Proresolving and tissue-protective actions of annexin A1-based cleavage-resistant peptides are mediated by formyl peptide receptor 2/lipoxin A4 receptor.

Endogenous mechanisms regulating the host response during inflammation resolution are critical in ensuring disposal of noxious stimuli and return to homeostasis. In this article, we engineered novel Annexin A1 (AnxA1)-based peptides, AnxA1(2-50), that displayed specific binding to the AnxA1 receptor (formyl peptide receptor 2/Lipoxin A4 receptor [FPR2/ALX]; IC50 ∼4 nM). Intravenous administration of AnxA1(2-50) markedly reduced (>60%) leukocyte adhesion to postcapillary venules in wild type and Fpr1(-/-), but not Fpr2/Alx(-/-), mice. Generation of a metabolically stable form of this peptide (CR-AnxA1(2-50)), engineered by substituting a cleavage site shared by human proteinase 3 and neutrophil elastase, yielded an agonist that was resistant to neutrophil-mediated cleavage and displayed enhanced proresolving actions: accelerated resolution of self-limited inflammation and enhanced macrophage efferocytosis after sterile injury, when compared with AnxA1(2-50). These actions were retained with human primary leukocytes where CR-AnxA1(2-50) decreased neutrophil-endothelial interactions (∼25-45%), and stimulated neutrophil apoptosis and macrophage efferocytosis (∼45%). In murine cardiac ischemia/reperfusion injury, CR-AnxA1(2-50) elicited tissue-protective actions reducing infarct size (∼60%) and incidence of 24-h death. These results identify AnxA1(2-50) and CR-AnxA1(2-50) as FPR2/ALX agonists that harness the proresolving actions of AnxA1, and thus may represent therapeutic tools for treatment of inflammatory conditions.

Production of salmon calcitonin by direct expression of a glycine-extended precursor in Escherichia coli.

The export of heterologous products into the conditioned medium of an Escherichia coli culture offers the advantages of a higher product yield, an increased probability of recovering an intact recombinant protein, proper folding for biological activity, and greater stability of a secreted product. In this report, we describe the development of an optimized direct expression system, designed to maximize the extracellular accumulation of recombinant glycine-extended salmon calcitonin peptide (sCTgly). We have used dual promoters, an ompA signal sequence, co-expression of homologous secretion factor genes, and multiple gene cartridges to express the sCTgly. High-density fermentation conditions have been developed that allow for the selective secretion and accumulation of the expressed sCTgly at very high levels. Purification and in vitro enzymatic conversion by peptidylglycine alpha-amidating monooxygenase yields authentic, biologically active salmon calcitonin. This recombinant production technology is applicable to a variety of amidated peptide hormones.