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Invariant chain controls the activity of extracellular cathepsin L.

Fiebiger E, Maehr R, Villadangos J, Weber E, Erickson A, Bikoff E, Ploegh HL, Lennon-Duménil AM - J. Exp. Med. (2002)

Bottom Line: A 65 amino acid segment of the p41 splice variant (p41(65aa)) of major histocompatibility complex class II-associated invariant chain (Ii) binds to the active site of CatL and permits the maintenance of a pool of mature enzyme in endosomal compartments of macro-phages and dendritic cells (DCs).Despite the neutral pH of the extracellular milieu, released CatL associated with p41(65aa) is catalytically active as demonstrated by active site labeling and elastin degradation assays.We propose that p41(65aa) stabilizes CatL in the extracellular environment and induces a local increase in the concentration of matrix-degrading enzymes during inflammation.

View Article: PubMed Central - PubMed

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

ABSTRACT
Secretion of proteases is critical for degradation of the extracellular matrix during an inflammatory response. Cathepsin (Cat) S and L are the major elastinolytic cysteine proteases in mouse macrophages. A 65 amino acid segment of the p41 splice variant (p41(65aa)) of major histocompatibility complex class II-associated invariant chain (Ii) binds to the active site of CatL and permits the maintenance of a pool of mature enzyme in endosomal compartments of macro-phages and dendritic cells (DCs). Here we show that interaction of p41(65aa) with mature CatL allows extracellular accumulation of the active enzyme. We detected mature CatL as a complex with p41(65aa) in culture supernatants from antigen-presenting cells (APCs). Extracellular accumulation of mature CatL is up-regulated by inflammatory stimuli as observed in interferon (IFN)-gamma-treated macrophages and lipopolysaccharide (LPS)-activated DCs. Despite the neutral pH of the extracellular milieu, released CatL associated with p41(65aa) is catalytically active as demonstrated by active site labeling and elastin degradation assays. We propose that p41(65aa) stabilizes CatL in the extracellular environment and induces a local increase in the concentration of matrix-degrading enzymes during inflammation. Through its interaction with CatL, Ii may therefore control the migratory response of APCs and/or the recruitment of effectors of the inflammatory response.

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Intra- and extracellular CatL. (A) Intracellular expression of CatL and CatS. Exposure time was adjusted to visualize mature, but not proCatL. The mature-25 kD enzyme is present in all samples but CatL−/−. Mature-24 kD CatL is coexpressed with p41. CatS is evenly detected in all samples. (B) Analysis of macrophage supernatants. ProCatL is detected in supernatants from all mouse strains. Mature CatL is detected only in wt and p41supernatants, but is absent from Ii−/− or p31 samples. Pro- and mature CatS are detected evenly in all samples irrespective of Ii expression. (C) Comparison of supernatants from resting and activated macrophages. Accumulation of extracellular mature CatL is up-regulated by IFN-γ.
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fig1: Intra- and extracellular CatL. (A) Intracellular expression of CatL and CatS. Exposure time was adjusted to visualize mature, but not proCatL. The mature-25 kD enzyme is present in all samples but CatL−/−. Mature-24 kD CatL is coexpressed with p41. CatS is evenly detected in all samples. (B) Analysis of macrophage supernatants. ProCatL is detected in supernatants from all mouse strains. Mature CatL is detected only in wt and p41supernatants, but is absent from Ii−/− or p31 samples. Pro- and mature CatS are detected evenly in all samples irrespective of Ii expression. (C) Comparison of supernatants from resting and activated macrophages. Accumulation of extracellular mature CatL is up-regulated by IFN-γ.

Mentions: The p41 isoform of Ii maintains a pool of mature enzyme in endocytic compartments of bone marrow-derived macrophages by means of a direct interaction (13, 20; Fig. 1 A). What functional role does this interaction between CatL and p41 play in APCs? One possibility is that CatL bound to p41 is packaged into vesicles of endosomal origin and released into extracellular space since, unlike free mature CatL, this complex can survive at neutral pH. We analyzed supernatants from IFN-γ–treated macrophages of WT, CatL (CatL−/−), and Ii knockout (Ii−/−) mice for the presence of CatL. Supernatants from cells that contain only the p31 or only the p41 splice variant of Ii were investigated (referred to as p31 and p41). Expression of proCatL is independent of p41, and anti-CatL immunoblotting experiments indeed showed presence of equal amounts of CatL zymogen in supernatants from all strains (Fig. 1 B). In contrast, the secreted 24 kD mature form of CatL was detected only in supernatants from WT and p41 macrophages, but not in Ii−/− or p31-derived samples (Fig. 1 B). As all supernatants contain large amounts of proCatL, extracellular activation of proforms can be ruled out as a mechanism for the generation of mature CatL. We performed an anti-CatS blot on the same supernatants and detected equal amounts of pro- and mature CatS in all samples (Fig. 1 B). Thus, accumulation of mature extracellular CatL is selectively regulated by the presence of p41. A comparison of supernatants prepared from untreated and stimulated macrophages shows the same pattern of accumulation, with considerably lower levels of mature CatL but larger amounts of proCatL in supernatants from untreated cells (Fig. 1 C).


Invariant chain controls the activity of extracellular cathepsin L.

Fiebiger E, Maehr R, Villadangos J, Weber E, Erickson A, Bikoff E, Ploegh HL, Lennon-Duménil AM - J. Exp. Med. (2002)

Intra- and extracellular CatL. (A) Intracellular expression of CatL and CatS. Exposure time was adjusted to visualize mature, but not proCatL. The mature-25 kD enzyme is present in all samples but CatL−/−. Mature-24 kD CatL is coexpressed with p41. CatS is evenly detected in all samples. (B) Analysis of macrophage supernatants. ProCatL is detected in supernatants from all mouse strains. Mature CatL is detected only in wt and p41supernatants, but is absent from Ii−/− or p31 samples. Pro- and mature CatS are detected evenly in all samples irrespective of Ii expression. (C) Comparison of supernatants from resting and activated macrophages. Accumulation of extracellular mature CatL is up-regulated by IFN-γ.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2194106&req=5

fig1: Intra- and extracellular CatL. (A) Intracellular expression of CatL and CatS. Exposure time was adjusted to visualize mature, but not proCatL. The mature-25 kD enzyme is present in all samples but CatL−/−. Mature-24 kD CatL is coexpressed with p41. CatS is evenly detected in all samples. (B) Analysis of macrophage supernatants. ProCatL is detected in supernatants from all mouse strains. Mature CatL is detected only in wt and p41supernatants, but is absent from Ii−/− or p31 samples. Pro- and mature CatS are detected evenly in all samples irrespective of Ii expression. (C) Comparison of supernatants from resting and activated macrophages. Accumulation of extracellular mature CatL is up-regulated by IFN-γ.
Mentions: The p41 isoform of Ii maintains a pool of mature enzyme in endocytic compartments of bone marrow-derived macrophages by means of a direct interaction (13, 20; Fig. 1 A). What functional role does this interaction between CatL and p41 play in APCs? One possibility is that CatL bound to p41 is packaged into vesicles of endosomal origin and released into extracellular space since, unlike free mature CatL, this complex can survive at neutral pH. We analyzed supernatants from IFN-γ–treated macrophages of WT, CatL (CatL−/−), and Ii knockout (Ii−/−) mice for the presence of CatL. Supernatants from cells that contain only the p31 or only the p41 splice variant of Ii were investigated (referred to as p31 and p41). Expression of proCatL is independent of p41, and anti-CatL immunoblotting experiments indeed showed presence of equal amounts of CatL zymogen in supernatants from all strains (Fig. 1 B). In contrast, the secreted 24 kD mature form of CatL was detected only in supernatants from WT and p41 macrophages, but not in Ii−/− or p31-derived samples (Fig. 1 B). As all supernatants contain large amounts of proCatL, extracellular activation of proforms can be ruled out as a mechanism for the generation of mature CatL. We performed an anti-CatS blot on the same supernatants and detected equal amounts of pro- and mature CatS in all samples (Fig. 1 B). Thus, accumulation of mature extracellular CatL is selectively regulated by the presence of p41. A comparison of supernatants prepared from untreated and stimulated macrophages shows the same pattern of accumulation, with considerably lower levels of mature CatL but larger amounts of proCatL in supernatants from untreated cells (Fig. 1 C).

Bottom Line: A 65 amino acid segment of the p41 splice variant (p41(65aa)) of major histocompatibility complex class II-associated invariant chain (Ii) binds to the active site of CatL and permits the maintenance of a pool of mature enzyme in endosomal compartments of macro-phages and dendritic cells (DCs).Despite the neutral pH of the extracellular milieu, released CatL associated with p41(65aa) is catalytically active as demonstrated by active site labeling and elastin degradation assays.We propose that p41(65aa) stabilizes CatL in the extracellular environment and induces a local increase in the concentration of matrix-degrading enzymes during inflammation.

View Article: PubMed Central - PubMed

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

ABSTRACT
Secretion of proteases is critical for degradation of the extracellular matrix during an inflammatory response. Cathepsin (Cat) S and L are the major elastinolytic cysteine proteases in mouse macrophages. A 65 amino acid segment of the p41 splice variant (p41(65aa)) of major histocompatibility complex class II-associated invariant chain (Ii) binds to the active site of CatL and permits the maintenance of a pool of mature enzyme in endosomal compartments of macro-phages and dendritic cells (DCs). Here we show that interaction of p41(65aa) with mature CatL allows extracellular accumulation of the active enzyme. We detected mature CatL as a complex with p41(65aa) in culture supernatants from antigen-presenting cells (APCs). Extracellular accumulation of mature CatL is up-regulated by inflammatory stimuli as observed in interferon (IFN)-gamma-treated macrophages and lipopolysaccharide (LPS)-activated DCs. Despite the neutral pH of the extracellular milieu, released CatL associated with p41(65aa) is catalytically active as demonstrated by active site labeling and elastin degradation assays. We propose that p41(65aa) stabilizes CatL in the extracellular environment and induces a local increase in the concentration of matrix-degrading enzymes during inflammation. Through its interaction with CatL, Ii may therefore control the migratory response of APCs and/or the recruitment of effectors of the inflammatory response.

Show MeSH
Related in: MedlinePlus