Antimicrobial peptides and enzymes

Phagolysosomal enzymes and cationic proteins

During phagocytosis, the destruction of microorganisms is carried out by more than 60 enzymes derived from neutrophil granules. Membrane fusion occurs in two stages and strongly depends on the pH of the medium:

1. Fusion with specific granules. Occurs at neutral or slightly alkaline pH. Enzymes active in this environment enter the phagosome: alkaline phosphatase and lysozyme.
2. Fusion with azurophilic granules. Occurs as the medium becomes acidified. The granules deliver acid hydrolases (myeloperoxidase, glycosidases, acid glycerophosphatase, $5'$-nucleotidase) and additional portions of lysozyme.

A special role is played by cationic proteins, whose positive charge is due to a high content of basic amino acids. These include enzymes (lysozyme, serprocidins) and competitive proteins.

Lysozyme (15 kDa) is an enzyme with muramidase activity. It depolymerizes peptidoglycans of the microbial cell wall. However, in an intact bacterium, the substrate is hidden by surface molecules, so lysozyme only works in combination with other phagolysosomal enzymes.

Serprocidins, lactoferrin, and BPI proteins

Serprocidins (Serine protease cidin) are a group of proteins with protease activity with a mass of 25–29 kDa (cathepsin G, elastase, azurocidin, proteinase-3). Their microbicidal effect in vitro does not correlate with enzymatic activity. Cathepsin G has the highest efficacy, functioning even in a fragmented state. Extracellularly, their activity is inhibited by special inhibitors — serpins.

Lactoferrin is a marker of specific granules. In the presence of bicarbonate ions, it tightly binds two $Fe^{3+}$ ions, which is why it was originally called the «red protein». Its main mechanism is bacteriostatic: it creates an iron deficiency without which bacteria cannot multiply.

BPI proteins (Bacteria permeability inducing) are eosinophil and neutrophil proteins that selectively target Gram-negative bacteria. They recognize lipopolysaccharide (LPS), block its activity, and sharply increase the permeability of the bacterial wall to proteins.

Bactericidal peptides: defensins and cathelicidins

These are small amphipathic molecules that kill pathogens independently of receptors.

Defensins are peptides (3–5 kDa) with a $\beta$-sheet structure. Their spatial framework is maintained by 3 disulfide bonds formed by 6 cysteine residues. Classification depends on the arrangement of these bonds:

• $\alpha$-defensins (cyclic due to the 1–6 bond). Make up to 5–7% of total neutrophil protein, present in macrophages and intestinal crypt epithelium.
• $\beta$-defensins (non-cyclic). Located in the respiratory epithelium. Important: they are absent in human leukocytes.
• $\theta$-defensins form a cycle via a peptide bond.

Cathelicidins are peptides whose structure is dominated by $\alpha$-helices. Homologous to cathepsin L. Human neutrophils contain only one cathelicidin variant — LL-37.

Mechanism of peptide action and degranulation

Antimicrobial peptides disrupt membranes via the «barrel-stave» model:

1. Due to electrostatic forces, the positively charged regions of the peptide bind to negative groups (LPS, peptidoglycans) on the bacterial surface.
2. Hydrophobic regions of the amphipathic molecule insert into the lipid bilayer of the microbe.
3. Monomers assemble to form a cyclic structure («barrel») — a transmembrane channel. Ions leak through it, and the bacterium dies.

The release of granule contents to the outside (e.g., upon completion of phagocytosis) is called degranulation. Phagolysosomes move along microtubules (via actomyosin) toward the cell membrane. A key stage is the recognition of membranes using proteins of the SNARE family. SNAP-23 and syntaxin-4 appear on the cell membrane, docking with syntaxin-6 and VAMP-2 on the granule membrane to ensure fusion.

Briefly: answers to topical questions

Class and exam questions

What else to study on this topic

• The effect of an acidic environment on the electrostatic membrane potential of a microbial cell.
• The role of serpins in suppressing the extracellular activity of serprocidins and defensins.
• Phagocyte secretion dependent on the Golgi apparatus (an alternative to degranulation).
• Chemokine-like and immunoregulatory properties of extracellular defensins.
• Features and role of degranulation in mast cells, eosinophils, and basophils.

Other topics in the "Innate Immunity" section

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