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Degradation of mouse invariant chain: roles of cathepsins S and D and the influence of major histocompatibility complex polymorphism.

Villadangos JA, Riese RJ, Peters C, Chapman HA, Ploegh HL - J. Exp. Med. (1997)

Bottom Line: Class II molecules are delivered to endocytic compartments by the class II accessory molecule invariant chain (Ii), which itself must be eliminated to allow peptide binding.The cellular location of Ii degradation, as well as the enzymology of this event, are important in determining the sets of antigenic peptides that will bind to class II molecules.These observations suggest that, first, class II molecules associated with larger Ii remnants can be converted efficiently to class II-peptide complexes and, second, that most class II-associated peptides can still be generated in cells treated with inhibitors of cysteine proteases.

View Article: PubMed Central - PubMed

Affiliation: Center for Cancer Research, Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.

ABSTRACT
Antigen-presenting cells (APC) degrade endocytosed antigens into peptides that are bound and presented to T cells by major histocompatibility complex (MHC) class II molecules. Class II molecules are delivered to endocytic compartments by the class II accessory molecule invariant chain (Ii), which itself must be eliminated to allow peptide binding. The cellular location of Ii degradation, as well as the enzymology of this event, are important in determining the sets of antigenic peptides that will bind to class II molecules. Here, we show that the cysteine protease cathepsin S acts in a concerted fashion with other cysteine and noncysteine proteases to degrade mouse Ii in a stepwise fashion. Inactivation of cysteine proteases results in incomplete degradation of Ii, but the extent to which peptide loading is blocked by such treatment varies widely among MHC class II allelic products. These observations suggest that, first, class II molecules associated with larger Ii remnants can be converted efficiently to class II-peptide complexes and, second, that most class II-associated peptides can still be generated in cells treated with inhibitors of cysteine proteases. Surprisingly, maturation of MHC class II in mice deficient in cathepsin D is unaffected, showing that this major aspartyl protease is not involved in degradation of Ii or in generation of the bulk of antigenic peptides.

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Role of Cat S on degradation  of mouse Ii. (A) LHVS is a specific inhibitor  of Cat S at the 1–10 nM range. Mouse splenocytes were incubated with the indicated  concentrations of LHVS or leupeptin (1 mM)  followed by addition of Cbz–[125I]–Tyr– Ala–CN2. The bands corresponding to Cat  S and to the high and low molecular weight  forms of Cat B are indicated. (B) A cys protease different from Cat S converts LIP22  into LIP10. H-2d splenocytes were pulse labeled for 30 min and chased for 240 min  without (control) or with 1 mM leupeptin or  3 nM LHVS. N22 immunoprecipitates  were loaded without boiling in 12.5% SDS-PAGE. (C) Cat S cleaves Ii NH2 terminally  of CLIP. H-2d splenocytes were pulse  chased in the presence of LHVS and immunoprecipitated with N22. The precipitate  was resuspended in Cat S buffer and incubated with or without Cat S for 1 h at 37°C.  After incubation, samples were boiled in 1%  SDS, 1/5 loaded directly on gel, and the remainder diluted in lysis buffer and reimmunoprecipitated as in Fig. 1 B. The arrow at  the right of the figure indicates the position  of CLIP-containing fragments devoid of the  NH2-terminal region of Ii.
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Figure 2: Role of Cat S on degradation of mouse Ii. (A) LHVS is a specific inhibitor of Cat S at the 1–10 nM range. Mouse splenocytes were incubated with the indicated concentrations of LHVS or leupeptin (1 mM) followed by addition of Cbz–[125I]–Tyr– Ala–CN2. The bands corresponding to Cat S and to the high and low molecular weight forms of Cat B are indicated. (B) A cys protease different from Cat S converts LIP22 into LIP10. H-2d splenocytes were pulse labeled for 30 min and chased for 240 min without (control) or with 1 mM leupeptin or 3 nM LHVS. N22 immunoprecipitates were loaded without boiling in 12.5% SDS-PAGE. (C) Cat S cleaves Ii NH2 terminally of CLIP. H-2d splenocytes were pulse chased in the presence of LHVS and immunoprecipitated with N22. The precipitate was resuspended in Cat S buffer and incubated with or without Cat S for 1 h at 37°C. After incubation, samples were boiled in 1% SDS, 1/5 loaded directly on gel, and the remainder diluted in lysis buffer and reimmunoprecipitated as in Fig. 1 B. The arrow at the right of the figure indicates the position of CLIP-containing fragments devoid of the NH2-terminal region of Ii.

Mentions: We have previously described the involvement of the cysteine protease Cat S in degradation of Ii in human B cells (37). Cat S can be specifically inactivated in human B cells with low concentrations of the irreversible inhibitor LHVS (37, 52). To assess whether Cat S plays a similar role in mouse APC, we first determined the content of active cysteine proteases in splenocytes treated with various concentrations of LHVS or with leupeptin. After this treatment, splenocytes were incubated with the iodinated form of Cbz–Tyr–Ala–CN2, an active site inhibitor that binds irreversibly to cysteine proteases in proportion to their activity (53). The amount of active cysteine proteases remaining can thus be visualized in SDS-PAGE as complexes bound to Cbz–[125I]–Tyr–Ala– CN2 (Fig. 2 A). Compared with untreated splenocytes, 1–10 nM LHVS completely inactivated Cat S without affecting Cat B. Thus, comparison of the effect of leupeptin (1 mM) and LHVS (3 nM) allows an assessment of the relative role of Cat S in maturation of class II molecules.


Degradation of mouse invariant chain: roles of cathepsins S and D and the influence of major histocompatibility complex polymorphism.

Villadangos JA, Riese RJ, Peters C, Chapman HA, Ploegh HL - J. Exp. Med. (1997)

Role of Cat S on degradation  of mouse Ii. (A) LHVS is a specific inhibitor  of Cat S at the 1–10 nM range. Mouse splenocytes were incubated with the indicated  concentrations of LHVS or leupeptin (1 mM)  followed by addition of Cbz–[125I]–Tyr– Ala–CN2. The bands corresponding to Cat  S and to the high and low molecular weight  forms of Cat B are indicated. (B) A cys protease different from Cat S converts LIP22  into LIP10. H-2d splenocytes were pulse labeled for 30 min and chased for 240 min  without (control) or with 1 mM leupeptin or  3 nM LHVS. N22 immunoprecipitates  were loaded without boiling in 12.5% SDS-PAGE. (C) Cat S cleaves Ii NH2 terminally  of CLIP. H-2d splenocytes were pulse  chased in the presence of LHVS and immunoprecipitated with N22. The precipitate  was resuspended in Cat S buffer and incubated with or without Cat S for 1 h at 37°C.  After incubation, samples were boiled in 1%  SDS, 1/5 loaded directly on gel, and the remainder diluted in lysis buffer and reimmunoprecipitated as in Fig. 1 B. The arrow at  the right of the figure indicates the position  of CLIP-containing fragments devoid of the  NH2-terminal region of Ii.
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Related In: Results  -  Collection

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Figure 2: Role of Cat S on degradation of mouse Ii. (A) LHVS is a specific inhibitor of Cat S at the 1–10 nM range. Mouse splenocytes were incubated with the indicated concentrations of LHVS or leupeptin (1 mM) followed by addition of Cbz–[125I]–Tyr– Ala–CN2. The bands corresponding to Cat S and to the high and low molecular weight forms of Cat B are indicated. (B) A cys protease different from Cat S converts LIP22 into LIP10. H-2d splenocytes were pulse labeled for 30 min and chased for 240 min without (control) or with 1 mM leupeptin or 3 nM LHVS. N22 immunoprecipitates were loaded without boiling in 12.5% SDS-PAGE. (C) Cat S cleaves Ii NH2 terminally of CLIP. H-2d splenocytes were pulse chased in the presence of LHVS and immunoprecipitated with N22. The precipitate was resuspended in Cat S buffer and incubated with or without Cat S for 1 h at 37°C. After incubation, samples were boiled in 1% SDS, 1/5 loaded directly on gel, and the remainder diluted in lysis buffer and reimmunoprecipitated as in Fig. 1 B. The arrow at the right of the figure indicates the position of CLIP-containing fragments devoid of the NH2-terminal region of Ii.
Mentions: We have previously described the involvement of the cysteine protease Cat S in degradation of Ii in human B cells (37). Cat S can be specifically inactivated in human B cells with low concentrations of the irreversible inhibitor LHVS (37, 52). To assess whether Cat S plays a similar role in mouse APC, we first determined the content of active cysteine proteases in splenocytes treated with various concentrations of LHVS or with leupeptin. After this treatment, splenocytes were incubated with the iodinated form of Cbz–Tyr–Ala–CN2, an active site inhibitor that binds irreversibly to cysteine proteases in proportion to their activity (53). The amount of active cysteine proteases remaining can thus be visualized in SDS-PAGE as complexes bound to Cbz–[125I]–Tyr–Ala– CN2 (Fig. 2 A). Compared with untreated splenocytes, 1–10 nM LHVS completely inactivated Cat S without affecting Cat B. Thus, comparison of the effect of leupeptin (1 mM) and LHVS (3 nM) allows an assessment of the relative role of Cat S in maturation of class II molecules.

Bottom Line: Class II molecules are delivered to endocytic compartments by the class II accessory molecule invariant chain (Ii), which itself must be eliminated to allow peptide binding.The cellular location of Ii degradation, as well as the enzymology of this event, are important in determining the sets of antigenic peptides that will bind to class II molecules.These observations suggest that, first, class II molecules associated with larger Ii remnants can be converted efficiently to class II-peptide complexes and, second, that most class II-associated peptides can still be generated in cells treated with inhibitors of cysteine proteases.

View Article: PubMed Central - PubMed

Affiliation: Center for Cancer Research, Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.

ABSTRACT
Antigen-presenting cells (APC) degrade endocytosed antigens into peptides that are bound and presented to T cells by major histocompatibility complex (MHC) class II molecules. Class II molecules are delivered to endocytic compartments by the class II accessory molecule invariant chain (Ii), which itself must be eliminated to allow peptide binding. The cellular location of Ii degradation, as well as the enzymology of this event, are important in determining the sets of antigenic peptides that will bind to class II molecules. Here, we show that the cysteine protease cathepsin S acts in a concerted fashion with other cysteine and noncysteine proteases to degrade mouse Ii in a stepwise fashion. Inactivation of cysteine proteases results in incomplete degradation of Ii, but the extent to which peptide loading is blocked by such treatment varies widely among MHC class II allelic products. These observations suggest that, first, class II molecules associated with larger Ii remnants can be converted efficiently to class II-peptide complexes and, second, that most class II-associated peptides can still be generated in cells treated with inhibitors of cysteine proteases. Surprisingly, maturation of MHC class II in mice deficient in cathepsin D is unaffected, showing that this major aspartyl protease is not involved in degradation of Ii or in generation of the bulk of antigenic peptides.

Show MeSH
Related in: MedlinePlus