α-defensin is a small cationic antimicrobial peptide, which was first isolated from rabbit lung macrophages in Lehrer laboratory at 1980, and then classified as α-defensin. It is mainly distributed in neutrophils of humans, rabbits, pigs and rats, alveolar macrophages of rabbits and Paneth cell of small intestine of humans and rodents. The disulfide bond sites in α -defensins are Cys 1-Cys6, Cys2-Cys4 and Cys3-Cys5 respectively. Cys 1-Cys6 connects the N-terminal and the C-terminal to form a large molecular ring.
β-defensins were first found in bovine airway epithelial cells by Diamond et al. (199 1), and then found in bovine granulocytes. 13 was highly similar to its sequence, but its homology was different from that of α-defensins, so it was named β-defensins. It is mainly distributed in bovine bone marrow and epithelial cells of gastrointestinal tract, respiratory tract, tongue, gums, kidneys and skin of human beings and various animals (cattle, sheep, pigs, camels, reindeer, mice and rats). Recently, short peptides have also been found in epithelial cells of sika deer tongue mucosa. Monocytes and macrophages usually lack defensins, but they can release messengers that induce epithelial cells to synthesize β -defensins The disulfide bond sites in the molecular chain of β -defensin are Cys 1-Cys5, Cys2- Cys4 and Cys3-Cys6, respectively.
θ-defensin is a 1 cyclic molecule isolated from rhesus monkey leukocytes by Trabi et al. in 2002, also known as rhesus monkey θ-defensin-1(RTD- 1), which is mainly distributed in macrophages. The structure of θ-defensins is different from that of α-defensins and β-defensins. Their precursors (three of which have been found) are α-defensins analogues. The fourth residue of the three cysteine carbon skeletons of α-defensins is truncated with 1 stop codon, and the 1 fragment of 9 amino acids is cut from the truncated α-defensins precursor, and then cut from beginning to end until the others are the same or the same. Mature θ-defensin is the product of modification and combination of two semi-defensins, and its precursor (called semi-defensin) is the coding product of mutant α-defensin gene and 1 immature stop codon, resulting in only 3 cysteine residues per 1 precursor. The disulfide bonds in the molecular chain of θ-defensins are connected at Cys 1-Cys4, Cys2-Cys5 and Cys3-Cys6, respectively, forming a cyclic structure. Defensins can effectively kill gram-negative bacteria and gram-positive bacteria. In vitro, the concentration of defensin is 10 ~ 100 mg/L, which can kill many kinds of bacteria, while the concentration of defensin in neutrophils is g/L, which far exceeds the above values, indicating that defensin may have stronger bactericidal activity in vivo. At present, it is found that defensins have stronger bactericidal ability against gram-positive bacteria than gram-negative bacteria. In vitro, the median lethal dose (LD50) of HBD-2 to Escherichia coli was 0.46nmol/ml, and the minimum inhibitory concentration (MIC) was 65438 0.5 μ g/ml, while the MIC to Pseudomonas aeruginosa and Staphylococcus aureus was 62 μg/ml. In vitro experiments showed that the minimum inhibitory concentration of most defensins was 0.5-10 μ mol/L.
Most researchers believe that the antibacterial mechanism of defensins is mainly related to the cell membrane structure of microorganisms. Defensins can play an antibacterial role in three stages:
(1) is attracted by static electricity. Defensin binds to the target cell membrane. Defensins are positively charged and can be combined with negatively charged bacterial membrane lipid layers through electrostatic interaction.
(2) channel formation. The positively charged defensin molecules or their polymers interact with negatively charged phospholipid heads and water molecules on bacterial plasma membranes, which significantly increases the permeability of biofilm. Defensins act on cell membranes to form stable multi-channels;
(3) Leakage of contents. After the channel is formed, defensins enter the cell, and other extracellular molecules also enter (such as peptides, protein or inorganic ions), while important substances (such as salt ions and macromolecules) of the target cell ooze out, causing irreversible damage and death of the target cell.
Defensins can also induce the release of cytokines and mediate the up-regulation of costimulatory molecules in immature dendritic cells, thus activating T cells, triggering specific immune responses and promoting the maturity of IDC. Defensins can kill some enveloped viruses, such as HIV, herpes virus and vesicular stomatitis virus, but they are ineffective against capsid-free viruses. Theta-defensins also have antiviral and antitoxin effects. In vivo experiments show that defensins can delay or eradicate syphilis in rabbits and make subgingival flora of periodontitis in rabbits return to normal. Defensins mainly bind to viral coat proteins, resulting in the loss of viral biological activity. This special mechanism also makes it difficult for microorganisms to resist them. Defensins can directly inhibit the virus, and the degree of inhibition depends on the concentration of defensins and the tightness of intramolecular disulfide bonds. Its antiviral effect is also affected by time, pH value, ionic strength, temperature and other factors. Defensins have strong antiviral activity under neutral and low ionic strength conditions, but adding serum or serum protein to the experimental system will greatly weaken the antiviral effect of defensins. The antiviral mechanism of defensins can be summarized as follows:
1, close the door-stop the virus from invading the host cell.
The outer membrane molecules of many cells and viruses are glycoproteins, which protrude like brushes (see figure 1).
The virus-infected cells adopt a "two-step" strategy: first, the coat of the virus, that is, the envelope, adheres to the outer membrane of the cell; Then, the virus envelope is fused with the cell membrane. After the two membranes are fused, 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 cannot enter the cell in the "closed" state. Viruses that fail to enter cells are subsequently destroyed by cells of the immune system.
2, breakthrough-kill the virus
Defensins usually have multiple positive charges, while virus envelopes and their surface glycoproteins usually have negative charges. This makes defensins adsorb to the negatively charged glycoprotein of the virus envelope like small magnets. In this way, the envelope virus is perforated, forming a breakthrough, and the contents leak out and die.
3, minefield-to prevent virus gene replication and transcription.
In case the virus enters the cell, defensins can combine with adrenocorticotropic hormone (ACTH), human heparin sulfate glycoprotein (HSPG) and low density lipoprotein receptor (LDLR) on the cell membrane surface, thus starting the cascade amplification reaction of G protein-coupled receptors and further activating phosphokinase C. These cell messengers are like mines embedded in the cell, which can prevent the virus complex from entering the nucleus before being integrated into the host genome, or prevent the transcription of virus genes. Viruses that failed to integrate the host genome were subsequently destroyed. Defensins can not only directly resist pathogenic microorganisms, but also have immunomodulatory effects. Defensins enhance the activity and chemotaxis of nonspecific immune cells, especially macrophages, through cell signal transmission. Defensins can also promote the chemotaxis and proliferation of T cells, enhance the ability of immune response, regulate specific immunity, and enhance the body's active defense function.
Defensins can be used as effector molecules to activate cell surface receptors such as macrophages, DC and tracheal epithelial cells, thus starting the acquired immune system and organically connecting innate immunity with acquired immunity. It has been proved that some α -defensins and β -defensins have chemotactic activity on T cells, monocytes and immature DC, and can induce monocytes and epithelial cells to produce cytokines.
Neutrophil defensins from human, mice, pigs and rabbits can induce mast cells to degranulate and release histamine. β -defensins can also bind to human chemokine receptor 6(CCR6), thus attracting immature dendritic cells (DC) and memory T cells (Tm) to the inflammatory site, and activating cellular immunity and humoral immunity. In addition, defensins can directly promote the replenishment and accumulation of neutrophils in infected areas.