Defensins are the main components of the defense system in animals. Most of them are composed of 29 to 42 amino acid residues, containing 3 pairs of intramolecular disulfide bonds, with a relative molecular mass of 2 to 6ku. According to their disulfide Different bond positions can be divided into three categories: α-defensins, β-defensins, and θ-defensins.
α-defensin is a highly cationic small-molecule antimicrobial peptide first isolated from rabbit lung macrophages in 1980 by the Lehrer Laboratory in the United States. It is called defensin and was later classified as α-defensin. - Defensins; mainly distributed in neutrophils of humans, rabbits, pigs, and mice, alveolar macrophages of rabbits, and Paneth cells of the small intestine of humans and rodents. The connection positions of the disulfide bonds in the α-defensin molecular chain are Cys1-Cys6, Cys2-Cys4, and Cys3-Cys5 respectively. Among them, Cys1-Cys6 connects the N-terminus and C-terminus to form a molecular macrocycle.
Beta-defensins were first discovered by Diamond et al. (1991) in bovine tracheal mucosal epithelial cells, and later found 13 species in bovine granulosa cells that were highly similar to their sequences, but their ** *Orderly is different from α-defensin, so it is named β-defensin; mainly distributed in the bone marrow of cattle and the stomach of humans and various animals (cattle, sheep, pigs, camels, reindeer, mice, rats) This short peptide has also been found in the epithelium of the intestine, respiratory tract, tongue, gums, kidneys, skin, and recently in the epithelial cells of the tongue mucosa of sika deer. Monocytes and macrophages normally lack defensins, but they can release messengers that induce epithelial cells to synthesize β-defensins. The connection positions of the disulfide bonds in the β-defensin molecular chain are Cys1-Cys5, Cys2-Cys4, and Cys3-Cys6 respectively.
Theta-defensin is a cyclic structure molecule isolated from the leukocytes of macaque monkeys by Trabi et al. in 2002 using reverse high-performance liquid chromatography. It is also known as the macaque theta-type. Defensin-1 (RTD-1) is mainly distributed in macrophages. The structure of θ-defensin is different from α-defensin and β-defensin. Its precursor (3 species have been discovered) is an α-defensin analogue, consisting of 1 stop codon and 3 cysteine ??carbons from α-defensin. The 4th residue of the skeleton is truncated, and a 9-amino acid fragment is cut from the truncated α-defensin precursor, and then sheared from beginning to end until other identical or similar nonapeptides appear. Mature θ-defensin is the product of modification and combination of two and a half defensins. Its precursor (called half-defensin) is encoded by a mutated α-defensin gene and an immature stop codon. The product, resulting in each precursor containing only 3 cysteine ??residues. The connection positions of the disulfide bonds in the θ-defensin molecular chain are Cys1-Cys4, Cys2-Cys5, and Cys3-Cys6, and the connections form a cyclic structure. Defensins can effectively kill Gram-negative bacteria and Gram-positive bacteria. Defensins at a concentration of 10 to 100 mg/L in vitro have a killing effect on a variety of bacteria, while the concentration of defensins in neutrophils is at the g/L level, far exceeding the above values, which indicates that in vivo defense Defensins may have stronger bactericidal activity. Current studies have found that defensins are significantly more capable of killing Gram-positive bacteria than Gram-negative bacteria. In vitro, the median lethal dose (LD50) of HBD-2 against Escherichia coli is 0.46 nmol/ml, the minimum inhibitory concentration (MIC) is 15 μg/ml, and the MIC against Pseudomonas aeruginosa and Staphylococcus aureus is 62 μg. /ml. In vitro experiments have shown that the MIC range of most defensins is 0.5-10 μmol/L.
As for the antibacterial activity mechanism of defensins, most researchers believe that it is mainly related to the cell membrane structure of microorganisms. The antibacterial effect of defensins can be divided into three stages:
(1) Attraction by electrostatic electricity. Defensins bind to target cell membranes. Defensins are positively charged and can bind to the negatively charged bacterial membrane lipid layer through electrostatic interaction;
(2) Channel formation. The positively charged defensin molecules or their polymers interact with the negatively charged phospholipid heads and water molecules on the bacterial plasma membrane, significantly increasing the permeability of the biological membrane. Defensins act on the membrane to form multiple stable channels;
(3) Content leakage.
After the channel is formed, when defensins enter the cells, other extracellular molecules also enter (such as peptides, proteins or inorganic ions), and important substances of the target cells (such as salt ions and macromolecules) leak out, causing the target cells to Death from irreversible damage.
Defensins can also promote the maturation of IDCs by inducing the release of cytokines and mediating the upregulation of costimulatory molecules in immature dendritic cells, thereby activating T cells and triggering specific immune responses. . Defensins can kill some enveloped viruses, such as HIV, herpes virus, and vesicular stomatitis virus, but they are not effective against non-capsid viruses. Theta-defensins also have anti-pathogen and anti-toxin effects. In vivo experiments show that defensins can delay or eradicate syphilis in rabbits and restore the subgingival flora of rabbits with periodontitis to normal. Defensins mainly cause the virus to lose biological activity by binding to the viral coat protein. This special mechanism of action also makes it difficult for microorganisms to develop resistance to it. Defensins can directly inhibit viruses. The degree of virus inhibition depends on the concentration of defensins and the tightness of intramolecular disulfide bonds. Its antiviral efficacy is also affected by factors such as time, pH value, ionic strength and temperature. In neutral Under conditions of low ionic strength and low ionic strength, defensins have strong antiviral activity, but adding serum or serum protein to the experimental system can greatly weaken the antiviral efficacy of defensins. The antiviral mechanism of defensins can generally be summarized as the following three points:
1. Closed door--preventing viruses from invading host cells
Many cells and viruses have external Membrane molecules are glycoproteins, and they protrude like brushes (see Figure 1).
Virus-infected cells adopt a "two-step" policy: first, the virus' coat, or envelope, adheres to the outer membrane of the cell; then, the virus envelope fuses with the cell membrane. Once the two membranes fuse, the virus inserts its genetic material into the cell. Defensins are inserted obliquely on the glycoprotein to prevent the virus from spreading to the cell glycoprotein (see Figure 2), so that the virus is blocked from entering the cell. Viruses that fail to enter cells are then destroyed by cells of the immune system.
2. Breakthrough - Killing Viruses
Defensins usually carry multiple positive net charges, while the virus envelope and its surface glycoprotein usually carry negative charges. This makes the defensins act like small magnets that attach to the negatively charged glycoproteins of the viral envelope. This will cause the enveloped virus to perforate, forming a breach, and the contents will leak out and cause death.
3. Mine array - prevent viral gene replication and transcription
In case the virus enters the cell, defensins can interact with adrenocorticotropic hormone (ACTH) on the cell membrane surface. Heparin sulfate glycoprotein (HSPG), low-density lipoprotein receptor (LDLR), etc. combine to initiate a cascade amplification reaction of G protein-coupled receptors and further activate phosphokinase C. These cellular messengers act like intracellular landmines that prevent viral complexes from entering the cell nucleus or preventing viral gene transcription before integrating into the host genome. Viruses that fail to integrate into the host genome are subsequently destroyed. Defensins not only directly resist pathogenic microorganisms, but also have immunomodulatory effects. Defensins enhance the activity and chemotaxis of non-specific immune cells, especially macrophages, through the action of cell signaling. Defensins can also promote the chemotaxis and proliferation of the body's T cells, enhance the body's immune response ability, regulate specific immunity, and enhance the body's active defense function.
Densins can act as effector molecules to activate cell surface receptors such as macrophages, DCs, and tracheal epithelial cells to activate the acquired immune system and organically connect innate immunity and acquired immunity. It has been demonstrated that some α-defensins and β-defensins have chemotactic activity on T cells, monocytes, and immature DCs and can induce the production of cytokines by monocytes and epithelial cells.
Neuttrophil defensins from humans, mice, pigs, and rabbits can induce mast cell degranulation and release histamine. β-defensins can also bind to human chemokine receptor 6 (CCR6), thereby attracting immature dendritic cells (DC) and memory T cells (Tm) to the inflammatory site, activating cellular immunity and humoral immunity. In addition, defensins can directly promote the recruitment and accumulation of neutrophils at the site of infection.