| 1.C.85 The Pore-Forming Beta-Defensin (β-Defensin) Family
β-defensins are small antimicrobial polypeptides that are mainly expressed by epithelial cells and play an important role in the antimicrobial innate immune response. In addition to the direct microbicidal effects of these polypeptides, certain members of the β-defensin superfamily have the capacity to promote local innate inflammatory and systemic adaptive immune responses by interacting with the CC-chemokine receptor CCR6. Rohrl et al. (Rohrl et al. 2008) have identified mouse β-defensin 14 (mBD14, Defb14) as an orthologue of human β-defensin 3 (hBD3 or DEFB103). Based on primary structural analysis, mBD14 demonstrates greater (68%) homology to its human orthologue, containing three conserved cysteine linkages, characteristic of the β-defensin super family. mBD14 is expressed in a wide variety of tissues including spleen, colon, and tissues of the upper and lower respiratory tract. Rohrl et al. (Rohrl et al. 2008) also detected mBD14 expression in immature CD11c+ bone marrow-derived dendritic cells. The expression of mBD14 could be induced by Toll-like receptor agonists such as lipopolysaccharide and poly(I:C) and by pro-inflammatory stimuli e.g. tumor necrosis factor and interferon-gamma. Furthermore, expression of mBD14 seemed to be regulated by activation of the intracellular pattern recognition receptor NOD2/CARD15 as revealed by reporter gene analysis. Both hBD3 and mBD14 were chemotactic for freshly isolated mouse resident peritoneal cells. Thus, based on structural and functional similarities, mBD14 appears to be an orthologue of hBD3 (Rohrl et al., 2008).
β-defensins are important in mammalian immunity, displaying both antimicrobial and chemoattractant activities. These glycopeptides are mainly expressed in epithelial cells and have been shown to form pores in biological membranes (Zanich et al, 2003). Their 3-D structures are known (2NLS_A; Hover et al, 2000, Hover et al, 2001). Three canonical disulfide intramolecular bonds are believed to be dispensable for antimicrobial activity but essential for chemoattractant ability. However, HBD3 (human beta-defensin 3) alkylated with iodoactemide and devoid of any disulfide bonds is still a potent chemoattractant (Taylor et al., 2008). Furthermore, when the canonical six cysteine residues are replaced with alanine, the peptide is no longer active as a chemoattractant. The chemoattractant activities of HBD3 are restored by introduction of a single cysteine in the fifth position (Cys V) of the beta-defensin six cysteine motif. In contrast, a peptide with a single cysteine at the first position is inactive.
A range of overlapping linear fragments do not act as chemoattractants, suggesting that the chemotactic activity of this peptide is not dependent solely on an epitope surrounding Cys V. Full-length peptides either with alkylated cysteine residues or with cysteine residues replaced with alanine are still strongly antimicrobial. Defb14 peptide fragments were also tested for antimicrobial activity, and peptides derived from the N-terminal region display potent antimicrobial activity. Thus, the chemoattractant and antimicrobial activities of beta-defensins can be separated (Taylor et al., 2008), and both of these functions are independent of intramolecular disulfide bonds. These findings are important for further understanding of the mechanism of action of defensins and for therapeutic design.
Shafee et al. 2016 have
suggested that defensins and small defensin-like proteins fall into two
superfamilies, which they call the cis-defensins (broadly distributed
in living organisms) and the trans-defensins (narrowly distrubuted).
They suggest that these two groups of proteins converged to show similar
sequences, secondary and tertiary structures, and disulfide
connectivities, with overlapping organismal sources and functions, in
spite of their independent origins. The functions of these short
proteins vary tremendously including pore formation, bacterial and
fungal toxicity, lipid targeting, toxic receptor and channel
interactions, fertilization, protease inhibiton and stress adaptation.
However, as noted by the authors, alternative pathways involving
divergent evolution from a common evolutionary source could have also
occurred although they consider this possibility less likely (Shafee et al. 2017).
The generalized transport reaction catalyzed by β-defensin is:
small molecules (in)  small molecules (out)
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This family belongs to the Defensin Superfamily.
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| References: |
Colavita, I., E. Nigro, D. Sarnataro, O. Scudiero, V. Granata, A. Daniele, A. Zagari, A. Pessi, and F. Salvatore. (2015). Membrane protein 4F2/CD98 is a cell surface receptor involved in the internalization and trafficking of human β-Defensin 3 in epithelial cells. Chem Biol 22: 217-228.
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da Silva, L.G., C.R.L. Costa-Júnior, C.A.S. Figueiredo-Júnior, T.C. Leal-Balbino, S. Crovella, D. Otranto, V.Q. Balbino, and F. Dantas-Torres. (2017). Canine β-defensin-1 (CBD1) gene as a possible marker for Leishmania infantum infection in dogs. Parasit Vectors 10: 199.
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Hayashi, M.A., F.D. Nascimento, A. Kerkis, V. Oliveira, E.B. Oliveira, A. Pereira, G. Rádis-Baptista, H.B. Nader, T. Yamane, I. Kerkis, and I.L. Tersariol. (2008). Cytotoxic effects of crotamine are mediated through lysosomal membrane permeabilization. Toxicon 52: 508-517.
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Hoover, D.M., K.R. Rajashankar, R. Blumenthal, A. Puri, J.J. Oppenheim, O. Chertov, and J. Lubkowski (2000). The structure of human β- defensin-2 shows evidence of higher order oligomerization. J. Biol. Chem. 275: 32911-32988.
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Hoover, D.M., O. Chertov, and J. Lubkowski. (2001). The structure of human β- defensin-1: new insights into structural properties of β- defensins. J. Biol. Chem. 276: 39021-39026.
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Huang, C., J.R. Morlighem, H. Zhou, &.#.2.0.1.;.P. Lima, P.B. Gomes, J. Cai, I. Lou, C.D. Pérez, S.M. Lee, and G. Rádis-Baptista. (2016). The Transcriptome of the Zoanthid Protopalythoa variabilis (Cnidaria, Anthozoa) Predicts a Basal Repertoire of Toxin-like and Venom-Auxiliary Polypeptides. Genome Biol Evol 8: 3045-3064.
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Kerkis, A., I. Kerkis, G. Rádis-Baptista, E.B. Oliveira, A.M. Vianna-Morgante, L.V. Pereira, and T. Yamane. (2004). Crotamine is a novel cell-penetrating protein from the venom of rattlesnake Crotalus durissus terrificus. FASEB J. 18: 1407-1409.
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Oguiura, N., M. Boni-Mitake, R. Affonso, and G. Zhang. (2011). In vitro antibacterial and hemolytic activities of crotamine, a small basic myotoxin from rattlesnake Crotalus durissus. J Antibiot (Tokyo) 64: 327-331.
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Peigneur, S., D.J. Orts, A.R. Prieto da Silva, N. Oguiura, M. Boni-Mitake, E.B. de Oliveira, A.J. Zaharenko, J.C. de Freitas, and J. Tytgat. (2012). Crotamine pharmacology revisited: novel insights based on the inhibition of KV channels. Mol Pharmacol 82: 90-96.
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Röhrl, J., D. Yang, J.J. Oppenheim, and T. Hehlgans. (2008). Identification and biological characterization of mouse β- defensin 14, the orthologue of human β- defensin 3. J. Biol. Chem. 283: 5414-5419.
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Sass, V., U. Pag, A. Tossi, G. Bierbaum, and H.G. Sahl. (2008). Mode of action of human β-defensin 3 against Staphylococcus aureus and transcriptional analysis of responses to defensin challenge. Int. J. Med. Microbiol. 298: 619-633.
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Shafee, T.M., F.T. Lay, M.D. Hulett, and M.A. Anderson. (2016). The Defensins Consist of Two Independent, Convergent Protein Superfamilies. Mol Biol Evol 33: 2345-2356.
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Shafee, T.M., F.T. Lay, T.K. Phan, M.A. Anderson, and M.D. Hulett. (2017). Convergent evolution of defensin sequence, structure and function. Cell Mol Life Sci 74: 663-682.
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Taylor, K., D.J. Clarke, B. McCullough, W. Chin, E. Seo, D. Yang, J. Oppenheim, D. Uhrin, J.R. Govan, D.J. Campopiano, D. MacMillan, P. Barran, and J.R. Dorin. (2008). Analysis and separation of residues important for the chemoattractant and antimicrobial activities of β- defensin 3. J. Biol. Chem. 283: 6631-6639.
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van Damme, C.M., T. Willemse, A. van Dijk, H.P. Haagsman, and E.J. Veldhuizen. (2009). Altered cutaneous expression of β-defensins in dogs with atopic dermatitis. Mol Immunol 46: 2449-2455.
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Zanich, A., J.C. Pascall, and R. Jones. (2003). Secreted epididymal glycoprotein 2D6 that binds to the sperm's plasma membrane is a member of the β- defensin superfamily of pore-forming glycopeptides. Biol. Reprod. 69: 1831-1842.
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Zhang, K., H. Zhang, C. Gao, R. Chen, and C. Li. (2020). Antimicrobial Mechanism of pBD2 against. Molecules 25:.
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Zhang, Y., Y. Zhao, H. Liu, W. Yu, F. Yang, W. Li, Z. Cao, and Y. Wu. (2018). Mouse β-Defensin 3, A Defensin Inhibitor of Both Its Endogenous and Exogenous Potassium Channels. Molecules 23:.
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| Examples: |
| TC# | Name | Organismal Type | Example |
| 1.C.85.1.1 | β-defensin-1 | Animals | β-defensin-1 of Homo sapiens (P60022) |
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| 1.C.85.1.10 | Canine β-defensin 107, cBD107, of 70 aas and 1 N-terminal TMS (van Damme et al. 2009). | | cBD107 of Canis lupus familiaris (Dog) (Canis familiaris) |
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| 1.C.85.1.11 | Porcine β-defensin-2 of 69 aas and 1 N-terminal TMS. Porcine beta defensin 2 (pBD2) caused the bacterial membranes to be broken, bulging, and perforated (Zhang et al. 2020). pBD2 may have multiple modes of action, but the main mechanism by which pBD2 kills S. aureus is the destruction of the membrane. It is 63% identical to human β-defensin-1. | | pBP2 of Sus scrofa |
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| 1.C.85.1.2 | β-defensin-2 | Animals | β-defensin 2 of Homo sapiens (O15263) |
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| 1.C.85.1.3 | β-defensin-3 of 67 aas and 1 N-terminal TMS. Canine BD103 (van Damme et al. 2009) is 79% identical. | Animals | β-defensin-3 of Homo sapiens (P81534) |
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| 1.C.85.1.4 | β-defensin-14 | Animals | β-defensin-14 of Mus musculus (Q7TNV9) |
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| 1.C.85.1.5 | Epididymus sperm-associated antigen (EP2E) | Mammals | EP2E of Homo sapiens (Q9H4P9) |
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| 1.C.85.1.6 | β-defensin-2 of 71 aas and 1 TMS | Animals | Defensin β2 of Mus musculus
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| 1.C.85.1.7 | β-defensin 11 of 69 aas and 1 TMS | Animals | Defb11 of Rattus norvegicus |
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| 1.C.85.1.8 | β-Defensin 3 (BD3) of 63 aas and 1 TMS (Colavita et al. 2015). Sass et al. 2008 have proposed that interference with the organisation of membrane-bound multienzyme complexes such as the electron transport chain and the cell wall biosynthetic complex rather than on formation of defined transmembrane
pores is responsible for death of Staphylococcus aureus (Sass et al. 2008). However, BD3 selectively inhibits mouse Kv1.6 and human KCNQ1/KCNE1 channels with IC50 values of 0.6 μM and 1.2 μM, respectively (Zhang et al. 2018).
| | BD3 pf Mus musculus |
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| 1.C.85.1.9 | Canine β-defensin-1, cBD1, of 41 aas and 1 N-terminal TMS. Production of beta-defensins constitutes an important role in skin
defense, and
variable expression of three cBDs in different organ systems of the dog has been observed. In
skin, three beta-defensins, cBD1, cBD103 and cBD107, were extensively
expressed (van Damme et al. 2009). There is a possible defect in the innate immune response of dogs with atopic dermatitis. cDB1 may be a marker for Leishmania infantum infection in dogs (da Silva et al. 2017). | | Defensin 1 of Canis lupus familiaris (Dog) (Canis familiaris) |
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| Examples: |
| TC# | Name | Organismal Type | Example |
| 1.C.85.2.1 | Myotoxin-4 or Crotamine-4. It specifically modifies voltage-sensitive Na+channels, inhibits K+ channels and exhibits analgesic effects. This snake myotoxin family member is a cationic peptide with multiple
functions. It acts as a cell-penetrating peptide (CPP), and has antimicrobial
activities, causes hind limb paralysis, and gives rise to severe muscle necrosis by a
non-enzymatic mechanism. As a cell-penetrating peptide, crotamine has
high specificity for actively proliferating cells, and it interacts inside
the cell with subcellular and subnuclear structures, like vesicular
compartments, chromosomes and centrioles (Hayashi et al. 2008). The toxin selectively inhibits Kv1.1/KCNA1, Kv1.2/KCNA2 and Kv1.3/KCNA3 channels with IC50 values of 369, 386 and 287 nM, respectively (Peigneur et al. 2012). Crotamine
shows antibacterial activity against E. coli and B. subtilis, and
antifungal activity against Candida spp., Trichosporon spp. and
C. neoformans. It kills bacteria through membrane permeabilization (Kerkis et al. 2004). | Snakes | Myotoxin-4 of Crotalus durissus terrificus (P24334) |
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| 1.C.85.2.2 | Crotamine-IV-2 toxin of 42 aas and 0 TMSs. Croamines are cationic peptides that possess multiple
functions. hey act as cell-penetrating peptides (CPPs), as potent
voltage-gated potassium channel inhibitors and as antimicrobial
agents (Hayashi et al. 2008). As an antimicrobial peptide, crotamine shows
antibacterial activity against E.coli and B.subtilis, and antifungal
activity against Candida spp., Trichosporon spp. and C. neoformans. It
kills bacteria through membrane permeabilization (Hayashi et al. 2008). It selectively inhibits Kv1.1/KCNA1, Kv1.2/KCNA2 and Kv1.3/KCNA3 channels (Peigneur et al. 2012). It is also hemolytic (Oguiura et al. 2011).
| Animals | Croamine-IV-2 of Crotalus durissus cumanensis |
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| 1.C.85.2.3 | β-defensin-like protein of 63 aas and 1 TMS | Animals | defensin-like protein of Bothrops matogrossensis (Pitviper) (Bothrops neuwiedi matogrossensis) |
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| 1.C.85.2.4 | Crotamine-like precursor of 76 aas and 1 TMS. | Animals | Crotamine-like peptide of Thamnodynastes strigatus (Coastal house snake) |
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| Examples: |
| TC# | Name | Organismal Type | Example |
| 1.C.85.3.1 | Epithelial Gallinacin-1α. The full length antimicrobial peptide precursor is CHP2. Attacks bacteria and fungi. | Birds | Gallinacin 1α of Gallus gallus (P46157) |
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| 1.C.85.3.2 | β-defensin prepropeptide of 59 aas and 2 TMSs. | Animals | β-defensin of Meleagris gallopavo (turkey) |
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| 1.C.85.3.3 | Avian beta-defensin, 5beta of 66 aas and 1 TMS.
| Animals | Beta-defensin of Columba livia (domestic pigeon) |
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| Examples: |
| TC# | Name | Organismal Type | Example |
| 1.C.85.4.1 | Helofensin-1 lethal toxin of 183 aas (PMID 19837656). This toxin possesses an inhibitory
effect on electrical stimulation of the isolated hemi-diaphragm
of mice. Neither hemorrhagic nor hemolytic activities were detected, but Huang et al. 2016 reported it to be a membrane active protein.
| | Helofensin-1 of Heloderma suspectum cinctum (Banded Gila monster) |
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| 1.C.85.4.2 | Helofensin-3 (90% identical to helofensin-1) of 182 aas. A lethal toxin. | | Helofensin-3 of Heloderma suspectum cinctum (Banded Gila monster) |
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| 1.C.85.4.3 | Uncharacterized protein of 172 aas | | UP of Nematostella vectensis (Starlet sea anemone) |
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