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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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Mutagenesis of consensus sequences for N-linked glycosylation reveals an atypical glycosylation siteA) Reducing SDS–PAGE analysis of mutants of one (N62S or N115S) or both (N62,115S) N-linked glycosylation sequences. Remaining glycosylation of cystatin F (Cyst. F) is revealed by comparing untreated samples (−) to PNGase treatment (+). (B) Human cystatin F amino acid residues 61–63 (NXC) represent a potential atypical N-linked glycosylation site. (C) Reducing SDS–PAGE comparing glycosylation of double (N61,62S) and triple (N61,62,115S) Asn mutants in the presence (+) or absence (−) of PNGase. Representative cell lysates (Lys) and culture medium (Med) are shown. (D) Immunofluorescence microscopy following transfection of 293T cells (as in C) with antibodies raised against full-length cystatin F (red) and markers of lysosomes (CD63) or the trans-Golgi network (TGN46) (green). Insets reveal areas of costaining. Bars, 10 μm.
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fig02: Mutagenesis of consensus sequences for N-linked glycosylation reveals an atypical glycosylation siteA) Reducing SDS–PAGE analysis of mutants of one (N62S or N115S) or both (N62,115S) N-linked glycosylation sequences. Remaining glycosylation of cystatin F (Cyst. F) is revealed by comparing untreated samples (−) to PNGase treatment (+). (B) Human cystatin F amino acid residues 61–63 (NXC) represent a potential atypical N-linked glycosylation site. (C) Reducing SDS–PAGE comparing glycosylation of double (N61,62S) and triple (N61,62,115S) Asn mutants in the presence (+) or absence (−) of PNGase. Representative cell lysates (Lys) and culture medium (Med) are shown. (D) Immunofluorescence microscopy following transfection of 293T cells (as in C) with antibodies raised against full-length cystatin F (red) and markers of lysosomes (CD63) or the trans-Golgi network (TGN46) (green). Insets reveal areas of costaining. Bars, 10 μm.

Mentions: We next assessed the possibility that glycosylation of cystatin F was important for targeting the endosomal/lysosomal system. In human cystatin F asparagines 62 and 115 carry carbohydrate chains, whereas murine cystatin F carries a single N-linked sugar at a site homologous to Asn62 in humans. We generated mutant forms of human cystatin F where one or both Asn residues were altered to Ser (N62S, N115S and N62,115S) and transfected these into 293T cells. Culture medium and cell lysates were then analyzed for the presence of cystatin F and digested with PNGase to assess its glycosylation status. Similar to wild-type cystatin F, all mutant forms of cystatin F migrated as a single band following treatment with PNGase. Puzzling, however, the gel mobility of the N62S mutant in the absence of PNGase digestion was similar to that of wild type (Figure 2A). Even more surprising is that the double mutant N62,115S, which we predicted would not be glycosylated, migrated as two species, one of which retained sensitivity to digestion with PNGase. Because N62 and N115 are the only Asn residues found in a conventional Asn-X-Thr/Ser glycosylation consensus sequence, these results pointed to the existence of an unusual glycosylation site on cystatin F. Glycosylation of Asn residues in the context an atypical Asn-X-Cys sequence has previously been observed, for example in the recombinant epidermal growth factor receptor, the T-cell activation antigen CD69 and a few other proteins (20,21). As shown in Figure 2B, Asn61 of cystatin F also occurs in this context. We therefore tested the hypothesis that mutagenesis of Asn62 might reveal a cryptic noncanonical glycosylation site at Asn61. Consistent with this possibility, the double mutant N61,62S now expressed a single PNGase-sensitive product, and the triple mutant N61,62,115S was fully resistant to the action of PNGase (Figure 2C). Thus, the Asn61-X-Cys63 sequence in cystatin F can be used by the glycosylation machinery.


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

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

Mutagenesis of consensus sequences for N-linked glycosylation reveals an atypical glycosylation siteA) Reducing SDS–PAGE analysis of mutants of one (N62S or N115S) or both (N62,115S) N-linked glycosylation sequences. Remaining glycosylation of cystatin F (Cyst. F) is revealed by comparing untreated samples (−) to PNGase treatment (+). (B) Human cystatin F amino acid residues 61–63 (NXC) represent a potential atypical N-linked glycosylation site. (C) Reducing SDS–PAGE comparing glycosylation of double (N61,62S) and triple (N61,62,115S) Asn mutants in the presence (+) or absence (−) of PNGase. Representative cell lysates (Lys) and culture medium (Med) are shown. (D) Immunofluorescence microscopy following transfection of 293T cells (as in C) with antibodies raised against full-length cystatin F (red) and markers of lysosomes (CD63) or the trans-Golgi network (TGN46) (green). Insets reveal areas of costaining. Bars, 10 μm.
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Related In: Results  -  Collection

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fig02: Mutagenesis of consensus sequences for N-linked glycosylation reveals an atypical glycosylation siteA) Reducing SDS–PAGE analysis of mutants of one (N62S or N115S) or both (N62,115S) N-linked glycosylation sequences. Remaining glycosylation of cystatin F (Cyst. F) is revealed by comparing untreated samples (−) to PNGase treatment (+). (B) Human cystatin F amino acid residues 61–63 (NXC) represent a potential atypical N-linked glycosylation site. (C) Reducing SDS–PAGE comparing glycosylation of double (N61,62S) and triple (N61,62,115S) Asn mutants in the presence (+) or absence (−) of PNGase. Representative cell lysates (Lys) and culture medium (Med) are shown. (D) Immunofluorescence microscopy following transfection of 293T cells (as in C) with antibodies raised against full-length cystatin F (red) and markers of lysosomes (CD63) or the trans-Golgi network (TGN46) (green). Insets reveal areas of costaining. Bars, 10 μm.
Mentions: We next assessed the possibility that glycosylation of cystatin F was important for targeting the endosomal/lysosomal system. In human cystatin F asparagines 62 and 115 carry carbohydrate chains, whereas murine cystatin F carries a single N-linked sugar at a site homologous to Asn62 in humans. We generated mutant forms of human cystatin F where one or both Asn residues were altered to Ser (N62S, N115S and N62,115S) and transfected these into 293T cells. Culture medium and cell lysates were then analyzed for the presence of cystatin F and digested with PNGase to assess its glycosylation status. Similar to wild-type cystatin F, all mutant forms of cystatin F migrated as a single band following treatment with PNGase. Puzzling, however, the gel mobility of the N62S mutant in the absence of PNGase digestion was similar to that of wild type (Figure 2A). Even more surprising is that the double mutant N62,115S, which we predicted would not be glycosylated, migrated as two species, one of which retained sensitivity to digestion with PNGase. Because N62 and N115 are the only Asn residues found in a conventional Asn-X-Thr/Ser glycosylation consensus sequence, these results pointed to the existence of an unusual glycosylation site on cystatin F. Glycosylation of Asn residues in the context an atypical Asn-X-Cys sequence has previously been observed, for example in the recombinant epidermal growth factor receptor, the T-cell activation antigen CD69 and a few other proteins (20,21). As shown in Figure 2B, Asn61 of cystatin F also occurs in this context. We therefore tested the hypothesis that mutagenesis of Asn62 might reveal a cryptic noncanonical glycosylation site at Asn61. Consistent with this possibility, the double mutant N61,62S now expressed a single PNGase-sensitive product, and the triple mutant N61,62,115S was fully resistant to the action of PNGase (Figure 2C). Thus, the Asn61-X-Cys63 sequence in cystatin F can be used by the glycosylation machinery.

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