Dataset Information

Biosynthesis and antimicrobial evaluation of backbone-cyclized ?-defensins.

ABSTRACT: Defensins are antimicrobial peptides that are important in the innate immune defense of mammals. Upon stimulation by bacterial antigens, enteric ?-defensins are secreted into the intestinal lumen where they have potent microbicidal activities. Cryptdin-4 (Crp4) is an ?-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 ?-sheet but lacks three amino acids between the fourth and fifth cysteine residues, making them distinct from other ?-defensins. The structure also reveals that the ?-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 ?-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.

SUBMITTER: Garcia AE

PROVIDER: S-EPMC3236816 | biostudies-literature | 2011 Dec

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Biosynthesis and antimicrobial evaluation of backbone-cyclized α-defensins.

Garcia Angie E AE Tai Kenneth P KP Puttamadappa Shadakshara S SS Shekhtman Alexander A Ouellette Andre J AJ Camarero Julio A JA

Biochemistry 20111109 48

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 ...[more]

PMID: 22040603

Similar Datasets

Project description:BackgroundDefensins represent an important class of antimicrobial peptides. These effector molecules of the innate immune system act as endogenous antibiotics to protect the organism against infections with pathogenic microorganisms. Mammalian defensins are classified into three distinct sub-families (alpha-, beta- and theta-defensins) according to their specific intramolecular disulfide-bond pattern. The peptides exhibit an antimicrobial activity against a broad spectrum of microorganisms including bacteria and fungi. Alpha-Defensins are primarily synthesised in neutrophils and intestinal Paneth cells. They play a role in the pathogenesis of intestinal diseases and may regulate the flora of the intestinal tract. An equine intestinal alpha-defensin (DEFA1), the first characterised in the Laurasiatheria, shows a broad antimicrobial spectrum against human and equine pathogens. Here we report a first investigation of the repertoire of equine intestinal alpha-defensins. The equine genome was screened for putative alpha-defensin genes by using known alpha-defensin sequences as matrices. Based on the obtained sequence information, a set of oligonucleotides specific to the alpha-defensin gene-family was designed. The products generated by reverse-transcriptase PCR with cDNA from the small intestine as template were sub-cloned and numerous clones were sequenced.ResultsThirty-eight equine intestinal alpha-defensin transcripts were determined. After translation it became evident that at least 20 of them may code for functional peptides. Ten transcripts lacked matching genomic sequences and for 14 alpha-defensin genes apparently present in the genome no appropriate transcript could be verified. In other cases the same genomic exons were found in different transcripts.ConclusionsThe large repertoire of equine alpha-defensins found in this study points to a particular importance of these peptides regarding animal health and protection from infectious diseases. Moreover, these findings make the horse an excellent species to study biological properties of alpha-defensins. Interestingly, the peptides were not found in other species of the Laurasiatheria to date. Comparison of the obtained transcripts with the genomic sequences in the current assembly of the horse (EquCab2.0) indicates that it is yet not complete and/or to some extent falsely assembled.

Dataset Information

Biosynthesis and antimicrobial evaluation of backbone-cyclized ?-defensins.

Publications

Biosynthesis and antimicrobial evaluation of backbone-cyclized α-defensins.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets