Annexin 1 Cleavage in Activated Neutrophils

Abstract

Annexin 1 is an anti-inflammatory protein that plays a key role in innate immunity by modulating the activation of several types of cells, including neutrophils. Here we have developed a cleavage assay using tagged annexin 1 and observed marked activity in the membrane fraction of activated neutrophils. A combination of inhibitors, transfected cells, and proteomic analyses allowed us to identify proteinase 3 as the main enzyme responsible for this cleavage in the N terminus region of the protein, at least in the context of neutrophil activation. Because annexin 1 is an important endogenous anti-inflammatory mediator, blocking its cleavage by proteinase 3 would augment its homeostatic pro-resolving actions and could represent an opportunity for innovative anti-inflammatory drug discovery. Annexin 1 is an anti-inflammatory protein that plays a key role in innate immunity by modulating the activation of several types of cells, including neutrophils. Here we have developed a cleavage assay using tagged annexin 1 and observed marked activity in the membrane fraction of activated neutrophils. A combination of inhibitors, transfected cells, and proteomic analyses allowed us to identify proteinase 3 as the main enzyme responsible for this cleavage in the N terminus region of the protein, at least in the context of neutrophil activation. Because annexin 1 is an important endogenous anti-inflammatory mediator, blocking its cleavage by proteinase 3 would augment its homeostatic pro-resolving actions and could represent an opportunity for innovative anti-inflammatory drug discovery. Annexin 1 (AnxA1) 5The abbreviations used are: AnxA1, annexin 1; HNE, human recombinant neutrophil elastase; PMN, polymorphonuclear; PR3, proteinase 3; TNF, tumor necrosis factor; HEK, human embryonic kidney; WL, whole lysate; CYT, cytosolic; MEM, membrane; IP, immunoprecipitate or immunoprecipitation; PMSF, phenylmethylsulfonyl fluoride; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; HMC1, human mast cell clone 1. 5The abbreviations used are: AnxA1, annexin 1; HNE, human recombinant neutrophil elastase; PMN, polymorphonuclear; PR3, proteinase 3; TNF, tumor necrosis factor; HEK, human embryonic kidney; WL, whole lysate; CYT, cytosolic; MEM, membrane; IP, immunoprecipitate or immunoprecipitation; PMSF, phenylmethylsulfonyl fluoride; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; HMC1, human mast cell clone 1. belongs to the superfamily of annexin proteins characterized by the presence of two principal domains, the N-terminal, which is believed to confer the particular biological effects of each annexin, and the large C-terminal-containing core (1Gerke V. Moss S.E. Physiol. Rev. 2002; 82: 331-371Crossref PubMed Scopus (1611) Google Scholar). Studies over the past 20 years have shown that as an endogenous of by in a activated PubMed Scopus Google PubMed Scopus Google Scholar). of and to to the of PubMed Scopus Google PubMed Scopus Google Scholar). that the actions of could in the of an activated and proteins to presence PubMed Scopus Google PubMed Scopus Google Scholar). cleavage human PubMed Scopus Google a that protein PubMed Scopus Google Scholar). the of human recombinant neutrophil V. V. PubMed Scopus Google 3 is in neutrophil including V. PubMed Google Scholar). have to PR3, its to neutrophil cell PubMed Scopus Google V. PubMed Scopus Google as tumor necrosis and and as Google Scholar). of the of in and to HNE, in PubMed Google V. 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V. PubMed Scopus Google Scholar). and as by by by using and by a laser as V. V. 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PMN, the of is to the PubMed Scopus Google PubMed Scopus Google and to a the cell membrane PubMed Scopus Google Scholar). we that the cell as a of activation the and a is by of that as a for that cell activation. of the and of the protein to of the the for to at the of the cell membrane in this that a to the membrane fraction of the PMN, responsible for the cleavage of the to the the we developed a cleavage assay a recombinant used as this we to the cleavage of by the PMN, that this in presence of the membrane and the an of proteins including the and to of the actions of of the cleavage activity in the we by the the cleavage we to cleavage at and of is of V. PubMed Scopus Google PubMed Scopus Google in of the N terminus at in of PubMed Scopus Google Scholar). the that we have to the we could that and the N is that in the context of and in the of an PMN, to and the as of the is the of the N is to that of to the in of to in V. V. 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Google PubMed Scopus Google Scholar). that membrane could have a and a for PubMed Scopus Google Scholar). a the of PR3, that of the endogenous of in would to this in have marked and cleavage PubMed Scopus Google we by that is responsible for the cleavage activity in membrane the is the (AnxA1) to the membrane PubMed Scopus Google for the enzyme this would as a in the of and in the and context of the activated a is in the for and as in PubMed Scopus Google PubMed Scopus Google PubMed Scopus Google Scholar). the of at the cleavage activity of endogenous and for as anti-inflammatory Annexin 1 (AnxA1) 5The abbreviations used are: AnxA1, annexin 1; HNE, human recombinant neutrophil elastase; PMN, polymorphonuclear; PR3, proteinase 3; TNF, tumor necrosis factor; HEK, human embryonic kidney; WL, whole lysate; CYT, cytosolic; MEM, membrane; IP, immunoprecipitate or immunoprecipitation; PMSF, phenylmethylsulfonyl fluoride; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; HMC1, human mast cell clone 1. 5The abbreviations used are: AnxA1, annexin 1; HNE, human recombinant neutrophil elastase; PMN, polymorphonuclear; PR3, proteinase 3; TNF, tumor necrosis factor; HEK, human embryonic kidney; WL, whole lysate; CYT, cytosolic; MEM, membrane; IP, immunoprecipitate or immunoprecipitation; PMSF, phenylmethylsulfonyl fluoride; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; HMC1, human mast cell clone 1. belongs to the superfamily of annexin proteins characterized by the presence of two principal domains, the N-terminal, which is believed to confer the particular biological effects of each annexin, and the large C-terminal-containing core (1Gerke V. Moss S.E. Physiol. Rev. 2002; 82: 331-371Crossref PubMed Scopus (1611) Google Scholar). Studies over the past 20 years have shown that as an endogenous of by in a activated PubMed Scopus Google PubMed Scopus Google Scholar). of and to to the of PubMed Scopus Google PubMed Scopus Google Scholar). that the actions of could in the of an activated and proteins to presence PubMed Scopus Google PubMed Scopus Google Scholar). cleavage human PubMed Scopus Google a that protein PubMed Scopus Google Scholar). the of human recombinant neutrophil V. V. PubMed Scopus Google Scholar). 3 is in neutrophil including V. PubMed Google Scholar). have to PR3, its to neutrophil cell PubMed Scopus Google V. PubMed Scopus Google as tumor necrosis and and as Google Scholar). of the of in and to HNE, in PubMed Google V. PubMed Scopus Google we the that could in the we have a cleavage assay to the effects of and and the actions of enzyme to to cleavage in the context of cell activation. and cell in 1 and human cell or human mast cell clone and human PR3, or as V. 2002; PubMed Scopus Google and of by as PubMed Scopus Google Scholar). activation for at of whole cell and in 20 for and membrane in 20 for at to a and for at and the in 20 at for by 3 of 1 for a of and to the for in the cleavage assay in the of of and using the and using to the tagged the N terminus and tagged the terminus using the in the and of transfected using of and of for each of a each in of and at cell transfected by using of in V. and in a for to cell or at of 20 and the of cleavage activity of cell or a of and to the at for and a of to at the in 20 of for and and to or to this in of or 1 to for at to to the cleavage in to and in 20 and at for 1 for 20 the at and and proteins using the and used and used and to of N membrane to the cleavage 20 and by of to of of in or at and in to or as V. V. PubMed Scopus Google Scholar). and as by by by using and by a laser as V. V. PubMed Scopus Google Scholar). and cell in 1 and human cell or human mast cell clone and human PR3, or as V. 2002; PubMed Scopus Google Scholar). and of by as PubMed Scopus Google Scholar). activation for at of whole cell and in 20 for and membrane in 20 for at to a and for at and the in 20 at for by 3 of 1 for a of and to the for in the cleavage assay in the of of and using the and using to the tagged the N terminus and tagged the terminus using the in the and of transfected using of and of for each of a each in of and at cell transfected by using of in V. and in a for to cell or at of 20 and the of cleavage activity of cell or a of and to the at for and a of to at the in 20 of for and and to or to this in of or 1 to for at to to the cleavage in to and in 20 and at for 1 for 20 the at and and proteins using the and used and used and to of N membrane to the cleavage 20 and by of to of of in or at and in to or as V. V. PubMed Scopus Google Scholar). and as by by by using and by a laser as V. V. PubMed Scopus Google Scholar). and of in activation or the membrane and in the PubMed Scopus Google Scholar). in in the activation the protein the membrane fraction and the the in the whole of the A and the the protein is we the and the protein the as a of two of and in and PMN, of the to in the as an to cell the protein to the membrane is and as an of over the past years have that is at the N terminus PubMed Scopus Google PubMed Scopus Google Scholar). and identify the responsible for this cleavage we developed an cleavage endogenous AnxA1, a protein a the N terminus and a the terminus in transfected and the cell by used as an in to identify the of the the by for the for the N terminus and for the shown in of and of the whole cell for the or a of of the protein the and in the and a of and in the to the and of AnxA1, of cell and or a N and or a and a that of the and of the of the cell shown in of in of and of the by or a to the that cleavage at the N of the fraction cleavage as the and in the of endogenous AnxA1, the of cleavage activity in the to the the N in the of the cleavage assay by of and A and that enzyme to a of the to of the N identify the and cleavage activity the membrane N terminus that to the of and 1 and of the of the N terminus the presence of cleavage at and by the membrane fraction and to terminus in a of by we the of to of the fraction to of the fraction at for fraction activity by PMSF, a of by inhibitors, including and PMSF, at a in blocking the cleavage of and the cleavage of the by by the of the A and of by a membrane in the or presence of 1 or of or of 1 or as in membrane 1 or and and by and at least the of the enzyme in by the of this in blocking HNE, actions the endogenous activity the of we used transfected PR3, the or V. 2002; PubMed Scopus Google Scholar). the to of the protein, which its N terminus in cells, as of in transfected in the of the and of the the of and of the cell in the in assay of cell or for and cell to a the N terminus of the of of the the the in and in in the this in the context of we analyses AnxA1, PR3, and of by in PR3, and transfected cell for and and PR3, and and and as at least and in and of endogenous and HNE, for in and which to membrane to proteins in the PMN, a of the for and in is to membrane of cell the to a of the two or or and of endogenous and in and activated in the or presence of for at to or as or is including the the a of the two proteins in of or and of in activation or the membrane and in the PubMed Scopus Google Scholar). in in the activation the protein the membrane fraction and the the in the whole of the A and the the protein is we the and the protein the as a of two of and in and PMN, of the to in the as an to cell the protein to the membrane is and as an of over the past years have that is at the N terminus PubMed Scopus Google PubMed Scopus Google Scholar). and identify the responsible for this cleavage we developed an cleavage endogenous AnxA1, a protein a the N terminus and a the terminus in transfected and the cell by used as an in to identify the of the the by for the for the N terminus and for the shown in of and of the whole cell for the or a of of the protein the and in the and a of and in the to the and of AnxA1, of cell and or a N and or a and a that of the and of the of the cell shown in of in of and of the by or a to the that cleavage at the N of the fraction cleavage as the and in the of endogenous AnxA1, the of cleavage activity in the to the the N in the of the cleavage assay by of and A and that enzyme to a of the to of the N identify the and cleavage activity the membrane N terminus that to the of and 1 and of the of the N terminus the presence of cleavage at and of by we the of to of the fraction to of the fraction at for fraction activity by PMSF, a of by inhibitors, including and PMSF, at a in blocking the cleavage of and the cleavage of the by by the of the A and A the of the enzyme in by the of this in blocking HNE, actions the endogenous activity the of we used transfected PR3, the or V. 2002; PubMed Scopus Google Scholar). the to of the protein, which its N terminus in cells, as of in transfected in the of the and of the the of and of the cell in the in assay of cell or for and cell to a the N terminus of the of of the the the in and in in the this in the context of we analyses AnxA1, PR3, and and in and of endogenous and HNE, for in and which to membrane to proteins in the PMN, a of the for and in is to membrane of cell the to a of the two or or and this we for the cleavage activity in activated of in and activated PMN, in to the protein and a N terminus cleavage and the of cell activation. least of to in a a to by as and a membrane to PubMed Scopus Google PubMed Scopus Google Scholar). 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