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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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Cystatin F subcellular localization and secretion does not depend on dimer formationA) SDS–PAGE separated cell lysates (L) and culture medium (M) under nonreducing conditions from 293T cells transfected with wild-type or C26,63S human cystatin F. Western blots were probed with an antibody raised against full-length cystatin F (FL Ab) (left and right panels) or an N-terminal peptide (N-term Ab) (center panel). Right panel shows change in mobility following deglycosylation with PNGase under reducing conditions. Open and closed arrowheads denote dimeric and monomeric cystatin F, respectively. Asterisk denotes N-terminal processing of the C26,63S mutant. B) Immunofluorescence microscopy of 293T cells shown in (A) expressing wild-type or C26,63S cystatin F (red) and counterstained with an antibody to the lysosomal marker CD63 (green). Insets demonstrate areas of colocalization at high magnification. Bars, 10 μm. C) Western blot analysis following size exclusion chromatography. Nonreducing SDS–PAGE gel analysis of equal sized aliquots of column fractions obtained following chromatography of wild-type (top panel) or C26,63S (bottom panel) cystatin F. D) The Stokes’ radius of wild-type (3.19 nm) and C26,63S (2.35 nm) cystatin F were calculated following size exclusion chromatography. Values were interpolated from the calibration curve obtained by the linear fitting of Stokes’ radius versus (−log Kav)1/2 for a series of proteins with known Stokes’ radii, as described. Protein calibration was obtained using ribonuclease A (a), carbonic anhydrase (b), ovalbumin (c) and conalbumin (d).
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fig01: Cystatin F subcellular localization and secretion does not depend on dimer formationA) SDS–PAGE separated cell lysates (L) and culture medium (M) under nonreducing conditions from 293T cells transfected with wild-type or C26,63S human cystatin F. Western blots were probed with an antibody raised against full-length cystatin F (FL Ab) (left and right panels) or an N-terminal peptide (N-term Ab) (center panel). Right panel shows change in mobility following deglycosylation with PNGase under reducing conditions. Open and closed arrowheads denote dimeric and monomeric cystatin F, respectively. Asterisk denotes N-terminal processing of the C26,63S mutant. B) Immunofluorescence microscopy of 293T cells shown in (A) expressing wild-type or C26,63S cystatin F (red) and counterstained with an antibody to the lysosomal marker CD63 (green). Insets demonstrate areas of colocalization at high magnification. Bars, 10 μm. C) Western blot analysis following size exclusion chromatography. Nonreducing SDS–PAGE gel analysis of equal sized aliquots of column fractions obtained following chromatography of wild-type (top panel) or C26,63S (bottom panel) cystatin F. D) The Stokes’ radius of wild-type (3.19 nm) and C26,63S (2.35 nm) cystatin F were calculated following size exclusion chromatography. Values were interpolated from the calibration curve obtained by the linear fitting of Stokes’ radius versus (−log Kav)1/2 for a series of proteins with known Stokes’ radii, as described. Protein calibration was obtained using ribonuclease A (a), carbonic anhydrase (b), ovalbumin (c) and conalbumin (d).

Mentions: To investigate the trafficking of cystatin F, we generated various mutant forms and analyzed these initially in the human kidney epithelial cell line 293T, which does not express cystatin F. The cells were transfected, and after 48 h, the relative amount of cystatin F in cell lysates (L) and in the culture medium (M) was assessed. Expression of wild-type cystatin F leads to significant secretion of the protein into the culture medium as we and others have previously reported (10,13,16). Nonetheless, a significant amount of cystatin F was also retained in 293T cells and was partially converted to the monomeric form (Figure 1A, left panel). Cystatin F recovered from the culture medium was dimeric, whereas cellular cystatin F comprised a mixture of dimeric and monomeric protein (Figure 1A, left panel). Furthermore, while the dimeric form reacted with antibodies raised against the N-terminus of cystatin F the monomeric form did not, indicating intracellular processing had occurred as we recently reported (Figure 1A, center panel) (16). As shown previously, heterogeneous N-glycosylation accounted for the resolution of distinct forms of cystatin F on SDS–PAGE (9). This is highlighted by the formation of a single form following treatment with PNGase (Figure 1A, right panel).


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

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

Cystatin F subcellular localization and secretion does not depend on dimer formationA) SDS–PAGE separated cell lysates (L) and culture medium (M) under nonreducing conditions from 293T cells transfected with wild-type or C26,63S human cystatin F. Western blots were probed with an antibody raised against full-length cystatin F (FL Ab) (left and right panels) or an N-terminal peptide (N-term Ab) (center panel). Right panel shows change in mobility following deglycosylation with PNGase under reducing conditions. Open and closed arrowheads denote dimeric and monomeric cystatin F, respectively. Asterisk denotes N-terminal processing of the C26,63S mutant. B) Immunofluorescence microscopy of 293T cells shown in (A) expressing wild-type or C26,63S cystatin F (red) and counterstained with an antibody to the lysosomal marker CD63 (green). Insets demonstrate areas of colocalization at high magnification. Bars, 10 μm. C) Western blot analysis following size exclusion chromatography. Nonreducing SDS–PAGE gel analysis of equal sized aliquots of column fractions obtained following chromatography of wild-type (top panel) or C26,63S (bottom panel) cystatin F. D) The Stokes’ radius of wild-type (3.19 nm) and C26,63S (2.35 nm) cystatin F were calculated following size exclusion chromatography. Values were interpolated from the calibration curve obtained by the linear fitting of Stokes’ radius versus (−log Kav)1/2 for a series of proteins with known Stokes’ radii, as described. Protein calibration was obtained using ribonuclease A (a), carbonic anhydrase (b), ovalbumin (c) and conalbumin (d).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2691902&req=5

fig01: Cystatin F subcellular localization and secretion does not depend on dimer formationA) SDS–PAGE separated cell lysates (L) and culture medium (M) under nonreducing conditions from 293T cells transfected with wild-type or C26,63S human cystatin F. Western blots were probed with an antibody raised against full-length cystatin F (FL Ab) (left and right panels) or an N-terminal peptide (N-term Ab) (center panel). Right panel shows change in mobility following deglycosylation with PNGase under reducing conditions. Open and closed arrowheads denote dimeric and monomeric cystatin F, respectively. Asterisk denotes N-terminal processing of the C26,63S mutant. B) Immunofluorescence microscopy of 293T cells shown in (A) expressing wild-type or C26,63S cystatin F (red) and counterstained with an antibody to the lysosomal marker CD63 (green). Insets demonstrate areas of colocalization at high magnification. Bars, 10 μm. C) Western blot analysis following size exclusion chromatography. Nonreducing SDS–PAGE gel analysis of equal sized aliquots of column fractions obtained following chromatography of wild-type (top panel) or C26,63S (bottom panel) cystatin F. D) The Stokes’ radius of wild-type (3.19 nm) and C26,63S (2.35 nm) cystatin F were calculated following size exclusion chromatography. Values were interpolated from the calibration curve obtained by the linear fitting of Stokes’ radius versus (−log Kav)1/2 for a series of proteins with known Stokes’ radii, as described. Protein calibration was obtained using ribonuclease A (a), carbonic anhydrase (b), ovalbumin (c) and conalbumin (d).
Mentions: To investigate the trafficking of cystatin F, we generated various mutant forms and analyzed these initially in the human kidney epithelial cell line 293T, which does not express cystatin F. The cells were transfected, and after 48 h, the relative amount of cystatin F in cell lysates (L) and in the culture medium (M) was assessed. Expression of wild-type cystatin F leads to significant secretion of the protein into the culture medium as we and others have previously reported (10,13,16). Nonetheless, a significant amount of cystatin F was also retained in 293T cells and was partially converted to the monomeric form (Figure 1A, left panel). Cystatin F recovered from the culture medium was dimeric, whereas cellular cystatin F comprised a mixture of dimeric and monomeric protein (Figure 1A, left panel). Furthermore, while the dimeric form reacted with antibodies raised against the N-terminus of cystatin F the monomeric form did not, indicating intracellular processing had occurred as we recently reported (Figure 1A, center panel) (16). As shown previously, heterogeneous N-glycosylation accounted for the resolution of distinct forms of cystatin F on SDS–PAGE (9). This is highlighted by the formation of a single form following treatment with PNGase (Figure 1A, right panel).

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