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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.

Show MeSH
Proposed model describing the requirement of cystatin F glycosylation for intracellular targeting and internalizationCystatin F is synthesized as an inactive glycosylated disulfide-linked dimer. M6P modified glycans direct cystatin F to lysosomes where N-terminal proteolysis induces its activation (processed cystatin F). A proportion of inactive dimeric cystatin F is secreted where it binds membrane receptors, such as the CI-MPR, on adjacent cells. Internalization of cystatin F leads to a M6P-dependent lysosomal localization where N-terminal processing results in its activation.
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fig07: Proposed model describing the requirement of cystatin F glycosylation for intracellular targeting and internalizationCystatin F is synthesized as an inactive glycosylated disulfide-linked dimer. M6P modified glycans direct cystatin F to lysosomes where N-terminal proteolysis induces its activation (processed cystatin F). A proportion of inactive dimeric cystatin F is secreted where it binds membrane receptors, such as the CI-MPR, on adjacent cells. Internalization of cystatin F leads to a M6P-dependent lysosomal localization where N-terminal processing results in its activation.

Mentions: In summary, our study sheds light on the unusual features of the cystatin F molecule. Glycosylation and more specifically M6P modification serve to target it to the endocytic pathway during biosynthesis, and for a proportion of molecules, by endocytosis from the external medium (Figure 7). In the event when Asn62 is not modified, a ‘backup’ noncanonical N-linked M6P-modified sugar can be added to Asn61. Although the dimeric form is not crucial for intracellular targeting, it appears to be necessary for endocytic uptake. Moreover, dimerization ensures the latency of the inhibitor, which only becomes active following activation by proteases. The possibility that cystatin F is activated by the same protease systems that it inhibits is currently under investigation.


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

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

Proposed model describing the requirement of cystatin F glycosylation for intracellular targeting and internalizationCystatin F is synthesized as an inactive glycosylated disulfide-linked dimer. M6P modified glycans direct cystatin F to lysosomes where N-terminal proteolysis induces its activation (processed cystatin F). A proportion of inactive dimeric cystatin F is secreted where it binds membrane receptors, such as the CI-MPR, on adjacent cells. Internalization of cystatin F leads to a M6P-dependent lysosomal localization where N-terminal processing results in its activation.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2691902&req=5

fig07: Proposed model describing the requirement of cystatin F glycosylation for intracellular targeting and internalizationCystatin F is synthesized as an inactive glycosylated disulfide-linked dimer. M6P modified glycans direct cystatin F to lysosomes where N-terminal proteolysis induces its activation (processed cystatin F). A proportion of inactive dimeric cystatin F is secreted where it binds membrane receptors, such as the CI-MPR, on adjacent cells. Internalization of cystatin F leads to a M6P-dependent lysosomal localization where N-terminal processing results in its activation.
Mentions: In summary, our study sheds light on the unusual features of the cystatin F molecule. Glycosylation and more specifically M6P modification serve to target it to the endocytic pathway during biosynthesis, and for a proportion of molecules, by endocytosis from the external medium (Figure 7). In the event when Asn62 is not modified, a ‘backup’ noncanonical N-linked M6P-modified sugar can be added to Asn61. Although the dimeric form is not crucial for intracellular targeting, it appears to be necessary for endocytic uptake. Moreover, dimerization ensures the latency of the inhibitor, which only becomes active following activation by proteases. The possibility that cystatin F is activated by the same protease systems that it inhibits is currently under investigation.

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