Design and characterization of a cleavage-resistant Annexin A1 mutant to control inflammation in the microvasculature

• Published in: Blood 2010-11, Vol.116 (20), p.4288-4296
• Main Authors: Pederzoli-Ribeil, Magali, Maione, Francesco, Cooper, Dianne, Al-Kashi, Adam, Dalli, Jesmond, Perretti, Mauro, D'Acquisto, Fulvio
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Annexin A1 - chemistry; Annexin A1 - isolation & purification; Annexin A1 - metabolism; Anti-Inflammatory Agents - metabolism; Biological and medical sciences; Cell Adhesion; Cell Communication; Cell Movement; Endothelial Cells - metabolism; Female; HEK293 Cells; Hematologic and hematopoietic diseases; Humans; Inflammation - pathology; Inflammation - prevention & control; Male; Medical sciences; Mice; Microvessels - metabolism; Microvessels - pathology; Molecular Sequence Data; Mutant Proteins - chemistry; Mutant Proteins - metabolism; Neutrophils - cytology; Neutrophils - metabolism; Protein Binding; Protein Engineering; Receptors, Formyl Peptide - metabolism; Receptors, Lipoxin - metabolism; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism
• ISSN: 0006-4971; 1528-0020
• DOI: 10.1182/blood-2010-02-270520

Human polymorphonuclear leukocytes adhesion to endothelial cells during the early stage of inflammation leads to cell surface externalization of Annexin A1 (AnxA1), an effector of endogenous anti-inflammation. The antiadhesive properties of AnxA1 become operative to finely tune polymorphonuclear leukocytes transmigration to the site of inflammation. Membrane bound proteinase 3 (PR3) plays a key role in this microenvironment by cleaving the N terminus bioactive domain of AnxA1. In the present study, we generated a PR3-resistant human recombinant AnxA1—named superAnxA1 (SAnxA1)—and tested its in vitro and in vivo properties in comparison to the parental protein. SAnxA1 bound and activated formyl peptide receptor 2 in a similar way as the parental protein, while showing a resistance to cleavage by recombinant PR3. SAnxA1 retained anti-inflammatory activities in the murine inflamed microcirculation (leukocyte adhesion being the readout) and in skin trafficking model. When longer-lasting models of inflammation were applied, SAnxA1 displayed stronger anti-inflammatory effect over time compared with the parental protein. Together these results indicate that AnxA1 cleavage is an important process during neutrophilic inflammation and that controlling the balance between AnxA1/PR3 activities might represent a promising avenue for the discovery of novel therapeutic approaches.