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Glycosylation directs targeting and activation of cystatin f from intracellular and extracellular sources.

Colbert JD, Plechanovová A, Watts C - Traffic (2009)

Bottom Line: Cystatin F is a cysteine protease inhibitor that is selectively expressed in immune cells and unlike other cystatin family members is targeted to a significant extent to intracellular compartments.We demonstrate the unusual addition of N-linked sugars to an Asn-X-Cys motif in cystatin F and provide evidence that the mannose 6-phosphate sorting machinery is used to divert cystatin F from the secretory pathway and to mediate its uptake from extracellular pools.These studies identify a function for the oligosaccharides on cystatin F and raise the possibility that cystatin F might regulate proteases in trans by secretion in an inactive form by one cell and subsequent internalization and activation by another cell.

View Article: PubMed Central - PubMed

Affiliation: Division of Cell Biology and Immunology, College of Life Sciences, University of Dundee, Dundee, UK. j.colbert@dundee.ac.uk

ABSTRACT
Cystatin F is a cysteine protease inhibitor that is selectively expressed in immune cells and unlike other cystatin family members is targeted to a significant extent to intracellular compartments. Initially made as an inactive glycosylated disulfide-linked dimer, cystatin F is converted to an active monomer by proteolytic cleavage following transport to the endosomal/lysosomal system. This active form of cystatin F targets cathepsin C/DPPI and probably other cathepsins in immune cells. We show that efficient targeting of cystatin F to the endocytic pathway is dependent not on its unique dimeric conformation but rather on its oligosaccharide chains. We demonstrate the unusual addition of N-linked sugars to an Asn-X-Cys motif in cystatin F and provide evidence that the mannose 6-phosphate sorting machinery is used to divert cystatin F from the secretory pathway and to mediate its uptake from extracellular pools. These studies identify a function for the oligosaccharides on cystatin F and raise the possibility that cystatin F might regulate proteases in trans by secretion in an inactive form by one cell and subsequent internalization and activation by another cell.

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Internalization and activation of cystatin F in primary cellsA) Lysates (L) and medium (M) from cultured primary murine CD8+ T lymphocytes (CTL) and dendritic cells (BMDC) isolated from spleen and bone marrow, respectively. B) Lysates from activated CD45.1+ (wild type) and CD45.2+ (knockout) CTLs were separated by nonreducing SDS–PAGE and immunoblotted for cystatin F (Cyst. F). Arrowheads denote dimeric (open) and monomeric (closed) cystatin F. Actin staining is used to confirm equivalent protein loading. C) CD45.1+ wild-type splenocytes were cocultured with CD45.2+ cystatin F knockout splenocytes for 48 h. Internalization of cystatin F into CD45.2+ cells was determined by intracellular immunostaining (after coculture). Numbers represent percentage of CD45.2+ cells staining for cystatin F before and after coculture. FACS data are representative of at least three independent experiments.
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fig06: Internalization and activation of cystatin F in primary cellsA) Lysates (L) and medium (M) from cultured primary murine CD8+ T lymphocytes (CTL) and dendritic cells (BMDC) isolated from spleen and bone marrow, respectively. B) Lysates from activated CD45.1+ (wild type) and CD45.2+ (knockout) CTLs were separated by nonreducing SDS–PAGE and immunoblotted for cystatin F (Cyst. F). Arrowheads denote dimeric (open) and monomeric (closed) cystatin F. Actin staining is used to confirm equivalent protein loading. C) CD45.1+ wild-type splenocytes were cocultured with CD45.2+ cystatin F knockout splenocytes for 48 h. Internalization of cystatin F into CD45.2+ cells was determined by intracellular immunostaining (after coculture). Numbers represent percentage of CD45.2+ cells staining for cystatin F before and after coculture. FACS data are representative of at least three independent experiments.

Mentions: Finally, we asked whether secretion of cystatin F by one cell and internalization by another could be demonstrated in primary immune cells. We found that significant amounts of dimeric cystatin F were secreted from primary CD8+ T lymphocytes and bone marrow-derived dendritic cells (BMDC). As expected, the cell lysates also contained cystatin F, much of which was converted to the active monomeric form (Figure 6A). We recently generated cystatin F-deficient mice that are currently being analyzed. These mice were used to generate a population of cystatin F-negative CD8+ T cells by in vitroexpansion. We incubated these cells with an equal number of wild-type CD8+ T cells and asked whether cystatin F was acquired by the cells. We used the CD45 marker and fluorescence-activated cell sorter (FACS) analysis to distinguish wild type (CD45.1+) from cystatin F (CD45.2+) cells (Figure 6B, top panel). As shown in Figure 6C, a small proportion of CD45.2+ cells became positive for cystatin F during the course of the experiment demonstrating that the protease inhibitor had been taken up by the cells (Figure 6C, bottom panel). This important result indicates that one cell may influence the protease activity of another through the agency of cystatin F.


Glycosylation directs targeting and activation of cystatin f from intracellular and extracellular sources.

Colbert JD, Plechanovová A, Watts C - Traffic (2009)

Internalization and activation of cystatin F in primary cellsA) Lysates (L) and medium (M) from cultured primary murine CD8+ T lymphocytes (CTL) and dendritic cells (BMDC) isolated from spleen and bone marrow, respectively. B) Lysates from activated CD45.1+ (wild type) and CD45.2+ (knockout) CTLs were separated by nonreducing SDS–PAGE and immunoblotted for cystatin F (Cyst. F). Arrowheads denote dimeric (open) and monomeric (closed) cystatin F. Actin staining is used to confirm equivalent protein loading. C) CD45.1+ wild-type splenocytes were cocultured with CD45.2+ cystatin F knockout splenocytes for 48 h. Internalization of cystatin F into CD45.2+ cells was determined by intracellular immunostaining (after coculture). Numbers represent percentage of CD45.2+ cells staining for cystatin F before and after coculture. FACS data are representative of at least three independent experiments.
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Related In: Results  -  Collection

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fig06: Internalization and activation of cystatin F in primary cellsA) Lysates (L) and medium (M) from cultured primary murine CD8+ T lymphocytes (CTL) and dendritic cells (BMDC) isolated from spleen and bone marrow, respectively. B) Lysates from activated CD45.1+ (wild type) and CD45.2+ (knockout) CTLs were separated by nonreducing SDS–PAGE and immunoblotted for cystatin F (Cyst. F). Arrowheads denote dimeric (open) and monomeric (closed) cystatin F. Actin staining is used to confirm equivalent protein loading. C) CD45.1+ wild-type splenocytes were cocultured with CD45.2+ cystatin F knockout splenocytes for 48 h. Internalization of cystatin F into CD45.2+ cells was determined by intracellular immunostaining (after coculture). Numbers represent percentage of CD45.2+ cells staining for cystatin F before and after coculture. FACS data are representative of at least three independent experiments.
Mentions: Finally, we asked whether secretion of cystatin F by one cell and internalization by another could be demonstrated in primary immune cells. We found that significant amounts of dimeric cystatin F were secreted from primary CD8+ T lymphocytes and bone marrow-derived dendritic cells (BMDC). As expected, the cell lysates also contained cystatin F, much of which was converted to the active monomeric form (Figure 6A). We recently generated cystatin F-deficient mice that are currently being analyzed. These mice were used to generate a population of cystatin F-negative CD8+ T cells by in vitroexpansion. We incubated these cells with an equal number of wild-type CD8+ T cells and asked whether cystatin F was acquired by the cells. We used the CD45 marker and fluorescence-activated cell sorter (FACS) analysis to distinguish wild type (CD45.1+) from cystatin F (CD45.2+) cells (Figure 6B, top panel). As shown in Figure 6C, a small proportion of CD45.2+ cells became positive for cystatin F during the course of the experiment demonstrating that the protease inhibitor had been taken up by the cells (Figure 6C, bottom panel). This important result indicates that one cell may influence the protease activity of another through the agency of cystatin F.

Bottom Line: Cystatin F is a cysteine protease inhibitor that is selectively expressed in immune cells and unlike other cystatin family members is targeted to a significant extent to intracellular compartments.We demonstrate the unusual addition of N-linked sugars to an Asn-X-Cys motif in cystatin F and provide evidence that the mannose 6-phosphate sorting machinery is used to divert cystatin F from the secretory pathway and to mediate its uptake from extracellular pools.These studies identify a function for the oligosaccharides on cystatin F and raise the possibility that cystatin F might regulate proteases in trans by secretion in an inactive form by one cell and subsequent internalization and activation by another cell.

View Article: PubMed Central - PubMed

Affiliation: Division of Cell Biology and Immunology, College of Life Sciences, University of Dundee, Dundee, UK. j.colbert@dundee.ac.uk

ABSTRACT
Cystatin F is a cysteine protease inhibitor that is selectively expressed in immune cells and unlike other cystatin family members is targeted to a significant extent to intracellular compartments. Initially made as an inactive glycosylated disulfide-linked dimer, cystatin F is converted to an active monomer by proteolytic cleavage following transport to the endosomal/lysosomal system. This active form of cystatin F targets cathepsin C/DPPI and probably other cathepsins in immune cells. We show that efficient targeting of cystatin F to the endocytic pathway is dependent not on its unique dimeric conformation but rather on its oligosaccharide chains. We demonstrate the unusual addition of N-linked sugars to an Asn-X-Cys motif in cystatin F and provide evidence that the mannose 6-phosphate sorting machinery is used to divert cystatin F from the secretory pathway and to mediate its uptake from extracellular pools. These studies identify a function for the oligosaccharides on cystatin F and raise the possibility that cystatin F might regulate proteases in trans by secretion in an inactive form by one cell and subsequent internalization and activation by another cell.

Show MeSH
Related in: MedlinePlus