title: Biosynthesis and antimicrobial evaluation of backbone-cyclized α-defensins.
abstract: Defensins are antimicrobial peptides that are important in the innate immune defense of mammals. Upon stimulation by bacterial antigens, enteric α-defensins are secreted into the intestinal lumen where they have potent microbicidal activities. Cryptdin-4 (Crp4) is an α-defensin expressed in Paneth cells of the mouse small intestine and the most bactericidal of the known cryptdin isoforms. The structure of Crp4 consists of a triple-stranded antiparallel β-sheet but lacks three amino acids between the fourth and fifth cysteine residues, making them distinct from other α-defensins. The structure also reveals that the α-amino and C-terminal carboxylic groups are in the proximity of each other (d ≈ 3 Å) in the folded structure. We present here the biosynthesis of backbone-cyclized Crp4 using a modified protein splicing unit or intein. Our data show that cyclized Crp4 can be biosynthesized by using this approach both in vitro and in vivo, although the expression yield was significantly lower when the protein was produced inside the cell. The resulting cyclic defensins retained the native α-defensin fold and showed equivalent or better microbicidal activities against several Gram-positive and Gram-negative bacteria when compared to native Crp4. No detectable hemolytic activity against human red blood cells was observed for either native Crp4 or its cyclized variants. Moreover, both forms of Crp4 also showed high stability to degradation when incubated with human serum. Altogether, these results indicate the potential for backbone-cyclized defensins in the development of novel peptide-based antimicrobial compounds.
authors: Department of Pharmacology and Pharmaceutical Sciences, University of Southern California, Los Angeles, Calofornia 90033, USA.
authors: Angie E
authors: AE
authors: Garcia
authors: Kenneth P
authors: KP
authors: Tai
authors: Shadakshara S
authors: SS
authors: Puttamadappa
authors: Alexander
authors: A
authors: Shekhtman
authors: Andre J
authors: AJ
authors: Ouellette
authors: Julio A
authors: JA
authors: Camarero
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000900:Anti-Bacterial Agents / Q000096:biosynthesis* / Q000097:blood / Q000494:pharmacology*; D006094:Gram-Positive Bacteria / Q000187:drug effects / Q000254:growth & development; D006461:Hemolysis / Q000187:drug effects; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D019879:Paneth Cells / Q000737:chemistry / Q000378:metabolism / Q000382:microbiology; D010456:Peptides, Cyclic / Q000096:biosynthesis* / Q000097:blood / Q000494:pharmacology*; D011487:Protein Conformation; D017510:Protein Folding; D055550:Protein Stability; D023084:alpha-Defensins / Q000096:biosynthesis* / Q000097:blood / Q000502:physiology*
chemical_list: D000900:Anti-Bacterial Agents; D010456:Peptides, Cyclic; D023084:alpha-Defensins; C401847:cryptdin 4, mouse