Limits...
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.

Show MeSH

Related in: MedlinePlus

Maturation of mouse MHC class II molecules in the  absence or the presence of leupeptin. (A) Fresh H-2d splenocytes  were pulse labeled for 30 min and chased for the times indicated in  the absence (control) or the presence of 1 mM leupeptin. MHC  class II molecules were immunoprecipitated with mAb N22, and  loaded on 12.5% reducing SDS-PAGE without (NB) or after (B)  boiling. The position of the MHC class II α and β subunits, SDS-stable mature αβ heterodimers, full-length Ii, and intermediate degradation products of Ii detected in the abscence (P10) or the presence of leupeptin (LIP25, LIP22, LIP18, and LIP10) are indicated.  Leupeptin-treated pulsed splenocytes showed the same pattern and  intensity of bands as the control sample (data not shown). (B) Reimmunoprecipitation of the leupeptin-induced polypeptides LIP22  and LIP10. H-2d splenocytes were pulse labeled for 30 min and  chased 240 min in the presence of 1 mM leupeptin. After immunoprecipitation with mAb N22, MHC class II molecules were  fully denatured by boiling in 1% SDS and the released polypeptides immunoprecipitated in parallel with rabbit sera for the NH2-terminal or the CLIP region of Ii, or with mAb P4H5. Samples  were loaded on 10% reducing SDS-PAGE with a fraction of the  boiled N22 immunoprecipitate. The lower part of the panel corresponds to a longer exposure of the same gel shown in the upper half. (C) Structure of mouse Ii and the degradation intermediates that accumulate in  mouse splenocytes treated with leupeptin (LIP22 and LIP10), as deduced from reimmunoprecipitations. The N-linked carbohydrates at positions 113 and  119, the region against which mAb P4H5 was raised, and the transmembrane, CLIP, and trimerization regions of Ii are indicated according to references  6, 7. The enzymes involved in each stage of degradation of Ii and the protease inhibitors that block those steps are indicated.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2199027&req=5

Figure 1: Maturation of mouse MHC class II molecules in the absence or the presence of leupeptin. (A) Fresh H-2d splenocytes were pulse labeled for 30 min and chased for the times indicated in the absence (control) or the presence of 1 mM leupeptin. MHC class II molecules were immunoprecipitated with mAb N22, and loaded on 12.5% reducing SDS-PAGE without (NB) or after (B) boiling. The position of the MHC class II α and β subunits, SDS-stable mature αβ heterodimers, full-length Ii, and intermediate degradation products of Ii detected in the abscence (P10) or the presence of leupeptin (LIP25, LIP22, LIP18, and LIP10) are indicated. Leupeptin-treated pulsed splenocytes showed the same pattern and intensity of bands as the control sample (data not shown). (B) Reimmunoprecipitation of the leupeptin-induced polypeptides LIP22 and LIP10. H-2d splenocytes were pulse labeled for 30 min and chased 240 min in the presence of 1 mM leupeptin. After immunoprecipitation with mAb N22, MHC class II molecules were fully denatured by boiling in 1% SDS and the released polypeptides immunoprecipitated in parallel with rabbit sera for the NH2-terminal or the CLIP region of Ii, or with mAb P4H5. Samples were loaded on 10% reducing SDS-PAGE with a fraction of the boiled N22 immunoprecipitate. The lower part of the panel corresponds to a longer exposure of the same gel shown in the upper half. (C) Structure of mouse Ii and the degradation intermediates that accumulate in mouse splenocytes treated with leupeptin (LIP22 and LIP10), as deduced from reimmunoprecipitations. The N-linked carbohydrates at positions 113 and 119, the region against which mAb P4H5 was raised, and the transmembrane, CLIP, and trimerization regions of Ii are indicated according to references 6, 7. The enzymes involved in each stage of degradation of Ii and the protease inhibitors that block those steps are indicated.

Mentions: To assess the role of cysteine proteases on breakdown of murine Ii, fresh H-2d splenocytes were pulse-labeled for 30 min and chased up to 240 min in the absence or presence of leupeptin. At each time point, class II molecules were recovered by immunoprecipitation with the mAb N22. The samples were then divided into two and displayed by SDS-PAGE with or without prior boiling to score for formation of peptide-bound SDS-stable αβ dimers (Fig. 1 A). The mAb N22 reacts with both I-Ad and I-Ed molecules, and allowed recovery of roughly similar amounts of α and β subunits at all time points of the pulse–chase experiment, indicating that N22 recognizes with similar efficiency Ii- and peptide-associated αβ dimers, but not any of the three individual subunits. This reactivity was confirmed by immunoprecipitation of in vitro translated I-Ad α and β subunits and of mouse Ii (Wolf, P., and H.L. Ploegh, unpublished observations). Pulse-labeled samples showed MHC class II α and β subunits, the p33 form of Ii, and a small amount of the p41 form of Ii (47). Maturation of MHC class II in control cells during the chase was accompanied by addition of sialic acids to both α and β chains, which is responsible for the increased complexity in the pattern of bands observed in the α region and for the reduced mobility of β. Furthermore, degradation of Ii occurred; the apparent increase in the amount of full-length Ii at 60 min of chase is due to association of excess labeled Ii to unlabeled αβ dimers. In addition, we observed the transient accumulation of a polypeptide of 10 kD at 60 min (P10) and the generation of αβ–peptide complexes, a fraction of which is resistant to dissociation in SDS at room temperature (48).


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)

Maturation of mouse MHC class II molecules in the  absence or the presence of leupeptin. (A) Fresh H-2d splenocytes  were pulse labeled for 30 min and chased for the times indicated in  the absence (control) or the presence of 1 mM leupeptin. MHC  class II molecules were immunoprecipitated with mAb N22, and  loaded on 12.5% reducing SDS-PAGE without (NB) or after (B)  boiling. The position of the MHC class II α and β subunits, SDS-stable mature αβ heterodimers, full-length Ii, and intermediate degradation products of Ii detected in the abscence (P10) or the presence of leupeptin (LIP25, LIP22, LIP18, and LIP10) are indicated.  Leupeptin-treated pulsed splenocytes showed the same pattern and  intensity of bands as the control sample (data not shown). (B) Reimmunoprecipitation of the leupeptin-induced polypeptides LIP22  and LIP10. H-2d splenocytes were pulse labeled for 30 min and  chased 240 min in the presence of 1 mM leupeptin. After immunoprecipitation with mAb N22, MHC class II molecules were  fully denatured by boiling in 1% SDS and the released polypeptides immunoprecipitated in parallel with rabbit sera for the NH2-terminal or the CLIP region of Ii, or with mAb P4H5. Samples  were loaded on 10% reducing SDS-PAGE with a fraction of the  boiled N22 immunoprecipitate. The lower part of the panel corresponds to a longer exposure of the same gel shown in the upper half. (C) Structure of mouse Ii and the degradation intermediates that accumulate in  mouse splenocytes treated with leupeptin (LIP22 and LIP10), as deduced from reimmunoprecipitations. The N-linked carbohydrates at positions 113 and  119, the region against which mAb P4H5 was raised, and the transmembrane, CLIP, and trimerization regions of Ii are indicated according to references  6, 7. The enzymes involved in each stage of degradation of Ii and the protease inhibitors that block those steps are indicated.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199027&req=5

Figure 1: Maturation of mouse MHC class II molecules in the absence or the presence of leupeptin. (A) Fresh H-2d splenocytes were pulse labeled for 30 min and chased for the times indicated in the absence (control) or the presence of 1 mM leupeptin. MHC class II molecules were immunoprecipitated with mAb N22, and loaded on 12.5% reducing SDS-PAGE without (NB) or after (B) boiling. The position of the MHC class II α and β subunits, SDS-stable mature αβ heterodimers, full-length Ii, and intermediate degradation products of Ii detected in the abscence (P10) or the presence of leupeptin (LIP25, LIP22, LIP18, and LIP10) are indicated. Leupeptin-treated pulsed splenocytes showed the same pattern and intensity of bands as the control sample (data not shown). (B) Reimmunoprecipitation of the leupeptin-induced polypeptides LIP22 and LIP10. H-2d splenocytes were pulse labeled for 30 min and chased 240 min in the presence of 1 mM leupeptin. After immunoprecipitation with mAb N22, MHC class II molecules were fully denatured by boiling in 1% SDS and the released polypeptides immunoprecipitated in parallel with rabbit sera for the NH2-terminal or the CLIP region of Ii, or with mAb P4H5. Samples were loaded on 10% reducing SDS-PAGE with a fraction of the boiled N22 immunoprecipitate. The lower part of the panel corresponds to a longer exposure of the same gel shown in the upper half. (C) Structure of mouse Ii and the degradation intermediates that accumulate in mouse splenocytes treated with leupeptin (LIP22 and LIP10), as deduced from reimmunoprecipitations. The N-linked carbohydrates at positions 113 and 119, the region against which mAb P4H5 was raised, and the transmembrane, CLIP, and trimerization regions of Ii are indicated according to references 6, 7. The enzymes involved in each stage of degradation of Ii and the protease inhibitors that block those steps are indicated.
Mentions: To assess the role of cysteine proteases on breakdown of murine Ii, fresh H-2d splenocytes were pulse-labeled for 30 min and chased up to 240 min in the absence or presence of leupeptin. At each time point, class II molecules were recovered by immunoprecipitation with the mAb N22. The samples were then divided into two and displayed by SDS-PAGE with or without prior boiling to score for formation of peptide-bound SDS-stable αβ dimers (Fig. 1 A). The mAb N22 reacts with both I-Ad and I-Ed molecules, and allowed recovery of roughly similar amounts of α and β subunits at all time points of the pulse–chase experiment, indicating that N22 recognizes with similar efficiency Ii- and peptide-associated αβ dimers, but not any of the three individual subunits. This reactivity was confirmed by immunoprecipitation of in vitro translated I-Ad α and β subunits and of mouse Ii (Wolf, P., and H.L. Ploegh, unpublished observations). Pulse-labeled samples showed MHC class II α and β subunits, the p33 form of Ii, and a small amount of the p41 form of Ii (47). Maturation of MHC class II in control cells during the chase was accompanied by addition of sialic acids to both α and β chains, which is responsible for the increased complexity in the pattern of bands observed in the α region and for the reduced mobility of β. Furthermore, degradation of Ii occurred; the apparent increase in the amount of full-length Ii at 60 min of chase is due to association of excess labeled Ii to unlabeled αβ dimers. In addition, we observed the transient accumulation of a polypeptide of 10 kD at 60 min (P10) and the generation of αβ–peptide complexes, a fraction of which is resistant to dissociation in SDS at room temperature (48).

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