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Analysis of protease activity in live antigen-presenting cells shows regulation of the phagosomal proteolytic contents during dendritic cell activation.

Lennon-Duménil AM, Bakker AH, Maehr R, Fiebiger E, Overkleeft HS, Rosemblatt M, Ploegh HL, Lagaudrière-Gesbert C - J. Exp. Med. (2002)

Bottom Line: Furthermore, the delivery of active proteases to the phagosome is significantly reduced after the activation of DCs with lipopolysaccharide.This observation is in agreement with the notion that DCs prevent the premature destruction of antigenic determinants to optimize T cell activation.Phagosomal maturation is therefore a tightly regulated process that varies according to the type and differentiation stage of the phagocyte.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Harvard Medical School, 200 Longwood Avenue, Boston, MA 02115, USA.

ABSTRACT
Here, we describe a new approach designed to monitor the proteolytic activity of maturing phagosomes in live antigen-presenting cells. We find that an ingested particle sequentially encounters distinct protease activities during phagosomal maturation. Incorporation of active proteases into the phagosome of the macrophage cell line J774 indicates that phagosome maturation involves progressive fusion with early and late endocytic compartments. In contrast, phagosome biogenesis in bone marrow-derived dendritic cells (DCs) and macrophages preferentially involves endocytic compartments enriched in cathepsin S. Kinetics of phagosomal maturation is faster in macrophages than in DCs. Furthermore, the delivery of active proteases to the phagosome is significantly reduced after the activation of DCs with lipopolysaccharide. This observation is in agreement with the notion that DCs prevent the premature destruction of antigenic determinants to optimize T cell activation. Phagosomal maturation is therefore a tightly regulated process that varies according to the type and differentiation stage of the phagocyte.

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Cysteine proteases recognized by the active site–directed probe DCG-04 in macrophage cell lysates. (A) Schematic overview of the approach designed to examine phagosomal proteolytic contents. To target phagosomal proteases, biotinylated active site–directed probes are coupled to streptavidin-coated latex beads. Upon phagocytosis of the beads, the probe reacts with the active proteases it encounters. Analysis of the modified enzymes at different time points allows a sampling of the proteolytic activities acquired by the phagosome upon fusion with the various endosomal compartments. (B–D) Proteins were separated by SDS-PAGE on a 12.5% gel and reactive proteases were visualized by streptavidin blotting. (B) Structure of the active site–directed probe JPM-565 and its biotinylated derivative DCG-04. (C) Titration of DCG-04 for the labeling of the total cell lysates from J774 cells (pH 5). Lysates were incubated with increasing concentrations of DCG-04. Only the bands in the 15–40-kD range are shown because the larger polypeptides have been shown to be contaminants (reference 18). (D) Identification of individual cysteine proteases labeled by DCG-04. Total extracts from J774 cells (pH 5) were incubated with 5 μM DCG-04 and subjected to immunoprecipitation using anti-CatB, anti-CatL, or anti-CatS antibodies.
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fig1: Cysteine proteases recognized by the active site–directed probe DCG-04 in macrophage cell lysates. (A) Schematic overview of the approach designed to examine phagosomal proteolytic contents. To target phagosomal proteases, biotinylated active site–directed probes are coupled to streptavidin-coated latex beads. Upon phagocytosis of the beads, the probe reacts with the active proteases it encounters. Analysis of the modified enzymes at different time points allows a sampling of the proteolytic activities acquired by the phagosome upon fusion with the various endosomal compartments. (B–D) Proteins were separated by SDS-PAGE on a 12.5% gel and reactive proteases were visualized by streptavidin blotting. (B) Structure of the active site–directed probe JPM-565 and its biotinylated derivative DCG-04. (C) Titration of DCG-04 for the labeling of the total cell lysates from J774 cells (pH 5). Lysates were incubated with increasing concentrations of DCG-04. Only the bands in the 15–40-kD range are shown because the larger polypeptides have been shown to be contaminants (reference 18). (D) Identification of individual cysteine proteases labeled by DCG-04. Total extracts from J774 cells (pH 5) were incubated with 5 μM DCG-04 and subjected to immunoprecipitation using anti-CatB, anti-CatL, or anti-CatS antibodies.

Mentions: We devised a strategy to sample the proteolytic environment encountered by phagocytosed antigens in professional APCs. For this purpose, we used the biotinylated active site–directed probe, DCG-04, coupled to streptavidin-coated latex beads. DCG-04 is a derivative of the peptide epoxide JPM-565 and specifically targets cysteine proteases (Fig. 1 B; reference 18). Probe-coated beads are internalized by APCs through phagocytosis. Bead-containing phagosomes undergo maturation by fusion with the different endocytic compartments of the APC. Once inside the cell, the active site–directed probe senses its proteolytic environment by reacting with active proteases incorporated into the phagosome (Fig. 1 A). This scenario presupposes that probes immobilized via biotin to the latex beads remain available for interaction with proteases via their COOH-terminal epoxide moiety. Because binding of the probe to the protease active site is covalent and irreversible, proteases labeled in vivo can then be visualized after direct lysis of the phagocytes in SDS sample buffer, followed by simple electrophoresis and streptavidin blotting. For a given protease, different labeling intensities correspond directly to differences in activity levels. This approach should therefore allow us to evaluate the activity of individual cysteine proteases delivered to the phagosome of APCs during its maturation.


Analysis of protease activity in live antigen-presenting cells shows regulation of the phagosomal proteolytic contents during dendritic cell activation.

Lennon-Duménil AM, Bakker AH, Maehr R, Fiebiger E, Overkleeft HS, Rosemblatt M, Ploegh HL, Lagaudrière-Gesbert C - J. Exp. Med. (2002)

Cysteine proteases recognized by the active site–directed probe DCG-04 in macrophage cell lysates. (A) Schematic overview of the approach designed to examine phagosomal proteolytic contents. To target phagosomal proteases, biotinylated active site–directed probes are coupled to streptavidin-coated latex beads. Upon phagocytosis of the beads, the probe reacts with the active proteases it encounters. Analysis of the modified enzymes at different time points allows a sampling of the proteolytic activities acquired by the phagosome upon fusion with the various endosomal compartments. (B–D) Proteins were separated by SDS-PAGE on a 12.5% gel and reactive proteases were visualized by streptavidin blotting. (B) Structure of the active site–directed probe JPM-565 and its biotinylated derivative DCG-04. (C) Titration of DCG-04 for the labeling of the total cell lysates from J774 cells (pH 5). Lysates were incubated with increasing concentrations of DCG-04. Only the bands in the 15–40-kD range are shown because the larger polypeptides have been shown to be contaminants (reference 18). (D) Identification of individual cysteine proteases labeled by DCG-04. Total extracts from J774 cells (pH 5) were incubated with 5 μM DCG-04 and subjected to immunoprecipitation using anti-CatB, anti-CatL, or anti-CatS antibodies.
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Related In: Results  -  Collection

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fig1: Cysteine proteases recognized by the active site–directed probe DCG-04 in macrophage cell lysates. (A) Schematic overview of the approach designed to examine phagosomal proteolytic contents. To target phagosomal proteases, biotinylated active site–directed probes are coupled to streptavidin-coated latex beads. Upon phagocytosis of the beads, the probe reacts with the active proteases it encounters. Analysis of the modified enzymes at different time points allows a sampling of the proteolytic activities acquired by the phagosome upon fusion with the various endosomal compartments. (B–D) Proteins were separated by SDS-PAGE on a 12.5% gel and reactive proteases were visualized by streptavidin blotting. (B) Structure of the active site–directed probe JPM-565 and its biotinylated derivative DCG-04. (C) Titration of DCG-04 for the labeling of the total cell lysates from J774 cells (pH 5). Lysates were incubated with increasing concentrations of DCG-04. Only the bands in the 15–40-kD range are shown because the larger polypeptides have been shown to be contaminants (reference 18). (D) Identification of individual cysteine proteases labeled by DCG-04. Total extracts from J774 cells (pH 5) were incubated with 5 μM DCG-04 and subjected to immunoprecipitation using anti-CatB, anti-CatL, or anti-CatS antibodies.
Mentions: We devised a strategy to sample the proteolytic environment encountered by phagocytosed antigens in professional APCs. For this purpose, we used the biotinylated active site–directed probe, DCG-04, coupled to streptavidin-coated latex beads. DCG-04 is a derivative of the peptide epoxide JPM-565 and specifically targets cysteine proteases (Fig. 1 B; reference 18). Probe-coated beads are internalized by APCs through phagocytosis. Bead-containing phagosomes undergo maturation by fusion with the different endocytic compartments of the APC. Once inside the cell, the active site–directed probe senses its proteolytic environment by reacting with active proteases incorporated into the phagosome (Fig. 1 A). This scenario presupposes that probes immobilized via biotin to the latex beads remain available for interaction with proteases via their COOH-terminal epoxide moiety. Because binding of the probe to the protease active site is covalent and irreversible, proteases labeled in vivo can then be visualized after direct lysis of the phagocytes in SDS sample buffer, followed by simple electrophoresis and streptavidin blotting. For a given protease, different labeling intensities correspond directly to differences in activity levels. This approach should therefore allow us to evaluate the activity of individual cysteine proteases delivered to the phagosome of APCs during its maturation.

Bottom Line: Furthermore, the delivery of active proteases to the phagosome is significantly reduced after the activation of DCs with lipopolysaccharide.This observation is in agreement with the notion that DCs prevent the premature destruction of antigenic determinants to optimize T cell activation.Phagosomal maturation is therefore a tightly regulated process that varies according to the type and differentiation stage of the phagocyte.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Harvard Medical School, 200 Longwood Avenue, Boston, MA 02115, USA.

ABSTRACT
Here, we describe a new approach designed to monitor the proteolytic activity of maturing phagosomes in live antigen-presenting cells. We find that an ingested particle sequentially encounters distinct protease activities during phagosomal maturation. Incorporation of active proteases into the phagosome of the macrophage cell line J774 indicates that phagosome maturation involves progressive fusion with early and late endocytic compartments. In contrast, phagosome biogenesis in bone marrow-derived dendritic cells (DCs) and macrophages preferentially involves endocytic compartments enriched in cathepsin S. Kinetics of phagosomal maturation is faster in macrophages than in DCs. Furthermore, the delivery of active proteases to the phagosome is significantly reduced after the activation of DCs with lipopolysaccharide. This observation is in agreement with the notion that DCs prevent the premature destruction of antigenic determinants to optimize T cell activation. Phagosomal maturation is therefore a tightly regulated process that varies according to the type and differentiation stage of the phagocyte.

Show MeSH