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Role for cathepsin F in invariant chain processing and major histocompatibility complex class II peptide loading by macrophages.

Shi GP, Bryant RA, Riese R, Verhelst S, Driessen C, Li Z, Bromme D, Ploegh HL, Chapman HA - J. Exp. Med. (2000)

Bottom Line: Comparison of cysteine proteases expressed by macrophages with those found in splenocytes and dendritic cells revealed two enzymes expressed exclusively in macrophages, cathepsins Z and F.Recombinant cathepsin Z did not generate CLIP from Ii-MHC class II complexes, whereas cathepsin F was as efficient as cathepsin S in CLIP generation.Different APCs can thus use distinct proteases to mediate MHC class II maturation and peptide loading.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Harvard Medical School, Boston, Massachusetts 02115, USA.

ABSTRACT
The major histocompatibility complex (MHC) class II-associated invariant chain (Ii) regulates intracellular trafficking and peptide loading of MHC class II molecules. Such loading occurs after endosomal degradation of the invariant chain to a approximately 3-kD peptide termed CLIP (class II-associated invariant chain peptide). Cathepsins L and S have both been implicated in degradation of Ii to CLIP in thymus and peripheral lymphoid organs, respectively. However, macrophages from mice deficient in both cathepsins S and L can process Ii and load peptides onto MHC class II dimers normally. Both processes are blocked by a cysteine protease inhibitor, indicating the involvement of an additional Ii-processing enzyme(s). Comparison of cysteine proteases expressed by macrophages with those found in splenocytes and dendritic cells revealed two enzymes expressed exclusively in macrophages, cathepsins Z and F. Recombinant cathepsin Z did not generate CLIP from Ii-MHC class II complexes, whereas cathepsin F was as efficient as cathepsin S in CLIP generation. Inhibition of cathepsin F activity and MHC class II peptide loading by macrophages exhibited similar specificity and activity profiles. These experiments show that cathepsin F, in a subset of antigen presenting cells (APCs), can efficiently degrade Ii. Different APCs can thus use distinct proteases to mediate MHC class II maturation and peptide loading.

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Isolation and expression of mouse Cats F and Z. (A) Northern blot analysis. Total RNA (20 μg/each) from mouse splenocytes, peritoneal macrophages, and flt-3–stimulated dendritic cells were separated on 1.2% agarose gel, blotted onto a nylon filter, and probed with full length mouse Cat F, Z, and S cDNAs. RNA loading control is shown by rRNAs (top panel). Both Cats F and Z can be detected from macrophages but not splenocytes or dendritic cells. In contrast, Cat S transcripts can be detected in all three cell types. (B) Amino acid sequence of mouse Cat Z. The active site amino acids are indicated with asterisks (*), and the potential sites for glycosylation are double underlined. Arrowheads indicate the potential cleavage sites of signal peptide and pro region of Cat Z. (C) Amino acid sequence of mouse Cat F. Arrowheads indicate the signal peptide and pro region cleavage sites. Three active site amino acids (Cys, His, and Asn) are indicated by asterisks (*), and three potential glycosylation sites are double underlined. The underlined region is the potential cystatin-like domain 32.
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Figure 3: Isolation and expression of mouse Cats F and Z. (A) Northern blot analysis. Total RNA (20 μg/each) from mouse splenocytes, peritoneal macrophages, and flt-3–stimulated dendritic cells were separated on 1.2% agarose gel, blotted onto a nylon filter, and probed with full length mouse Cat F, Z, and S cDNAs. RNA loading control is shown by rRNAs (top panel). Both Cats F and Z can be detected from macrophages but not splenocytes or dendritic cells. In contrast, Cat S transcripts can be detected in all three cell types. (B) Amino acid sequence of mouse Cat Z. The active site amino acids are indicated with asterisks (*), and the potential sites for glycosylation are double underlined. Arrowheads indicate the potential cleavage sites of signal peptide and pro region of Cat Z. (C) Amino acid sequence of mouse Cat F. Arrowheads indicate the signal peptide and pro region cleavage sites. Three active site amino acids (Cys, His, and Asn) are indicated by asterisks (*), and three potential glycosylation sites are double underlined. The underlined region is the potential cystatin-like domain 32.

Mentions: We next compared the set of cysteine proteases expressed by macrophages, B cells, and dendritic cells. EST databases were screened for murine cysteine protease cDNAs as a source of DNA probes. Sequences were obtained for murine Cats O, W, K, Z, and F in addition to the major Cats H, B, L, and S. PCRs of reverse-transcribed mRNA and Northern blot analyses were used to compare the expression of these enzymes in macrophages, B cells, and dendritic cells. Cat W and K mRNA was not seen in these cells, consistent with prior reports 2627. Low levels of Cat O were found in both B cells and macrophages. Because this enzyme was comparable in both cell types, Cat O was not further studied. Two enzymes, Cat Z and Cat F, were found to be expressed in macrophages but not splenocytes or dendritic cells by Northern blot analysis (Fig. 3 A). Comparison of mRNA levels of Cats Z and F with levels of Cat S in macrophages shows the relative level of Cat S to be considerably higher. Whereas Cat S mRNA is also evident in splenocytes and dendritic cells, longer exposure of the Northern blot shown in Fig. 3 A did not reveal a signal for either Cat F or Z mRNA in these cell types (not shown). Also unlike macrophages, PCR amplification of reverse-transcribed RNA failed to reveal Cats Z and F in splenocytes. Cats Z and F were therefore characterized in more detail.


Role for cathepsin F in invariant chain processing and major histocompatibility complex class II peptide loading by macrophages.

Shi GP, Bryant RA, Riese R, Verhelst S, Driessen C, Li Z, Bromme D, Ploegh HL, Chapman HA - J. Exp. Med. (2000)

Isolation and expression of mouse Cats F and Z. (A) Northern blot analysis. Total RNA (20 μg/each) from mouse splenocytes, peritoneal macrophages, and flt-3–stimulated dendritic cells were separated on 1.2% agarose gel, blotted onto a nylon filter, and probed with full length mouse Cat F, Z, and S cDNAs. RNA loading control is shown by rRNAs (top panel). Both Cats F and Z can be detected from macrophages but not splenocytes or dendritic cells. In contrast, Cat S transcripts can be detected in all three cell types. (B) Amino acid sequence of mouse Cat Z. The active site amino acids are indicated with asterisks (*), and the potential sites for glycosylation are double underlined. Arrowheads indicate the potential cleavage sites of signal peptide and pro region of Cat Z. (C) Amino acid sequence of mouse Cat F. Arrowheads indicate the signal peptide and pro region cleavage sites. Three active site amino acids (Cys, His, and Asn) are indicated by asterisks (*), and three potential glycosylation sites are double underlined. The underlined region is the potential cystatin-like domain 32.
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Related In: Results  -  Collection

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Figure 3: Isolation and expression of mouse Cats F and Z. (A) Northern blot analysis. Total RNA (20 μg/each) from mouse splenocytes, peritoneal macrophages, and flt-3–stimulated dendritic cells were separated on 1.2% agarose gel, blotted onto a nylon filter, and probed with full length mouse Cat F, Z, and S cDNAs. RNA loading control is shown by rRNAs (top panel). Both Cats F and Z can be detected from macrophages but not splenocytes or dendritic cells. In contrast, Cat S transcripts can be detected in all three cell types. (B) Amino acid sequence of mouse Cat Z. The active site amino acids are indicated with asterisks (*), and the potential sites for glycosylation are double underlined. Arrowheads indicate the potential cleavage sites of signal peptide and pro region of Cat Z. (C) Amino acid sequence of mouse Cat F. Arrowheads indicate the signal peptide and pro region cleavage sites. Three active site amino acids (Cys, His, and Asn) are indicated by asterisks (*), and three potential glycosylation sites are double underlined. The underlined region is the potential cystatin-like domain 32.
Mentions: We next compared the set of cysteine proteases expressed by macrophages, B cells, and dendritic cells. EST databases were screened for murine cysteine protease cDNAs as a source of DNA probes. Sequences were obtained for murine Cats O, W, K, Z, and F in addition to the major Cats H, B, L, and S. PCRs of reverse-transcribed mRNA and Northern blot analyses were used to compare the expression of these enzymes in macrophages, B cells, and dendritic cells. Cat W and K mRNA was not seen in these cells, consistent with prior reports 2627. Low levels of Cat O were found in both B cells and macrophages. Because this enzyme was comparable in both cell types, Cat O was not further studied. Two enzymes, Cat Z and Cat F, were found to be expressed in macrophages but not splenocytes or dendritic cells by Northern blot analysis (Fig. 3 A). Comparison of mRNA levels of Cats Z and F with levels of Cat S in macrophages shows the relative level of Cat S to be considerably higher. Whereas Cat S mRNA is also evident in splenocytes and dendritic cells, longer exposure of the Northern blot shown in Fig. 3 A did not reveal a signal for either Cat F or Z mRNA in these cell types (not shown). Also unlike macrophages, PCR amplification of reverse-transcribed RNA failed to reveal Cats Z and F in splenocytes. Cats Z and F were therefore characterized in more detail.

Bottom Line: Comparison of cysteine proteases expressed by macrophages with those found in splenocytes and dendritic cells revealed two enzymes expressed exclusively in macrophages, cathepsins Z and F.Recombinant cathepsin Z did not generate CLIP from Ii-MHC class II complexes, whereas cathepsin F was as efficient as cathepsin S in CLIP generation.Different APCs can thus use distinct proteases to mediate MHC class II maturation and peptide loading.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Harvard Medical School, Boston, Massachusetts 02115, USA.

ABSTRACT
The major histocompatibility complex (MHC) class II-associated invariant chain (Ii) regulates intracellular trafficking and peptide loading of MHC class II molecules. Such loading occurs after endosomal degradation of the invariant chain to a approximately 3-kD peptide termed CLIP (class II-associated invariant chain peptide). Cathepsins L and S have both been implicated in degradation of Ii to CLIP in thymus and peripheral lymphoid organs, respectively. However, macrophages from mice deficient in both cathepsins S and L can process Ii and load peptides onto MHC class II dimers normally. Both processes are blocked by a cysteine protease inhibitor, indicating the involvement of an additional Ii-processing enzyme(s). Comparison of cysteine proteases expressed by macrophages with those found in splenocytes and dendritic cells revealed two enzymes expressed exclusively in macrophages, cathepsins Z and F. Recombinant cathepsin Z did not generate CLIP from Ii-MHC class II complexes, whereas cathepsin F was as efficient as cathepsin S in CLIP generation. Inhibition of cathepsin F activity and MHC class II peptide loading by macrophages exhibited similar specificity and activity profiles. These experiments show that cathepsin F, in a subset of antigen presenting cells (APCs), can efficiently degrade Ii. Different APCs can thus use distinct proteases to mediate MHC class II maturation and peptide loading.

Show MeSH
Related in: MedlinePlus