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Reawakening retrocyclins: ancestral human defensins active against HIV-1.

ABSTRACT: Human alpha and beta defensins contribute substantially to innate immune defenses against microbial and viral infections. Certain nonhuman primates also produce theta-defensins-18 residue cyclic peptides that act as HIV-1 entry inhibitors. Multiple human theta-defensin genes exist, but they harbor a premature termination codon that blocks translation. Consequently, the theta-defensins (retrocyclins) encoded within the human genome are not expressed as peptides. In vivo production of theta-defensins in rhesus macaques involves the post-translational ligation of two nonapeptides, each derived from a 12-residue "demidefensin" precursor. Neither the mechanism of this unique process nor its existence in human cells is known. To ascertain if human cells retained the ability to process demidefensins, we transfected human promyelocytic cells with plasmids containing repaired retrocyclin-like genes. The expected peptides were isolated, their sequences were verified by mass spectrometric analyses, and their anti-HIV-1 activity was confirmed in vitro. Our study reveals for the first time, to our knowledge, that human cells have the ability to make cyclic theta-defensins. Given this evidence that human cells could make theta-defensins, we attempted to restore endogenous expression of retrocyclin peptides. Since human theta-defensin genes are transcribed, we used aminoglycosides to read-through the premature termination codon found in the mRNA transcripts. This treatment induced the production of intact, bioactive retrocyclin-1 peptide by human epithelial cells and cervicovaginal tissues. The ability to reawaken retrocyclin genes from their 7 million years of slumber using aminoglycosides could provide a novel way to secure enhanced resistance to HIV-1 infection.

SUBMITTER: Venkataraman N

PROVIDER: S-EPMC2672613 | biostudies-literature | 2009 Apr

REPOSITORIES: biostudies-literature

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Reawakening retrocyclins: ancestral human defensins active against HIV-1.

Venkataraman Nitya N Cole Amy L AL Ruchala Piotr P Waring Alan J AJ Lehrer Robert I RI Stuchlik Olga O Pohl Jan J Cole Alexander M AM

PLoS biology 20090401 4

Human alpha and beta defensins contribute substantially to innate immune defenses against microbial and viral infections. Certain nonhuman primates also produce theta-defensins-18 residue cyclic peptides that act as HIV-1 entry inhibitors. Multiple human theta-defensin genes exist, but they harbor a premature termination codon that blocks translation. Consequently, the theta-defensins (retrocyclins) encoded within the human genome are not expressed as peptides. In vivo production of theta-defens ...[more]

PMID: 19402752

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Project description:Big defensins, ancestors of β-defensins, are composed of a β-defensin-like C-terminal domain and a globular hydrophobic ancestral N-terminal domain. This unique structure is found in a limited number of phylogenetically distant species, including mollusks, ancestral chelicerates, and early-branching cephalochordates, mostly living in marine environments. One puzzling evolutionary issue concerns the advantage for these species of having maintained a hydrophobic domain lost during evolution toward β-defensins. Using native ligation chemistry, we produced the oyster Crassostrea gigas BigDef1 (Cg-BigDef1) and its separate domains. Cg-BigDef1 showed salt-stable and broad-range bactericidal activity, including against multidrug-resistant human clinical isolates of Staphylococcus aureus We found that the ancestral N-terminal domain confers salt-stable antimicrobial activity to the β-defensin-like domain, which is otherwise inactive. Moreover, upon contact with bacteria, the N-terminal domain drives Cg-BigDef1 assembly into nanonets that entrap and kill bacteria. We speculate that the hydrophobic N-terminal domain of big defensins has been retained in marine phyla to confer salt-stable interactions with bacterial membranes in environments where electrostatic interactions are impaired. Those remarkable properties open the way to future drug developments when physiological salt concentrations inhibit the antimicrobial activity of vertebrate β-defensins.IMPORTANCE β-Defensins are host defense peptides controlling infections in species ranging from humans to invertebrates. However, the antimicrobial activity of most human β-defensins is impaired at physiological salt concentrations. We explored the properties of big defensins, the β-defensin ancestors, which have been conserved in a number of marine organisms, mainly mollusks. By focusing on a big defensin from oyster (Cg-BigDef1), we showed that the N-terminal domain lost during evolution toward β-defensins confers bactericidal activity to Cg-BigDef1, even at high salt concentrations. Cg-BigDef1 killed multidrug-resistant human clinical isolates of Staphylococcus aureus Moreover, the ancestral N-terminal domain drove the assembly of the big defensin into nanonets in which bacteria are entrapped and killed. This discovery may explain why the ancestral N-terminal domain has been maintained in diverse marine phyla and creates a new path of discovery to design β-defensin derivatives active at physiological and high salt concentrations.

Project description:BACKGROUND:X-tox proteins are a family of immune-related proteins only found in Lepidoptera and characterized by imperfectly conserved tandem repeats of several defensin-like motifs. Previous phylogenetic analysis of X-tox genes supported the hypothesis that X-tox have evolved from defensins in a lineage-specific gene evolution restricted to Lepidoptera. In this paper, we performed a protein study in which we asked whether X-tox proteins have conserved the antimicrobial functions of their ancestral defensins and have evolved as defensin reservoirs. METHODOLOGY/PRINCIPAL FINDINGS:We followed the outcome of Spod-11-tox, an X-tox protein characterized in Spodoptera frugiperda, in bacteria-challenged larvae using both immunochemistry and antimicrobial assays. Three hours post infection, the Spod-11-tox protein was expressed in 80% of the two main classes of circulating hemocytes (granulocytes and plasmatocytes). Located in secretory granules of hemocytes, Spod-11-tox was never observed in contact with microorganisms entrapped within phagolyzosomes showing that Spod-11-tox is not involved in intracellular pathogen killing. In fact, the Spod-11-tox protein was found to be secreted into the hemolymph of experimentally challenged larvae. In order to determine antimicrobial properties of the Spod-11-tox protein, it was consequently fractionated according to a protocol frequently used for antimicrobial peptide purification. Over the course of purification, the anti-Spod-11-tox immunoreactivity was found to be dissociated from the antimicrobial activity. This indicates that Spod-11-tox is not processed into bioactive defensins in response to a microbial challenge. CONCLUSIONS/SIGNIFICANCE:Altogether, our results show that X-tox proteins have not evolved as defensin reservoirs and have lost the antimicrobial properties of the ancestral insect defensins. The lepidopteran X-tox protein family will provide a valuable and tractable model to improve our knowledge on the molecular evolution of defensins, a class of innate immune effectors largely distributed over the three eukaryotic kingdoms.

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Reawakening retrocyclins: ancestral human defensins active against HIV-1.

Publications

Reawakening retrocyclins: ancestral human defensins active against HIV-1.

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