through its cytolytic and proinflammatory effects. While MAC is essential for eliminating pathogens, dysregulated or excessive MAC activity can lead to tissue damage and contribute to autoimmune, inflammatory, and neurological disorders.

Table 3 The roles of MAC in the pathogenesis of various diseases.

	Disease	Mechanism of Action
Autoimmune and Inflammatory Disorders	Lupus Nephritis	Glomerular MAC deposition: Causes podocyte injury and proteinuria, contributing to renal dysfunction. Immune complex activation: Predominantly involves the classical pathway, with MAC formation exacerbating inflammation.
Autoimmune and Inflammatory Disorders	Rheumatoid Arthritis	Synovial MAC activation: Triggers fibroblast-mediated joint destruction and cytokine release, promoting inflammation. Inflammatory signaling: Sublytic MAC deposition activates NF-κB pathways, enhancing local inflammation.
Neurological Disorders	Multiple Sclerosis	MAC contributes to demyelination by disrupting oligodendrocyte membranes and inducing inflammatory responses.
Neurological Disorders	Neuronal damage	MAC triggers excitotoxicity and apoptosis in neurons, contributing to neurodegenerative diseases.
Infection Susceptibility	Neisseria Infections	MAC deficiency: Increases susceptibility to Neisseria meningitidis infections due to impaired pathogen lysis. Evolutionary trade-offs: Reduced MAC activity may confer protection against autoimmune diseases but increases infection risk.
Kidney Diseases	C3 Glomerulonephritis and Dense Deposit Disease	Activation of the alternative pathway leads to C5b-9 formation, contributing to glomerular injury.
Kidney Diseases	Primary Membranous Nephropathy	Involves the lectin pathway, with MAC deposition contributing to disease progression.

Therapeutic Targeting of MAC

Therapeutic targeting of the MAC has emerged as a promising strategy for managing diseases associated with complement system dysregulation. By inhibiting MAC assembly or its downstream effects, treatments aim to reduce tissue damage while preserving immune defense mechanisms.

Inhibiting MAC assembly

Prevent C5 cleavage, blocking MAC initiation
Disrupt pore formation without affecting opsonization
Accelerate MAC clearance from host cells

Targeting Complement Pathways

Block C3 activation, preventing downstream complement activation, including MAC formation.
Target factor B to reduce alternative pathway activation, which is a major contributor to MAC formation.

As pioneers in complement-targeted therapies, Creative Biolabs offers:

If you want more information, please feel free to contact us.

References

Girardi, Guillermina, et al. "Essential role of complement in pregnancy: from implantation to parturition and beyond." Frontiers in immunology 11 (2020): 1681. https://doi.org/10.3389/fimmu.2020.01681
Bayly-Jones, Charles, Doryen Bubeck, and Michelle A. Dunstone. "The mystery behind membrane insertion: a review of the complement membrane attack complex." Philosophical Transactions of the Royal Society B: Biological Sciences 372.1726 (2017): 20160221. https://doi.org/10.1098/rstb.2016.0221
Andrade, Fabiana A., et al. "Serine proteases in the lectin pathway of the complement system." Proteases in Physiology and Pathology (2017): 397-420. https://doi.org/10.1007/978-981-10-2513-6_18
under Open Access license CC BY 4.0, without modification

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